Publications | The Sorek Lab

2025

Phage therapy with nebulized cocktail BX004-A for chronic Pseudomonas aeruginosa infections in cystic fibrosis: a randomized first-in-human trial

Weiner I., Kahan-Hanum M., Buchstab N., Zelcbuch L., Navok S., Sherman I., Nicenboim J., Axelrod T., Berko-Ashur D., Olshina M., Mordoch R., Jablonska J., Gera-Inbar T., Vainberg-Slutskin I., Blumenfeld B., Sberro-Livnat H., Moses A., Nevenzel H., Levy-Saar I., Gold J., Goldberg M., Tchernorudsky N., Ben-Yishay N., Cohen T., Kario E., Levovich I., Safonov R., Tzur Y., Zarchin Y., Lev V., Elharar Y., Safyon-Gartman E., Gahali-Sass I., Puttagunta S., Ussery X., Vilchez R., Rappo U., Zak N., Golembo M., Cohen A., Koff J., Kerem E., Sorek R. & Bassan M. Nature Communications, 16, 5579 (2025)

Cystic fibrosis is a monogenetic disease complicated by recurrent bacterial lung infections that require chronic antibiotics. Pseudomonas aeruginosa is an increasingly antibiotic-resistant pathogen associated with cystic fibrosis morbidity and mortality. Here, we describe the development of a three-phage cocktail (BX004-A) designed to target a wide range of P. aeruginosa strains. We evaluated BX004-A in Part 1 of a first-in-human double-blind placebo-controlled phase 1b/2a clinical trial, which included nine adult cystic fibrosis patients chronically infected with P. aeruginosa (NCT05010577). BX004-A met the primary endpoints of safety and tolerability. Exploratory endpoints included pharmacokinetics and Pseudomonas aeruginosa sputum density reduction. Efficient phage delivery to the lower respiratory tract was observed, and a potential reduction in P. aeruginosa sputum burden was noted in the phage arm. However, due to the studys small sample size, definitive conclusions regarding efficacy are limited. These data pave the way toward further development of novel phage-based therapeutics in antibiotic-resistant pulmonary bacterial infections.

A widespread family of viral sponge proteins reveals specific inhibition of nucleotide signals in anti-phage defense

Chang RB, Toyoda HC, Hobbs SJ, Hadary R, Richmond-Buccola D, Wein T, Osterman I, Burger N, Chouchani ET, Sorek R & Kranzusch PJ. Molecular Cell, 85, 16, p. 3151-3165.e6 (2025)

Cyclic oligonucleotide-based anti-phage signaling systems (CBASSs) are bacterial anti-phage defense operons that use nucleotide signals to control immune activation. Here, we biochemically screen 57 diverse E. coli and Bacillus phages for the ability to disrupt CBASS immunity and discover anti-CBASS 4 (Acb4) from the Bacillus phage SPO1 as the founding member of a large family of >1,300 immune evasion proteins. A 2.1 Å crystal structure of Acb4 in complex with 33-cyclic guanosine monophosphate (GMP)-AMP (33-cGAMP) reveals a tetrameric assembly that functions as a sponge to sequester CBASS signals and inhibit immune activation. We demonstrate that Acb4 alone is sufficient to disrupt CBASS activation in vitro and enable immune evasion in vivo. Analyzing phages that infect diverse bacteria, we explain how Acb4 selectively targets nucleotide signals in host defense and avoids disruption of cellular homeostasis. Together, our results reveal principles of immune evasion protein evolution and explain a major mechanism phages use to inhibit host immunity.

Manipulation of the nucleotide pool in human, bacterial and plant immunity

Hochhauser D, Sorek R. Manipulation of the nucleotide pool in human, bacterial and plant immunity. Nature Reviews Immunology , 26:7–22 (2026)

Hochhauser_Nat_Rev_Immunol_2026.pdf

The cell-autonomous innate immune system is responsible for sensing and mitigating viral infection at the level of individual cells. Many of the mechanisms used by the cell-autonomous innate immune system in eukaryotic cells are ancient and have evolutionary roots in bacterial systems that defend against phage infection. Studies from recent years have shown that modification of the free nucleotide pool is central to many of these conserved immune mechanisms. In this Review, we explain how immune pathways manipulate the available pool of nucleotides to deprive viruses of molecules essential for their replication, how immune proteins chemically modify nucleotides to generate immune signalling molecules, and how cell-autonomous innate immune mechanisms produce altered nucleotides that poison viral replication. We also discuss the mechanisms used by viruses to antagonize nucleotide-based immunity. Finally, we explore the evolutionary logic of using nucleotides as building blocks for immune responses.

A DNA-gated molecular guard controls bacterial Hailong anti-phage defence

Tan JM, Melamed S, Cofsky JC, Syangtan D, Hobbs SJ, del Mármol J, Jost M, Kruse AC, Sorek R & Kranzusch PJ. Nature, 643(8072):794-800 (2025)

Animal and bacterial cells use nucleotidyltransferase (NTase) enzymes to respond to viral infection and control major forms of immune signalling including cGAS-STING innate immunity and CBASS anti-phage defence^{1, 2, 34}. Here we discover a family of bacterial defence systems, which we name Hailong, that use NTase enzymes to constitutively synthesize DNA signals and guard against phage infection. Hailong protein B (HalB) is an NTase that converts deoxy-ATP into single-stranded DNA oligomers. A series of X-ray crystal structures define a stepwise mechanism of HalB DNA synthesis initiated by a C-terminal tyrosine residue that enables de novo enzymatic priming. We show that HalB DNA signals bind to and repress activation of a partnering Hailong protein A (HalA) effector complex. A 2.0-Å cryo-electron microscopy structure of the HalADNA complex reveals a membrane protein with a conserved ion channel domain and a unique crown domain that binds the DNA signal and gates activation. Analysing Hailong defence in vivo, we demonstrate that viral DNA exonucleases required for phage replication trigger release of the primed HalA complex and induce protective host cell growth arrest. Our results explain how inhibitory nucleotide immune signals can serve as molecular guards against phage infection and expand the mechanisms NTase enzymes use to control antiviral immunity.

TIR domains produce histidine-ADPR as an immune signal in bacteria

Sabonis D, Avraham C, Chang RB, Lu A, Herbst E, Silanskas A, Vilutis D, Leavitt A, Yirmiya E, Toyoda HC, Ruksenaite A, Zaremba M, Osterman I, Amitai G, Kranzusch PJ, Sorek R, Tamulaitiene G. Nature, 642:467–473 (2025)

Sabonis_Nature_2025.pdf

Toll/interleukin-1 receptor (TIR) domains are central components of pattern recognition immune proteins across all domains of life^1,2. In bacteria and plants, TIR-domain proteins recognize pathogen invasion and then produce immune signalling molecules exclusively comprising nucleotide moieties^{2, 3, 45}. Here we show that the TIR-domain protein of the type II Thoeris defence system in bacteria produces a unique signalling molecule comprising the amino acid histidine conjugated to ADP-ribose (His-ADPR). His-ADPR is generated in response to phage infection and activates the cognate Thoeris effector by binding a Macro domain located at the C terminus of the effector protein. By determining the crystal structure of a ligand-bound Macro domain, we describe the structural basis for His-ADPR and its recognition and show its role by biochemical and mutational analyses. Our analyses furthermore reveal a family of phage proteins that bind and sequester His-ADPR signalling molecules, enabling phages to evade TIR-mediated immunity. These data demonstrate diversity in bacterial TIR signalling and reveal a new class of TIR-derived immune signalling molecules that combine nucleotide and amino acid moieties.

Structure-guided discovery of viral proteins that inhibit host immunity

Yirmiya E., Hobbs S. J., Leavitt A., Osterman I., Avraham C., Hochhauser D., Madhala B., Skovorodka M., Tan J. M., Toyoda H. C., Chebotar I., Itkin M., Malitsky S., Amitai G., Kranzusch P. J. & Sorek R. Cell, 188(6):1681-1692.e17 (2025)

Yirmiya_Cell_2025.pdf

Viruses encode proteins that inhibit host defenses, but sifting through the millions of available viral sequences for immune-modulatory proteins has been so far impractical. Here, we develop a process to systematically screen virus-encoded proteins for inhibitors that physically bind host immune proteins. Focusing on Thoeris and CBASS, bacterial defense systems that are the ancestors of eukaryotic Toll/interleukin-1 receptor (TIR) and cyclic GMP-AMP synthase (cGAS) immunity, we discover seven families of Thoeris and CBASS inhibitors, encompassing thousands of genes widespread in phages. Verified inhibitors exhibit extensive physical interactions with the respective immune protein counterpart, with all inhibitors blocking the active site of the immune protein. Remarkably, a phage-encoded inhibitor of bacterial TIR proteins can bind and inhibit distantly related human and plant immune TIRs, and a phage-derived inhibitor of bacterial cGAS-like enzymes can inhibit the human cGAS. Our results demonstrate that phages are a reservoir for immune-modulatory proteins capable of inhibiting bacterial, animal, and plant immunity.

CARD domains mediate anti-phage defence in bacterial gasdermin systems

Wein T, Millman A, Lange K, Yirmiya E, Hadary R, Garb J, Melamed S, Amitai G, Dym O, Steinruecke F, Hill AB, Kranzusch PJ, Sorek R. Nature, 639(8055):727-734 (2025)

Wein_Nature_2025.pdf

Caspase recruitment domains (CARDs) and pyrin domains are important facilitators of inflammasome activity and pyroptosis¹. Following pathogen recognition by nucleotide binding-domain, leucine-rich, repeat-containing (NLR) proteins, CARDs recruit and activate caspases, which, in turn, activate gasdermin pore-forming proteins to induce pyroptotic cell death². Here we show that CARD domains are present in defence systems that protect bacteria against phage. The bacterial CARD domain is essential for protease-mediated activation of certain bacterial gasdermins, which promote cell death once phage infection is recognized. We further show that multiple anti-phage defence systems use CARD domains to activate a variety of cell death effectors, and that CARD domains mediate proteinprotein interactions in these systems. We find that these systems are triggered by a conserved immune-evasion protein used by phages to overcome the bacterial defence system RexAB³, demonstrating that phage proteins inhibiting one defence system can activate another. Our results suggest that CARD domains represent an ancient component of innate immune systems conserved from bacteria to humans, and that CARD-dependent activation of gasdermins is shared in organisms across the tree of life.

TIR signaling activates caspase-like immunity in bacteria

Rousset F., Osterman I., Scherf T., Falkovich A. H., Leavitt A., Amitai G., Shir S., Malitsky S., Itkin M., Savidor A. & Sorek R. Science, 387(6733):510-516 (2025)

Rousset_Science_2025.pdf

Caspase family proteases and Toll/interleukin-1 receptor (TIR)-domain proteins have central roles in innate immunity and regulated cell death in humans. We describe a bacterial immune system comprising both a caspase-like protease and a TIR-domain protein. We found that the TIR protein, once it recognizes phage invasion, produces the previously unknown immune signaling molecule adenosine 5-diphosphate-cyclo[N7:1]-ribose (N7-cADPR). This molecule specifically activates the bacterial caspase-like protease, which then indiscriminately degrades cellular proteins to halt phage replication. The TIR-caspase defense system, which we denote as type IV Thoeris, is abundant in bacteria and efficiently protects against phage propagation. Our study highlights the diversity of TIR-produced immune signaling molecules and demonstrates that cell death regulated by proteases of the caspase family is an ancient mechanism of innate immunity.

2024

Diversification of molecular pattern recognition in bacterial NLR-like proteins

Béchon N., Tal N., Stokar-Avihail A., Savidor A., Kupervaser M., Melamed S., Amitai G. & Sorek R. Nature Communications , 15:9860 (2024)

Bechon_Nature_Communication_2024.pdf

Antiviral STANDs (Avs) are bacterial anti-phage proteins evolutionarily related to immune pattern recognition receptors of the NLR family. Type 2 Avs proteins (Avs2) were suggested to recognize the phage large terminase subunit as a signature of phage infection. Here, we show that Avs2 from Klebsiella pneumoniae (KpAvs2) can recognize several different phage proteins as signature for infection. While KpAvs2 recognizes the large terminase subunit of Seuratvirus phages, we find that to protect against Dhillonvirus phages, KpAvs2 recognizes a different phage protein named KpAvs2-stimulating protein 1 (Ksap1). KpAvs2 directly binds Ksap1 to become activated, and phages mutated in Ksap1 escape KpAvs2 defense despite encoding an intact terminase. We further show that KpAvs2 protects against a third group of phages by recognizing another protein, Ksap2. Our results exemplify the evolutionary diversification of molecular pattern recognition in bacterial Avs2, and show that a single pattern recognition receptor evolved to recognize different phage-encoded proteins.

Tricking phages with a reverse move: An antiviral gene is absent in DNA but expressed by rolling circle reverse transcription.

Osterman I. & Sorek R. Science, 386(6717):25-26 (2024)

Osterman_Science_2024.pdf

The textbook definition of a protein-coding gene requires it to be linearly encoded along the DNA axis so that the beginning of the gene is always upstream of the genes end. On pages 40 and 41 of this issue, Tang et al. (1) and Wilkinson et al. (2), respectively, challenge this paradigm and reveal peculiar antiphage proteins in bacteria that are not directly encoded in the bacterial DNA. To produce these proteins, DNA is first transcribed into a noncoding RNA (ncRNA), which is converted back to DNA by a rolling circle reverse transcription reaction. The resulting DNA is subsequently transcribed into an mRNA that encodes the protein. The protein assists bacteria to overcome infection by viruses (phage).

Phages reconstitute NAD⁺ to counter bacterial immunity

Osterman I., Samra H., Rousset F., Loseva E., Itkin M., Malitsky S., Yirmiya E., Millman A. & Sorek R. Nature, 634(8036):1160-1167 (2024)

Accepted version

Osterman_Nature_2024.pdf

Bacteria defend against phage infection through a variety of antiphage defence systems¹. Many defence systems were recently shown to deplete cellular nicotinamide adenine dinucleotide (NAD⁺) in response to infection, by cleaving NAD⁺ into ADP-ribose (ADPR) and nicotinamide²⁷. It was demonstrated that NAD⁺ depletion during infection deprives the phage of this essential molecule and impedes phage replication. Here we show that a substantial fraction of phages possess enzymatic pathways allowing reconstitution of NAD⁺ from its degradation products in infected cells. We describe NAD⁺ reconstitution pathway 1 (NARP1), a two-step pathway in which one enzyme phosphorylates ADPR to generate ADPR pyrophosphate (ADPR-PP), and the second enzyme conjugates ADPR-PP and nicotinamide to generate NAD⁺. Phages encoding NARP1 can overcome a diverse set of defence systems, including Thoeris, DSR1, DSR2, SIR2HerA and SEFIR, all of which deplete NAD⁺ as part of their defensive mechanism. Phylogenetic analyses show that NARP1 is primarily encoded on phage genomes, suggesting a phage-specific function in countering bacterial defences. A second pathway, NARP2, allows phages to overcome bacterial defences by building NAD⁺ using metabolites different from ADPR-PP. Our findings reveal a unique immune evasion strategy in which viruses rebuild molecules depleted by defence systems, thus overcoming host immunity.

Phage genome engineering with retrons

Osterman I. & Sorek R. Nature Biotechnology, doi: 10.1038/s41587-024-02392-z (2024)

Osterman_Nat_Biotech_2024.pdf

Bacteria conjugate ubiquitin-like proteins to interfere with phage assembly

Hör J., Wolf S. G. & Sorek R. Nature, 631:850-856 (2024)

Accepted version

Hoer_Nature_2024.pdf

Several immune pathways in humans conjugate ubiquitin-like proteins to virus and host molecules as a means of antiviral defence¹⁵. Here we studied an antiphage defence system in bacteria, comprising a ubiquitin-like protein, ubiquitin-conjugating enzymes E1 and E2, and a deubiquitinase. We show that during phage infection, this system specifically conjugates the ubiquitin-like protein to the phage central tail fibre, a protein at the tip of the tail that is essential for tail assembly as well as for recognition of the target host receptor. Following infection, cells encoding this defence system release a mixture of partially assembled, tailless phage particles and fully assembled phages in which the central tail fibre is obstructed by the covalently attached ubiquitin-like protein. These phages show severely impaired infectivity, explaining how the defence system protects the bacterial population from the spread of phage infection. Our findings demonstrate that conjugation of ubiquitin-like proteins is an antiviral strategy conserved across the tree of life.

The SARM1 TIR domain produces glycocyclic ADPR molecules as minor products

Garb J., Amitai G., Lu A., Ofir G., Brandis A., Mehlman T., Kranzusch P. J. & Sorek R. PLoS ONE, 19, 4 , e0302251 (2024).

Sterile alpha and TIR motif-containing 1 (SARM1) is a protein involved in programmed death of injured axons. Following axon injury or a drug-induced insult, the TIR domain of SARM1 degrades the essential molecule nicotinamide adenine dinucleotide (NAD+), leading to a form of axonal death called Wallerian degeneration. Degradation of NAD+ by SARM1 is essential for the Wallerian degeneration process, but accumulating evidence suggest that other activities of SARM1, beyond the mere degradation of NAD+, may be necessary for programmed axonal death. In this study we show that the TIR domains of both human and fruit fly SARM1 produce 10 0-20 and 10 0-30 glycocyclic ADP-ribose (gcADPR) molecules as minor products. As previously reported, we observed that SARM1 TIR domains mostly convert NAD+ to ADPR (for human SARM1) or cADPR (in the case of SARM1 from Drosophila melanogaster). However, we now show that human and Drosophila SARM1 additionally convert ∼0.1-0.5% of NAD+ into gcADPR molecules. We find that SARM1 TIR domains produce gcADPR molecules both when purified in vitro and when expressed in bacterial cells. Given that gcADPR is a second messenger involved in programmed cell death in bacteria and likely in plants, we propose that gcADPR may play a role in SARM1- induced programmed axonal death in animals.

Activation of Thoeris antiviral system via SIR2 effector filament assembly

Tamulaitiene G., Sabonis D., Sasnauskas G., Ruksenaite A., Silanskas A., Avraham C., Ofir G., Sorek R., Zaremba M. & Siksnys V. Nature, 627:431-436 (2024)

Tamulaitiene et al. 2024.pdf

To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems¹⁷. Some of them (for example, type III CRISPRCas, CBASS, Pycsar and Thoeris) consist of two modules: a sensor responsible for infection recognition and an effector that stops viral replication by destroying key cellular components⁸¹². In the Thoeris system, a Toll/interleukin-1 receptor (TIR)-domain protein, ThsB, acts as a sensor that synthesizes an isomer of cyclic ADP ribose, 1−3 glycocyclic ADP ribose (gcADPR), which is bound in the Smf/DprA-LOG (SLOG) domain of the ThsA effector and activates the silent information regulator 2 (SIR2)-domain-mediated hydrolysis of a key cell metabolite, NAD⁺ (refs. ¹²¹⁴). Although the structure of ThsA has been solved¹⁵, the ThsA activation mechanism remained incompletely understood. Here we show that 1−3 gcADPR, synthesized in vitro by the dimeric ThsB protein, binds to the ThsA SLOG domain, thereby activating ThsA by triggering helical filament assembly of ThsA tetramers. The cryogenic electron microscopy (cryo-EM) structure of activated ThsA revealed that filament assembly stabilizes the active conformation of the ThsA SIR2 domain, enabling rapid NAD⁺ depletion. Furthermore, we demonstrate that filament formation enables a switch-like response of ThsA to the 1−3 gcADPR signal.

Structural basis of Gabija anti-phage defence and viral immune evasion

Antine S. P., Johnson A. G., Mooney S. E., Leavitt A., Mayer M. L., Yirmiya E., Amitai G., Sorek R. & Kranzusch P. J. Nature, 625:360-365 (2024).

Antine et al. 2023.pdf

Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation¹⁵. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes^1,6,7, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajAGajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.

Phages overcome bacterial immunity via diverse anti-defence proteins

Yirmiya E., Leavitt A., Lu A., Ragucci A. E., Avraham C., Osterman I., Garb J., Antine S. P., Mooney S. E., Hobbs S. J., Kranzusch P. J., Amitai G. & Sorek R. Nature, 625:352-359 (2024).

Accepted version

Yirmiya et al. 2023.pdf

It was recently shown that bacteria use, apart from CRISPRCas and restriction systems, a considerable diversity of phage resistance systems¹⁴, but it is largely unknown how phages cope with this multilayered bacterial immunity. Here we analysed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defence systems, and discovered four distinct families of anti-defence proteins that inhibit the Gabija, Thoeris and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2 and Had1 efficiently cancel the defensive activity when co-expressed with the respective defence system or introduced into phage genomes. Homologues of these anti-defence proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defence complex to cleave phage-derived DNA. Our data further reveal that the anti-Thoeris protein Tad2 is a sponge that sequesters the immune signalling molecules produced by Thoeris TIR-domain proteins in response to phage infection. Our results demonstrate that phages encode an arsenal of anti-defence proteins that can disable a variety of bacterial defence mechanisms.

2023

A conserved family of immune effectors cleaves cellular ATP upon viral infection

Rousset F., Yirmiya E., Nesher S., Brandis A., Mehlman T., Itkin M., Malitsky S., Millman A., Melamed S. & Sorek R. Cell, 186:3619-3631.e13 (2023).

Submitted version

Rousset et al 2023.pdf

During viral infection, cells can deploy immune strategies that deprive viruses of molecules essential for their replication. Here, we report a family of immune effectors in bacteria that, upon phage infection, degrade cellular adenosine triphosphate (ATP) and deoxyadenosine triphosphate (dATP) by cleaving the N-glycosidic bond between the adenine and sugar moieties. These ATP nucleosidase effectors are widely distributed within multiple bacterial defense systems, including cyclic oligonucleotide-based antiviral signaling systems (CBASS), prokaryotic argonautes, and nucleotide-binding leucine-rich repeat (NLR)-like proteins, and we show that ATP and dATP degradation during infection halts phage propagation. By analyzing homologs of the immune ATP nucleosidase domain, we discover and characterize Detocs, a family of bacterial defense systems with a two-component phosphotransfer-signaling architecture. The immune ATP nucleosidase domain is also encoded within diverse eukaryotic proteins with immune-like architectures, and we show biochemically that eukaryotic homologs preserve the ATP nucleosidase activity. Our findings suggest that ATP and dATP degradation is a cell-autonomous innate immune strategy conserved across the tree of life.

The evolutionary success of regulated cell death in bacterial immunity

Rousset F. & Sorek R. Current Opinion in Microbiology. 74, 102312 (2023).

Rousset Sorek 2023.pdf

Bacteria employ a complex arsenal of immune mechanisms to defend themselves against phages. Recent studies demonstrate that these immune mechanisms frequently involve regulated cell death in response to phage infection. By sacrificing infected cells, this strategy prevents the spread of phages within the surrounding population. In this review, we discuss the principles of regulated cell death in bacterial defense, and show that over 70% of sequenced prokaryotes employ this strategy as part of their defensive arsenals. We highlight the modularity of defense systems involving regulated cell death, explaining how shuffling between phage-sensing and cell-killing protein domains dominates their evolution. Some of these defense systems are the evolutionary ancestors of key components of eukaryotic immunity, highlighting their importance in shaping the evolutionary trajectory of immune systems across the tree of life.

The defense island repertoire of the Escherichia coli pan-genome

Hochhauser D., Millman A. & Sorek R. PLoS Genetics, 19:4, e1010694(2023).

Hochhauser et al 2023.pdf

It has become clear in recent years that anti-phage defense systems cluster non-randomly within bacterial genomes in so-called "defense islands". Despite serving as a valuable tool for the discovery of novel defense systems, the nature and distribution of defense islands themselves remain poorly understood. In this study, we comprehensively mapped the defense system repertoire of >1,300 strains of Escherichia coli, the most widely studied organism for phage-bacteria interactions. We found that defense systems are usually carried on mobile genetic elements including prophages, integrative conjugative elements and transposons, which preferentially integrate at several dozens of dedicated hotspots in the E. coli genome. Each mobile genetic element type has a preferred integration position but can carry a diverse variety of defensive cargo. On average, an E. coli genome has 4.7 hotspots occupied by defense system-containing mobile elements, with some strains possessing up to eight defensively occupied hotspots. Defense systems frequently co-localize with other systems on the same mobile genetic element, in agreement with the observed defense island phenomenon. Our data show that the overwhelming majority of the E. coli panimmune system is carried on mobile genetic elements, explaining why the immune repertoire varies substantially between different strains of the same species.

Discovery of phage determinants that confer sensitivity to bacterial immune systems

Stokar-Avihail A., Fedorenko T., Hör J., Garb J., Leavitt A., Millman A., Shulman G., Wojtania N., Melamed S., Amitai G. & Sorek R. Cell, 186:1863-1876 (2023).

Submitted version

Stokar et al 2023.pdf

Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. Although the mechanism of defense for some of these systems is understood, a major unanswered question is how these systems sense phage infection. To systematically address this question, we isolated 177 phage mutants that escape 15 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders.

Cryo-EM structure of the RADAR supramolecular anti-phage defense complex

Duncan-Lowey B., Tal N., Johnson A. G., Rawson S., Mayer M. L., Doron S., Millman A., Melamed S., Fedorenko T., Kacen A., Brandis A., Mehlman T., Amitai G., Sorek R. & Kranzusch P. J. Cell, 186:987-998 (2023).

Duncan-Lowey et al 2023.pdf

RADAR is a two-protein bacterial defense system that was reported to defend against phage by \u201cediting\u201d messenger RNA. Here, we determine cryo-EM structures of the RADAR defense complex, revealing RdrA as a heptameric, two-layered AAA+ ATPase and RdrB as a dodecameric, hollow complex with twelve surface-exposed deaminase active sites. RdrA and RdrB join to form a giant assembly up to 10 MDa, with RdrA docked as a funnel over the RdrB active site. Surprisingly, our structures reveal an RdrB active site that targets mononucleotides. We show that RdrB catalyzes ATP-to-ITP conversion in vitro and induces the massive accumulation of inosine mononucleotides during phage infection in vivo, limiting phage replication. Our results define ATP mononucleotide deamination as a determinant of RADAR immunity and reveal supramolecular assembly of a nucleotide-modifying machine as a mechanism of anti-phage defense.

2022

An expanded arsenal of immune systems that protect bacteria from phages

Millman A., Melamed S., Leavitt A., Doron S., Bernheim A., Hör J., Garb J., Bechon N., Brandis A., Lopatina A., Ofir G., Hochhauser D., Stokar-Avihail A., Tal N., Sharir S., Voichek M., Erez Z., Ferrer J. L. M., Dar D., Kacen A., Amitai G. & Sorek R. (2022) Cell Host and Microbe. 30, 11, p. 1556-1569.e5

Accepted version

Millman et al 2022.pdf

Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection.

Viruses inhibit TIR gcADPR signalling to overcome bacterial defence

Leavitt A., Yirmiya E., Amitai G., Lu A., Garb J., Herbst E., Morehouse B. R., Hobbs S. J., Antine S. P., Sun Z. J., Kranzusch P. J. & Sorek R. Nature , 611(7935):326-331 (2022)

Submitted version

Leavitt et al 2022.pdf

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants and animals¹³. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signalling molecule whose molecular structure remains elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function¹. We identified a large family of phage-encoded proteins, denoted here as Thoeris anti-defence 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are sponges that bind and sequester the immune signalling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. Tad1 can also efficiently sequester molecules derived from a plant TIR-domain protein, and a high-resolution crystal structure of Tad1 bound to a plant-derived molecule showed a unique chemical structure of 1 2 glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 13 gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 12 gcADPR. Our results define the chemical structure of a central immune signalling molecule and show a new mode of action by which pathogens can suppress host immunity.

Multiple phage resistance systems inhibit infection via SIR2-dependent NAD⁺ depletion

Garb J., Lopatina A., Bernheim A., Zaremba M., Siksnys V., Melamed S., Leavitt A., Millman A., Amitai G. & Sorek R. Nature Microbiology, 7(11):1849-1856 (2022)

Submitted version

Accepted version

Garb et al 2022.pdf

Defence-associated sirtuins (DSRs) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbour an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD⁺) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defence systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages.

Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD⁺ depletion

Zaremba M., Dakineviciene D., Golovinas E., Zagorskaite E., Stankunas E., Lopatina A., Sorek R., Manakova E., Ruksenaite A., Silanskas A., Asmontas S., Grybauskas A., Tylenyte U., Jurgelaitis E., Grigaitis R., Timinskas K., Venclovas C. & Siksnys V. (2022) Nature Microbiology. 7, 11, p. 1857-1869

Submitted version

Zaremba et al 2022.pdf

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). The majority (~60%) of pAgos identified bioinformatically are shorter (comprising only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here we show that Geobacter sulfurreducens short pAgo and the NAD ⁺-bound Sir2 protein form a stable heterodimeric complex. The GsSir2/Ago complex presumably recognizes invading plasmid or phage DNA and activates the Sir2 subunit, which triggers endogenous NAD ⁺ depletion and cell death, and prevents the propagation of invading DNA. We reconstituted NAD ⁺ depletion activity in vitro and showed that activated GsSir2/Ago complex functions as a NADase that hydrolyses NAD ⁺ to ADPR. Thus, short Sir2-associated pAgos provide defence against phages and plasmids, underscoring the diversity of mechanisms of prokaryotic Agos.

Bacterial origins of human cell-autonomous innate immune mechanisms

Wein T. & Sorek R. Nature Reviews in Immunology, 22:629-638 (2022)

Wein Sorek 2022.pdf

The cell-autonomous innate immune system enables animal cells to resist viral infection. This system comprises an array of sensors that, after detecting viral molecules, activate the expression of antiviral proteins and the interferon response. The repertoire of immune sensors and antiviral proteins has long been considered to be derived from extensive evolutionary innovation in vertebrates, but new data challenge this dogma. Recent studies show that central components of the cell-autonomous innate immune system have ancient evolutionary roots in prokaryotic genes that protect bacteria from phages. These include the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, Toll/IL-1 receptor (TIR) domain-containing pathogen receptors, the viperin family of antiviral proteins, SAMHD1-like nucleotide-depletion enzymes, gasdermin proteins and key components of the RNA interference pathway. This Perspective details current knowledge of the elements of antiviral immunity that are conserved from bacteria to humans, and presents possible evolutionary scenarios to explain the observed conservation.

Targeted suppression of human IBD-associated gut microbiota commensals by phage consortia for treatment of intestinal inflammation

Federici S., Kredo-Russo S., Valdes Mas R., Kviatcovsky D., Weinstock E., Matiuhin Y., Silberberg Y., Atarashi K., Furuichi M., Oka A., Liu B., Fibelman M., Weiner I., Khabra E., Cullin N., Ben-Yishai N., Inbar D., Ben David H., Nicenboim J., Kowalsman N., Lieb W., Kario E., Cohen T., Friedman Geffen Y., Zelcbuch L., Cohen A., Rappo U., Gahali-Sass I., Golembo M., Lev Goldman V., Dori-Bachash M., Shapiro H., Moresi C., Cuevas Sierra A., Mohapatra G., Kern L., Zheng D., Nobs S. P., Suez J., Harmelin A., Stettner N., Zak N., Puttagunta S., Bassan M., Honda K., Sokol H., Bang C., Franke A., Schramm C., Maharshak N., Balfour Sartor R., Sorek R. & Elinav E. Cell, 185(16):2879-2898.e24 (2022)

Human gut commensals are increasingly suggested to impact non-communicable diseases, such as inflammatory bowel diseases (IBD), yet their targeted suppression remains a daunting unmet challenge. In four geographically distinct IBD cohorts (n = 537), we identify a clade of Klebsiella pneumoniae (Kp) strains, featuring a unique antibiotics resistance and mobilome signature, to be strongly associated with disease exacerbation and severity. Transfer of clinical IBD-associated Kp strains into colitis-prone, germ-free, and colonized mice enhances intestinal inflammation. Stepwise generation of a lytic five-phage combination, targeting sensitive and resistant IBD-associated Kp clade members through distinct mechanisms, enables effective Kp suppression in colitis-prone mice, driving an attenuated inflammation and disease severity. Proof-of-concept assessment of Kp-targeting phages in an artificial human gut and in healthy volunteers demonstrates gastric acid-dependent phage resilience, safety, and viability in the lower gut. Collectively, we demonstrate the feasibility of orally administered combination phage therapy in avoiding resistance, while effectively inhibiting non-communicable disease-contributing pathobionts.

Bacteria deplete deoxynucleotides to defend against bacteriophage infection

Tal N., Millman A., Stokar Avihail A., Fedorenko T., Leavitt A., Melamed S., Yirmiya E., Avraham C., Brandis A., Mehlman T., Amitai G. & Sorek R. (2022) Nature Microbiology. 7, 8, p. 1200-1209

Accepted version

Tal et al 2022.pdf

DNA viruses and retroviruses consume large quantities of deoxynucleotides (dNTPs) when replicating. The human antiviral factor SAMHD1 takes advantage of this vulnerability in the viral lifecycle, and inhibits viral replication by degrading dNTPs into their constituent deoxynucleosides and inorganic phosphate. Here, we report that bacteria use a similar strategy to defend against bacteriophage infection. We identify a family of defensive bacterial deoxycytidine triphosphate (dCTP) deaminase proteins that convert dCTP into deoxyuracil nucleotides in response to phage infection. We also identify a family of phage resistance genes that encode deoxyguanosine triphosphatase (dGTPase) enzymes, which degrade dGTP into phosphate-free deoxyguanosine and are distant homologues of human SAMHD1. Our results suggest that bacterial defensive proteins deplete specific deoxynucleotides (either dCTP or dGTP) from the nucleotide pool during phage infection, thus starving the phage of an essential DNA building block and halting its replication. Our study shows that manipulation of the dNTP pool is a potent antiviral strategy shared by both prokaryotes and eukaryotes.

The DarTG toxin-antitoxin system provides phage defence by ADP-ribosylating viral DNA

LeRoux M., Srikant S., Teodoro G. I., Zhang T., Littlehale M. L., Doron S., Badiee M., Leung A. K., Sorek R. & Laub M. T. (2022) Nature Microbiology. 7, 7, p. 1028-1040

Accepted version

LeRoux et al 2022.pdf

Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, bacteriophages. To identify such TA systems, we searched bioinformatically for those frequently encoded near known phage defence genes in bacterial genomes. This search identified homologues of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. Representatives from two different subfamilies, DarTG1 and DarTG2, strongly protected E. coli MG1655 against different phages. We demonstrate that for each system, infection with either RB69 or T5 phage, respectively, triggers release of the DarT toxin, a DNA ADP-ribosyltransferase, that then modifies viral DNA and prevents replication, thereby blocking the production of mature virions. Further, we isolated phages that have evolved to overcome DarTG defence either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defence may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.

Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity

Hobbs S. J., Wein T., Lu A., Morehouse B. R., Schnabel J., Leavitt A., Yirmiya E., Sorek R. & Kranzusch P. J. (2022) Nature. 605, 7910, p. 522-526

Hobbs et al 2022.pdf

The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation(1,2). Phages use several strategies to defeat host CRISPR and restriction-modification systems(3-10), but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3'3'-cyclic GMP-AMP define a mechanism of metal-independent hydrolysis 3' of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.

SnapShot: Bacterial immunity

Tal N. & Sorek R. Cell, 185(3):578-578.e1 (2022).

Accepted version

Tal Sorek 2022.pdf

Bacterial gasdermins reveal an ancient mechanism of cell death

Johnson A. G., Wein T., Mayer M. L., Duncan-Lowey B., Yirmiya E., Oppenheimer-Shaanan Y., Amitai G., Sorek R. & Kranzusch P. J. (2022) Science. 375, 6577, p. 221-225

Accepted version

Johnson et al 2022.pdf

Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. We discovered gasdermin homologs encoded in bacteria that defended against phages and executed cell death. Structures of bacterial gasdermins revealed a conserved pore-forming domain that was stabilized in the inactive state with a buried lipid modification. Bacterial gasdermins were activated by dedicated caspase-like proteases that catalyzed site-specific cleavage and the removal of an inhibitory C-terminal peptide. Release of autoinhibition induced the assembly of large and heterogeneous pores that disrupted membrane integrity. Thus, pyroptosis is an ancient form of regulated cell death shared between bacteria and animals.

2021

Antiviral activity of bacterial TIR domains via immune signalling molecules

Ofir G., Herbst E., Baroz M., Cohen D., Millman A., Doron S., Tal N., Malheiro D. B. A., Malitsky S., Amitai G. & Sorek R. Nature, 600(7887):116-120 (2021)

Submitted version

Ofir et al 2021.pdf

The Toll/interleukin-1 receptor (TIR) domain is a canonical component of animal and plant immune systems^1,2. In plants, intracellular pathogen sensing by immune receptors triggers their TIR domains to generate a molecule that is a variant of cyclic ADP-ribose^3,4. This molecule is hypothesized to mediate plant cell death through a pathway that has yet to be resolved⁵. TIR domains have also been shown to be involved in a bacterial anti-phage defence system called Thoeris⁶, but the mechanism of Thoeris defence remained unknown. Here we show that phage infection triggers Thoeris TIR-domain proteins to produce an isomer of cyclic ADP-ribose. This molecular signal activates a second protein, ThsA, which then depletes the cell of the essential molecule nicotinamide adenine dinucleotide (NAD) and leads to abortive infection and cell death. We also show that, similar to eukaryotic innate immune systems, bacterial TIR-domain proteins determine the immunological specificity to the invading pathogen. Our results describe an antiviral signalling pathway in bacteria, and suggest that the generation of intracellular signalling molecules is an ancient immunological function of TIR domains that is conserved in both plant and bacterial immunity.

Effector-mediated membrane disruption controls cell death in CBASS antiphage defense

Duncan-Lowey B., McNamara-Bordewick N. K., Tal N., Sorek R. & Kranzusch P. J. Molecular Cell, 81(24):5039 -5051.e5 (2021)

Duncan et al 2021.pdf

Cyclic oligonucleotide-based antiphage signaling systems (CBASS) are antiviral defense operons that protect bacteria from phage replication. Here, we discover a widespread class of CBASS transmembrane (TM) effector proteins that respond to antiviral nucleotide signals and limit phage propagation through direct membrane disruption. Crystal structures of the Yersinia TM effector Cap15 reveal a compact 8-stranded β-barrel scaffold that forms a cyclic dinucleotide receptor domain that oligomerizes upon activation. We demonstrate that activated Cap15 relocalizes throughout the cell and specifically induces rupture of the inner membrane. Screening for active effectors, we identify the function of distinct families of CBASS TM effectors and demonstrate that cell death via disruption of inner-membrane integrity is a common mechanism of defense. Our results reveal the function of the most prominent class of effector protein in CBASS immunity and define disruption of the inner membrane as a widespread strategy of abortive infection in bacterial phage defense.

Cyclic CMP and cyclic UMP mediate bacterial immunity against phages

Tal N., Morehouse B. R., Millman A., Stokar-Avihail A., Avraham C., Fedorenko T., Yirmiya E., Herbst E., Brandis A., Mehlman T., Oppenheimer-Shaanan Y., Keszei A. F., Shao S., Amitai G., Kranzusch P. J. & Sorek R. Cell, 184(23):5728-5739 (2021)

Accepted version

Tal et al 2021.pdf

The cyclic pyrimidines 3,5-cyclic cytidine monophosphate (cCMP) and 3,5-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3,5-cyclic adenosine monophosphate (cAMP) and 3,5-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria.

A treasure trove of molecular scissors: Ancestral RNA-guided nucleases greatly expand the toolbox for gene editing

Rousset F. & Sorek R. Science, 374(6563):37-38 (2021)

Rousset Sorek 2021.pdf

Prokaryotic viperins produce diverse antiviral molecules

Bernheim A., Millman Dayan A. J., Ofir G., Meitav G., Avraham C., Shomar H., Rosenberg M. M., Tal N., Melamed S., Amitai G. & Sorek R. Nature, 589(7840):120-124 (2021)

Accepted version

Bernheim et al 2020.pdf

Viperin is an interferon-induced cellular protein that is conserved in animals ¹. It has previously been shown to inhibit the replication of multiple viruses by producing the ribonucleotide 3-deoxy-3,4-didehydro (ddh)-cytidine triphosphate (ddhCTP), which acts as a chain terminator for viral RNA polymerase ². Here we show that eukaryotic viperin originated from a clade of bacterial and archaeal proteins that protect against phage infection. Prokaryotic viperins produce a set of modified ribonucleotides that include ddhCTP, ddh-guanosine triphosphate (ddhGTP) and ddh-uridine triphosphate (ddhUTP). We further show that prokaryotic viperins protect against T7 phage infection by inhibiting viral polymerase-dependent transcription, suggesting that it has an antiviral mechanism of action similar to that of animal viperin. Our results reveal a class of potential natural antiviral compounds produced by bacterial immune systems.

2020

Bacterial Retrons Function In Anti-Phage Defense

Millman A., Bernheim A., Stokar-Avihail A., Fedorenko T., Voichek M., Leavitt A., Oppenheimer-Shaanan Y. & Sorek R. Cell, 183(6):551-1561 (2020)

Submitted version

Accepted version

Millman et al Cell 2020.pdf

Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection. Focusing on retron Ec48, we show evidence that it \u201cguards\u201d RecBCD, a complex with central anti-phage functions in bacteria. Inhibition of RecBCD by phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed.

Diversity and classification of cyclic-oligonucleotide-based anti-phage signalling systems

Millman A., Melamed S., Amitai G. & Sorek R. (2020) Nature Microbiology. 5, 12, p. 1608-1615

Accepted version

Millman et al 2020.pdf

Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS) are a family of defence systems against bacteriophages (hereafter phages) that share ancestry with the cGAS-STING innate immune pathway in animals. CBASS systems are composed of an oligonucleotide cyclase, which generates signalling cyclic oligonucleotides in response to phage infection, and an effector that is activated by the cyclic oligonucleotides and promotes cell death. Cell death occurs before phage replication is completed, therefore preventing the spread of phages to nearby cells. Here, we analysed 38,000 bacterial and archaeal genomes and identified more than 5,000 CBASS systems, which have diverse architectures with multiple signalling molecules, effectors and ancillary genes. We propose a classification system for CBASS that groups systems according to their operon organization, signalling molecules and effector function. Four major CBASS types were identified, sharing at least six effector subtypes that promote cell death by membrane impairment, DNA degradation or other means. We observed evidence of extensive gain and loss of CBASS systems, as well as shuffling of effector genes between systems. We expect that our classification and nomenclature scheme will guide future research in the developing CBASS field.

A genome assembly and the somatic genetic and epigenetic mutation rate in a wild long-lived perennial Populus trichocarpa

Hofmeister B. T., Denkena J., Colomé-Tatché M., Shahryary Y., Hazarika R., Grimwood J., Mamidi S., Jenkins J., Grabowski P. P., Sreedasyam A., Shu S., Barry K., Lail K., Adam C., Lipzen A., Sorek R., Kudrna D., Talag J., Wing R., Hall D. W., Jacobsen D., Tuskan G. A., Schmutz J., Johannes F. & Schmitz R. J. (2020) GENOME BIOLOGY. 21, 1, 259.

Background: Plants can transmit somatic mutations and epimutations to offspring, which in turn can affect fitness. Knowledge of the rate at which these variations arise is necessary to understand how plant development contributes to local adaption in an ecoevolutionary context, particularly in long-lived perennials. Results: Here, we generate a new high-quality reference genome from the oldest branch of a wild Populus trichocarpa tree with two dominant stems which have been evolving independently for 330 years. By sampling multiple, age-estimated branches of this tree, we use a multi-omics approach to quantify age-related somatic changes at the genetic, epigenetic, and transcriptional level. We show that the per-year somatic mutation and epimutation rates are lower than in annuals and that transcriptional variation is mainly independent of age divergence and cytosine methylation. Furthermore, a detailed analysis of the somatic epimutation spectrum indicates that transgenerationally heritable epimutations originate mainly from DNA methylation maintenance errors during mitotic rather than during meiotic cell divisions. Conclusion: Taken together, our study provides unprecedented insights into the origin of nucleotide and functional variation in a long-lived perennial plant.

STING cyclic dinucleotide sensing originated in bacteria

Morehouse B. R., Govande A. A., Millman A., Keszei A. F., Lowey B., Ofir G., Shao S., Sorek R. & Kranzusch P. J. (2020) Nature. 586, 7829, p. 429-433

Morehouse et al 2020.pdf

Stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides that are released during bacterial infection and in endogenous cyclic GMPAMP signalling during viral infection and anti-tumour immunity¹⁵. STING shares no structural homology with other known signalling proteins⁶⁹, which has limited attempts at functional analysis and prevented explanation of the origin of cyclic dinucleotide signalling in mammalian innate immunity. Here we reveal functional STING homologues encoded within prokaryotic defence islands, as well as a conserved mechanism of signal activation. Crystal structures of bacterial STING define a minimal homodimeric scaffold that selectively responds to cyclic di-GMP synthesized by a neighbouring cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme. Bacterial STING domains couple the recognition of cyclic dinucleotides with the formation of protein filaments to drive oligomerization of TIR effector domains and rapid NAD⁺ cleavage. We reconstruct the evolutionary events that followed the acquisition of STING into metazoan innate immunity, and determine the structure of a full-length TIRSTING fusion from the Pacific oyster Crassostrea gigas. Comparative structural analysis demonstrates how metazoan-specific additions to the core STING scaffold enabled a switch from direct effector function to regulation of antiviral transcription. Together, our results explain the mechanism of STING-dependent signalling and reveal the conservation of a functional cGASSTING pathway in prokaryotic defence against bacteriophages.

Peptide-based quorum sensing systems in Paenibacillus polymyxa

Voichek M., Maaß S., Kroniger T., Becher D. & Sorek R. (2020) Life Science Alliance. 3, 10, 202000847.

Voichek et al 2020.pdf

Paenibacillus polymyxa is an agriculturally important plant growth-promoting rhizobacterium. Many Paenibacillus species are known to be engaged in complex bacteria-bacteria and bacteria-host interactions, which in other species were shown to necessitate quorum sensing communication. However, to date, no quorum sensing systems have been described in Paenibacillus. Here, we show that the type strain P. polymyxa ATCC 842 encodes at least 16 peptide-based communication systems. Each of these systems is comprised of a pro-peptide that is secreted to the growth medium and processed to generate a mature short peptide. Each peptide has a cognate intracellular receptor of the RRNPP family, and we show that external addition of P. polymyxa communication peptides leads to reprogramming of the transcriptional response. We found that these quorum sensing systems are conserved across hundreds of species belonging to the Paenibacillaceae family, with some species encoding more than 25 different peptide-receptor pairs, representing a record number of quorum sensing systems encoded in a single genome.

Abortive Infection: Bacterial Suicide as an Antiviral Immune Strategy

Lopatina A., Tal N. & Sorek R. (2020) Annual Review of Virology. 7, p. 371-384

Accepted version

Lopatina Tal Sorek 2020.pdf

Facing frequent phage challenges, bacteria have evolved numerous mechanisms to resist phage infection. A commonly used phage resistance strategy is abortive infection (Abi), in which the infected cell commits suicide before the phage can complete its replication cycle. Abi prevents the phage epidemic from spreading to nearby cells, thus protecting the bacterial colony. The Abi strategy is manifested by a plethora of mechanistically diverse defense systems that are abundant in bacterial genomes. In turn, phages have developed equally diverse mechanisms to overcome bacterial Abi. This review summarizes the current knowledge on bacterial defense via cell suicide. It describes the principles of Abi, details how these principles are implemented in a variety of natural defense systems, and discusses phage counter-defense mechanisms.

The pan-immune system of bacteria: antiviral defence as a community resource

Bernheim A. & Sorek R. (2020) Nature Reviews Microbiology. 18, 2, p. 113-119

Accepted version

Bernheim Sorek 2019_0.pdf

Viruses and their hosts are engaged in a constant arms race leading to the evolution of antiviral defence mechanisms. Recent studies have revealed that the immune arsenal of bacteria against bacteriophages is much more diverse than previously envisioned. These discoveries have led to seemingly contradictory observations: on one hand, individual microorganisms often encode multiple distinct defence systems, some of which are acquired by horizontal gene transfer, alluding to their fitness benefit. On the other hand, defence systems are frequently lost from prokaryotic genomes on short evolutionary time scales, suggesting that they impose a fitness cost. In this Perspective article, we present the 'pan-immune system' model in which we suggest that, although a single strain cannot carry all possible defence systems owing to their burden on fitness, it can employ horizontal gene transfer to access immune defence mechanisms encoded by closely related strains. Thus, the 'effective' immune system is not the one encoded by the genome of a single microorganism but rather by its pan-genome, comprising the sum of all immune systems available for a microorganism to horizontally acquire and use.In this Perspective article, Bernheim and Sorek present the 'pan-immune system' model in which bacteria employ horizontal gene transfer to access immune defence mechanisms encoded by closely related strains, and conclude by discussing the implications on the evolution of anti-defence strategies in phages.

2019

Cyclic GMP-AMP signalling protects bacteria against viral infection

Cohen D., Melamed S., Millman A., Shulman G., Oppenheimer-Shaanan Y., Kacen A., Doron S., Amitai G. & Sorek R. Nature, 574(7780):691-695 (2019)

Accepted version

Cohen et al 2019.pdf

The cyclic GMP-AMP synthase (cGAS)-STING pathway is a central component of the cell-autonomous innate immune system in animals(1,2). The cGAS protein is a sensor of cytosolic viral DNA and, upon sensing DNA, it produces a cyclic GMP-AMP (cGAMP) signalling molecule that binds to the STING protein and activates the immune response(3-5). The production of cGAMP has also been detected in bacteria(6), and has been shown, in Vibrio cholerae, to activate a phospholipase that degrades the inner bacterial membrane(7). However, the biological role of cGAMP signalling in bacteria remains unknown. Here we show that cGAMP signalling is part of an antiphage defence system that is common in bacteria. This system is composed of a four-gene operon that encodes the bacterial cGAS and the associated phospholipase, as well as two enzymes with the eukaryotic-like domains E1, E2 and JAB. We show that this operon confers resistance against a wide variety of phages. Phage infection triggers the production of cGAMP, which-in turn-activates the phospholipase, leading to a loss of membrane integrity and to cell death before completion of phage reproduction. Diverged versions of this system appear in more than 10% of prokaryotic genomes, and we show that variants with effectors other than phospholipase also protect against phage infection. Our results suggest that the eukaryotic cGAS-STING antiviral pathway has ancient evolutionary roots that stem from microbial defences against phages.

Widespread Utilization of Peptide Communication in Phages Infecting Soil and Pathogenic Bacteria

Stokar-Avihail A., Tal N., Erez Z., Lopatina A. & Sorek R. (2019) Cell Host & Microbe. 25, 5, p. 746-755.e5

Accepted version

Stokar et al 2019.pdf

Temperate phages can adopt either a lytic or lysogenic lifestyle within their host bacteria. It was recently shown that Bacillus-subtilis-infecting phages of the SPbeta group utilize a peptide-based communication system called arbitrium to coordinate the lysogeny decision. The occurrence of peptide-based communication systems among phages more broadly remains to be explored. Here, we uncover a wide array of peptide-based communication systems utilized by phages for lysogeny decisions. These arbitrium-like systems show diverse peptide codes and can be detected in numerous genetically distant phage types and conjugative elements. The pathogens Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are commonly infected by arbitrium-carrying mobile elements, which often carry toxins essential for pathogenicity. Experiments with phages containing these arbitrium-like systems demonstrate their involvement in lysogeny decisions. Finally, our results suggest that the peptidebased decision is executed by an antisense RNA that controls the regulator of the lysogenic state.

Consent insufficient for data release−Response

Amann R. I., Baichoo S., Blencowe B. J., Bork P., Borodovsky M., Brooksbank C., Chain P. S., Colwell R. R., Daffonchio D. G., Danchin A., de Lorenzo V., Dorrestein P. C., Finn R. D., Fraser C. M., Gilbert J. A., Hallam S. J., Hugenholtz P., Ioannidis J. P., Jansson J. K., Kim J. F., Klenk H. P., Klotz M. G., Knight R., Konstantinidis K. T., Kyrpides N. C., Mason C. E., McHardy A. C., Meyer F., Ouzounis C. A., Patrinos A. A., Podar M., Pollard K. S., Ravel J., Muñoz A. R., Roberts R. J., Rosselló-Móra R., Sansone S. A., Schloss P. D., Schriml L. M., Setubal J. C., Sorek R., Stevens R. L., Tiedje J. M., Turjanski A., Tyson G. W., Ussery D. W., Weinstock G. M., White O., Whitman W. B. & Xenarios I. (2019) Science. 364, 6439, p. 446

Successful Brincidofovir Treatment of Metagenomics-detected Adenovirus Infection in a Severely Ill Signal Transducer and Activator of Transcription-1-deficient Patient

Averbuch D., Safadi R., Dar D., Wolf D., Cherniak M., Sorek R. & Amit S. (2019) Pediatric Infectious Disease Journal. 38, 3, p. 297-299

A signal transducer and activator of transcription-1-deficient patient presented with prolonged fever, cachexia, anemia, hypoalbuminemia and finally relapsing debilitating mycobacterial osteomyelitis while receiving a previously effective antimycobacterial treatment. Progression despite rigorous workup and multiple antibiotics prompted shotgun metagenomics revealing adenovirus in liver samples. Brincidofovir led to a complete, sustained clinical recovery, including osteomyelitis, probably attributed to reversal of adenovirus-induced immune dysregulation.

Data access: Toward unrestricted use of public genomic data

Amann R. I., Baichoo S., Blencowe B. J., Bork P., Borodovsky M., Brooksbank C., Chain P. S., Colwell R. R., Daffonchio D. G., Danchin A., De Lorenzo V., Dorrestein P. C., Finn R. D., Fraser C. M., Gilbert J. A., Hallam S. J., Hugenholtz P., Ioannidis J. P., Jansson J. K., Kim J. F., Klenk H. P., Klotz M. G., Knight R., Konstantinidis K. T., Kyrpides N. C., Mason C. E., McHardy A. C., Meyer F., Ouzounis C. A., Patrinos A. A., Podar M., Pollard K. S., Ravel J., Muñoz A. R., Roberts R. J., Rosselló-Móra R., Sansone S. A., Schloss P. D., Schriml L. M., Setubal J. C., Sorek R., Stevens R. L., Tiedje J. M., Turjanski A., Tyson G. W., Ussery D. W., Weinstock G. M., White O., Whitman W. B. & Xenarios I. (2019) Science. 363, 6425, p. 350-352

Despite some notable progress in data sharing policies and practices, restrictions are still often placed on the open and unconditional use of various genomic data after they have received official approval for release to the public domain or to public databases. These restrictions, which often conflict with the terms and conditions of the funding bodies who supported the release of those data for the benefit of the scientific community and society, are perpetuated by the lack of clear guiding rules for data usage. Existing guidelines for data released to the public domain recognize but fail to resolve tensions between the importance of free and unconditional use of these data and the \u201cright\u201d of the data producers to the first publication. This self-contradiction has resulted in a loophole that allows different interpretations and a continuous debate between data producers and data users on the use of public data. We argue that the publicly available data should be treated as open data, a shared resource with unrestricted use for analysis, interpretation, and publication.

A rhli 5= UTR-derived sRNA regulates RhlR-dependent quorum sensing in Pseudomonas aeruginosa

Thomason M. K., Voichek M., Dar D., Addis V., Fitzgerald D., Gottesman S., Sorek R. & Greenberga E. P. (2019) mBio. 10, 5, e02253-19.

N-Acyl homoserine lactone (AHL) quorum sensing (QS) controls expression of over 200 genes in Pseudomonas aeruginosa. There are two AHL regulatory systems: The LasR-LasI circuit and the RhlR-RhlI system. We mapped transcription termination sites affected by AHL QS in P. aeruginosa, and in doing so we identiﬁed AHL-regulated small RNAs (sRNAs). Of interest, we noted that one particular sRNA was located within the rhlI locus. We found that rhlI, which encodes the enzyme that produces the AHL N-butanoyl-homoserine lactone (C4-HSL), is controlled by a 5 = untranslated region (UTR)-derived sRNA we name RhlS. We also identiﬁed an antisense RNA encoded opposite the beginning of the rhlI open reading frame, which we name asRhlS. RhlS accumulates as wild-type cells enter stationary phase and is required for the production of normal levels of C4-HSL through activation of rhlI translation. RhlS also directly posttranscriptionally regulates at least one other unlinked gene, fpvA. The asRhlS appears to be expressed at maximal levels during logarithmic growth, and we suggest RhlS may act antagonistically to the asRhlS to regulate rhlI translation. The rhlI-encoded sRNAs represent a novel aspect of RNAmediated tuning of P. aeruginosa QS. IMPORTANCE The opportunistic human pathogen Pseudomonas aeruginosa possesses multiple quorum sensing systems that regulate and coordinate production of virulence factors and adaptation to different environments. Despite extensive research, the regulatory elements that play a role in this complex network are still not fully understood. By using several RNA sequencing techniques, we were able to identify a small regulatory RNA we named RhlS. RhlS increases translation of RhlI, a key enzyme in the quorum sensing pathway, and represses the fpvA mRNA encoding one of the siderophore pyoverdine receptors. Our results highlight a new regulatory layer of P. aeruginosa quorum sensing and contribute to the growing understanding of the role regulatory RNAs play in bacterial physiology.

2018

HflXr, a homolog of a ribosome-splitting factor, mediates antibiotic resistance

Duval M., Dar D., Carvalho F., Rocha E. P., Sorek R. & Cossart P. (2018) Proceedings of the National Academy of Sciences of the United States of America. 115, 52, p. 13359-13364

Duval et al 2019.pdf

To overcome the action of antibiotics, bacteria have evolved a variety of different strategies, such as drug modification, target mutation, and efflux pumps. Recently, we performed a genome-wide analysis of Listeria monocytogenes gene expression after growth in the presence of antibiotics, identifying genes that are up-regulated upon antibiotic treatment. One of them, lmo0762, is a homolog of hflX, which encodes a heat shock protein that rescues stalled ribosomes by separating their two subunits. To our knowledge, ribosome splitting has never been described as an antibiotic resistance mechanism. We thus investigated the role of lmo0762 in antibiotic resistance. First, we demonstrated that lmo0762 is an antibiotic resistance gene that confers protection against lincomycin and erythromycin, and that we renamed hflXr (hflX resistance). We show that hflXr expression is regulated by a transcription attenuation mechanism relying on the presence of alternative RNA structures and a small ORF encoding a 14 amino acid peptide containing the RLR motif, characteristic of macrolide resistance genes. We also provide evidence that HflXr is involved in ribosome recycling in presence of antibiotics. Interestingly, L. monocytogenes possesses another copy of hflX, lmo1296, that is not involved in antibiotic resistance. Phylogenetic analysis shows several events of hflXr duplication in prokaryotes and widespread presence of hflXr in Firmicutes. Overall, this study reveals the Listeria hflXr as the founding member of a family of antibiotic resistance genes. The resistance conferred by this gene is probably of importance in the environment and within microbial communities.

Bacterial noncoding RNAs excised from within protein-coding transcripts

Dar D. & Sorek R. (2018) mBio. 9, 5, e01730-18.

Dar mbio 2018.pdf

Prokaryotic genomes encode a plethora of small noncoding RNAs (ncRNAs) that fine-tune the expression of specific genes. The vast majority of known bacterial ncRNAs are encoded from within intergenic regions, where their expression is controlled by promoter and terminator elements, similarly to protein-coding genes. In addition, recent studies have shown that functional ncRNAs can also be derived from gene 3' untranslated regions (3'UTRs) via an alternative biogenesis pathway, in which the ncRNA segment is separated from the mRNA via RNase cleavage. Here, we report the detection of a large set of decay-generated noncoding RNAs (decRNAs), many of which are completely embedded within protein-coding mRNA regions rather than in the UTRs. We show that these decRNAs are \u201ccarved out\u201d of the mRNA through the action of RNase E and that they are predicted to fold into highly stable RNA structures, similar to those of known ncRNAs. A subset of these decRNAs is predicted to interact with Hfq or ProQ or both, which act as ncRNA chaperones, and some decRNAs display evolutionarily conserved sequences and conserved expression patterns between different species. These results suggest that mRNA protein-coding regions may harbor a large set of potentially functional small RNAs. IMPORTANCE Bacteria and archaea utilize regulatory small noncoding RNAs (ncRNAs) to control the expression of specific genetic programs. These ncRNAs are almost exclusively encoded within intergenic regions and are independently transcribed. Here, we report on a large set ncRNAs that are \u201ccarved out\u201d from within the protein-coding regions of Escherichia coli mRNAs by cellular RNases. These protected mRNA fragments fold into energetically stable RNA structures, reminiscent of those of intergenic regulatory ncRNAs. In addition, a subset of these ncRNAs coprecipitate with the major ncRNA chaperones Hfq and ProQ and display evolutionarily conserved sequences and conserved expression patterns between different bacterial species. Our data suggest that protein-coding genes can potentially act as a reservoir of regulatory ncRNAs.

High-resolution RNA 3'-ends mapping of bacterial Rho-dependent transcripts

Dar D. & Sorek R. (2018) Nucleic Acids Research. 46, 13, p. 6797-6805

Dar Sorek NAR 2018.pdf

Transcription termination in bacteria can occur either via Rho-dependent or independent (intrinsic) mechanisms. Intrinsic terminators are composed of a stem-loop RNA structure followed by a uridine stretch and are known to terminate in a precise manner. In contrast, Rho-dependent terminators have more loosely defined characteristics and are thought to terminate in a diffuse manner. While transcripts ending in an intrinsic terminator are protected from 3'-5' exonuclease digestion due to the stem-loop structure of the terminator, it remains unclear what protects Rho-dependent transcripts from being degraded. In this study, we mapped the exact steady-state RNA 3' ends of hundreds of Escherichia coli genes terminated either by Rho-dependent or independent mechanisms. We found that transcripts generated from Rho-dependent termination have precise 3'-ends at steady state. These termini were localized immediately downstream of energetically stable stem-loop structures, which were not followed by uridine rich sequences. We provide evidence that these structures protect Rho-dependent transcripts from 3'-5' exonucleases such as PNPase and RNase II, and present data localizing the Rho-utilization (rut) sites immediately downstream of these protective structures. This study represents the first extensive in-vivo map of exact RNA 3'-ends of Rho-dependent transcripts in E. coli.

Viruses cooperate to defeat bacteria

Bernheim A. & Sorek R. (2018) Nature. 559, 7715, p. 482-484

Bernheim Sorek Nature 2018.pdf

Extensive reshaping of bacterial operons by programmed mRNA decay

Dar D. & Sorek R. (2018) PLoS Genetics. 14, 4, e1007354.

Dar Sorek 2018.pdf

Bacterial operons synchronize the expression of multiple genes by placing them under the control of a shared promoter. It was previously shown that polycistronic transcripts can undergo differential RNA decay, leaving some genes within the polycistron more stable than others, but the extent of regulation by differential mRNA decay or its evolutionary conservation remains unknown. Here, we find that a substantial fraction of E. coli genes display non-uniform mRNA stoichiometries despite being coded from the same operon. We further show that these altered operon stoichiometries are shaped post-transcriptionally by differential mRNA decay, which is regulated by RNA structures that protect specific regions in the transcript from degradation. These protective RNA structures are generally coded within the protein-coding regions of the regulated genes and are frequently evolutionarily conserved. Furthermore, we provide evidence that differences in ribosome densities across polycistronic transcript segments, together with the conserved structural RNA elements, play a major role in the differential decay process. Our results highlight a major role for differential mRNA decay in shaping bacterial transcriptomes.

Contemporary Phage Biology: From Classic Models to New Insights

Ofir G. & Sorek R. (2018) Cell. 172, 6, p. 1260-1270

Ofir Sorek 2018.pdf

Bacteriophages, discovered about a century ago, have been pivotal as models for understanding the fundamental principles of molecular biology. While interest in phage biology declined after the phage "golden era,'' key recent developments, including advances in phage genomics, microscopy, and the discovery of the CRISPR-Cas anti-phage defense system, have sparked a renaissance in phage research in the past decade. This review highlights recently discovered unexpected complexities in phage biology, describes a new arsenal of phage genes that help them overcome bacterial defenses, and discusses advances toward documentation of the phage biodiversity on a global scale.

Systematic discovery of antiphage defense systems in the microbial pangenome

Doron S., Melamed S., Ofir G., Leavitt A., Lopatina A., Keren M., Amitai G. & Sorek R. Science, 359(6379):eaar4120 (2018)

Accepted version

Doron et al 2018.pdf

The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in "defense islands" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria.

Quantitative species-level ecology of reef fish larvae via metabarcoding

Kimmerling N., Zuqert O., Amitai G., Gurevich T., Armoza-Zvuloni R., Kolesnikov I., Berenshtein I., Melamed S., Gilad S., Benjamin S., Rivlin A., Ohavia M., Paris C. B., Holzman R., Kiflawi M. & Sorek R. (2018) Nature ecology & evolution. 2, 2, p. 306-316

Kimmerling et al 2017.pdf

The larval pool of coral reef fish has a crucial role in the dynamics of adult fish populations. However, large-scale species-level monitoring of species-rich larval pools has been technically impractical. Here, we use high-throughput metabarcoding to study larval ecology in the Gulf of Aqaba, a region that is inhabited by >500 reef fish species. We analysed 9,933 larvae from 383 samples that were stratified over sites, depth and time. Metagenomic DNA extracted from pooled larvae was matched to a mitochondrial cytochrome c oxidase subunit I barcode database compiled for 77% of known fish species within this region. This yielded species-level reconstruction of the larval community, allowing robust estimation of larval spatio-temporal distributions. We found significant correlations between species abundance in the larval pool and in local adult assemblages, suggesting a major role for larval supply in determining local adult densities. We documented larval flux of species whose adults were never documented in the region, suggesting environmental filtering as the reason for the absence of these species. Larvae of several deep-sea fishes were found in shallow waters, supporting their dispersal over shallow bathymetries, potentially allowing Lessepsian migration into the Mediterranean Sea. Our method is applicable to any larval community and could assist coral reef conservation and fishery management efforts.

DISARM is a widespread bacterial defence system with broad anti-phage activities

Ofir G., Melamed S., Sberro H., Mukamel Z., Silverman S., Yaakov G., Doron S. & Sorek R. (2018) Nature Microbiology. 3, 1, p. 90-98

Accepted version

Ofir et al 2017.pdf

The evolutionary pressure imposed by phage predation on bacteria and archaea has resulted in the development of effective anti-phage defence mechanisms, including restriction-modification and CRISPR-Cas systems. Here, we report on a new defence system, DISARM (defence island system associated with restriction-modification), which is widespread in bacteria and archaea. DISARM is composed of five genes, including a DNA methylase and four other genes annotated as a helicase domain, a phospholipase D (PLD) domain, a DUF1998 domain and a gene of unknown function. Engineering the Bacillus parali-cheniformis 9945a DISARM system into Bacillus subtilis has rendered the engineered bacteria protected against phages from all three major families of tailed double-stranded DNA phages. Using a series of gene deletions, we show that four of the five genes are essential for DISARM-mediated defence, with the fifth (PLD) being redundant for defence against some of the phages. We further show that DISARM restricts incoming phage DNA and that the B. paralicheniformis DISARM methylase modifies host CCWGG motifs as a marker of self DNA akin to restriction-modification systems. Our results suggest that DISARM is a new type of multi-gene restriction-modification module, expanding the arsenal of defence systems known to be at the disposal of prokaryotes against their viruses.

2017

Intracellular signaling in CRISPR-Cas defense

Amitai G. & Sorek R. (2017) Science. 357, 6351, p. 550-551

Amitai Sorek Science 2017.pdf

Evidence for a cytoplasmic pool of ribosome-free mRNAs encoding inner membrane proteins in Escherichia coli

Benhalevy D., Biran I., Bochkareva E. S., Sorek R. & Bibi E. (2017) PLoS ONE. 12, 8, e0183862.

Translation-independent mRNA localization represents an emerging concept in cell biology. In Escherichia coli, mRNAs encoding integral membrane proteins (MPRs) are targeted to the membrane where they are translated by membrane associated ribosomes and the produced proteins are inserted into the membrane co-translationally. In order to better understand aspects of the biogenesis and localization of MPRs, we investigated their subcellular distribution using cell fractionation, RNA-seq and qPCR. The results show that MPRs are overrepresented in the membrane fraction, as expected, and depletion of the signal recognition particle-receptor, FtsY reduced the amounts of all mRNAs on the membrane. Surprisingly, however, MPRs were also found relatively abundant in the soluble ribosome-free fraction and their amount in this fraction is increased upon overexpression of CspE, which was recently shown to interact with MPRs. CspE also conferred a positive effect on the membrane-expression of integral membrane proteins. We discuss the possibility that the effects of CspE overexpression may link the intriguing subcellular localization of MPRs to the cytosolic ribosome-free fraction with their translation into membrane proteins and that the ribosome-free pool of MPRs may represent a stage during their targeting to the membrane, which precedes translation.

Regulation of antibiotic-resistance by non-coding RNAs in bacteria

Dar D. & Sorek R. (2017) Current Opinion in Microbiology. 36, p. 111-117

Dar Sorek Curr_Opinion 2017.pdf

Antibiotic resistance genes are commonly regulated by sophisticated mechanisms that activate gene expression in response to antibiotic exposure. Growing evidence suggest that cis-acting non-coding RNAs play a major role in regulating the expression of many resistance genes, specifically those which counteract the effects of translation-inhibiting antibiotics. These ncRNAs reside in the 5'UTR of the regulated gene, and sense the presence of the antibiotics by recruiting translating ribosomes onto short upstream open reading frames (uORFs) embedded in the ncRNA. In the presence of translation-inhibiting antibiotics ribosomes arrest over the uORF, altering the RNA structure of the regulator and switching the expression of the resistance gene to 'ON'. The specificity of these riboregulators is tuned to sense-specific classes of antibiotics based on the length and composition of the respective uORF. Here we review recent work describing new types of antibiotic-sensing RNA-based regulators and elucidating the molecular mechanisms by which they function to control antibiotic resistance in bacteria.

Communication between viruses guides lysis-lysogeny decisions

Erez Z., Steinberger-Levy I., Shamir M., Doron S., Stokar Avihail A. A., Peleg Y., Melamed S., Leavitt A., Savidor A., Albeck S., Amitai G. & Sorek R. Nature, 541(7638):488-493 (2017)

Accepted version

Erez et al 2017.pdf

Temperate viruses can become dormant in their host cells, a process called lysogeny. In every infection, such viruses decide between the lytic and the lysogenic cycles, that is, whether to replicate and lyse their host or to lysogenize and keep the host viable. Here we show that viruses (phages) of the SPbeta group use a small-molecule communication system to coordinate lysis-lysogeny decisions. During infection of its Bacillus host cell, the phage produces a six aminoacids-long communication peptide that is released into the medium. In subsequent infections, progeny phages measure the concentration of this peptide and lysogenize if the concentration is sufficiently high. We found that different phages encode different versions of the communication peptide, demonstrating a phage-specific peptide communication code for lysogeny decisions. We term this communication system the 'arbitrium' system, and further show that it is encoded by three phage genes: aimP, which produces the peptide; aimR, the intracellular peptide receptor; and aimX, a negative regulator of lysogeny. The arbitrium system enables a descendant phage to 'communicate' with its predecessors, that is, to estimate the amount of recent previous infections and hence decide whether to employ the lytic or lysogenic cycle.

Optimality and sub-optimality in a bacterial growth law

Towbin B. D., Korem Y., Bren A., Doron S., Sorek R. & Alon U. (2017) Nature Communications. 8, 14123.

Towbin et al 2017.pdf

Organisms adjust their gene expression to improve fitness in diverse environments. But finding the optimal expression in each environment presents a challenge. We ask how good cells are at finding such optima by studying the control of carbon catabolism genes in Escherichia coli. Bacteria show a growth law: growth rate on different carbon sources declines linearly with the steady-state expression of carbon catabolic genes. We experimentally modulate gene expression to ask if this growth law always maximizes growth rate, as has been suggested by theory. We find that the growth law is optimal in many conditions, including a range of perturbations to lactose uptake, but provides sub-optimal growth on several other carbon sources. Combining theory and experiment, we genetically re-engineer E. coli to make sub-optimal conditions into optimal ones and vice versa. We conclude that the carbon growth law is not always optimal, but represents a practical heuristic that often works but sometimes fails.

Vesicles Spread Susceptibility to Phages

Ofir G. & Sorek R. (2017) Cell. 168, 1-2, p. 13-15

Ofir Cell 2017.pdf

Computational prediction of regulatory, premature transcription termination in bacteria

Millman A., Dar D., Shamir M. & Sorek R. (2017) Nucleic Acids Research. 45, 2, p. 886-893

Millman NAR 2016.pdf

A common strategy for regulation of gene expression in bacteria is conditional transcription termination. This strategy is frequently employed by 5'UTR cis-acting RNA elements (riboregulators), including riboswitches and attenuators. Such riboregulators can assume two mutually exclusive RNA structures, one of which forms a transcriptional terminator and results in premature termination, and the other forms an antiterminator that allows read-through into the coding sequence to produce a full-length mRNA. We developed a machine-learning based approach, which, given a 5'UTR of a gene, predicts whether it can form the two alternative structures typical to riboregulators employing conditional termination. Using a large positive training set of riboregulators derived from 89 human microbiome bacteria, we show high specificity and sensitivity for our classifier. We further show that our approach allows the discovery of previously unidentified riboregulators, as exemplified by the detection of new LeuA leaders and T-boxes in Streptococci. Finally, we developed PASIFIC (www.weizmann.ac.il/molgen/Sorek/PASIFIC/), an online web-server that, given a user-provided 5'UTR sequence, predicts whether this sequence can adopt two alternative structures conforming with the conditional termination paradigm. This webserver is expected to assist in the identification of new riboswitches and attenuators in the bacterial pan-genome.

2016

Repeat Size Determination by Two Molecular Rulers in the Type I-E CRISPR Array

Goren M. G., Doron S., Globus R., Amitai G., Sorek R. & Qimron U. (2016) Cell Reports. 16, 11, p. 2811-2818

Prokaryotic adaptive immune systems are composed of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. These systems adapt to new threats by integrating short nucleic acids, termed spacers, into the CRISPR array. The functional motifs in the repeat and the mechanism by which a constant repeat size is maintained are still elusive. Here, through a series of mutations within the repeat of the CRISPR-Cas type I-E, we identify motifs that are crucial for adaptation and show that they serve as anchor sites for two molecular rulers determining the size of the new repeat. Adaptation products from various repeat mutants support a model in which two motifs in the repeat bind to two different sites in the adaptation complex that are 8 and 16 bp away from the active site. This model significantly extends our understanding of the adaptation process and broadens the scope of its applications.

Widespread formation of alternative 3' UTR isoforms via transcription termination in archaea

Dar D., Prasse D., Schmitz R. A. & Sorek R. (2016) Nature Microbiology. 1, 10, 16143.

Transcription termination sets the 3' end boundaries of RNAs and plays key roles in gene regulation. Although termination has been well studied in bacteria, the signals that mediate termination in archaea remain poorly understood. Here, we applied term-seq to comprehensively map RNA 3' termini, with single-base precision, in two phylogenetically distant archaea: Methanosarcina mazei and Sulfolobus acidocaldarius. Comparison of RNA 3' ends across hundreds of genes revealed the sequence composition of transcriptional terminators in each organism, highlighting both common and divergent characteristics between the different archaeal phyla. We find that, in contrast to bacteria, a considerable portion of archaeal genes are controlled by multiple consecutive terminators, generating several alternative 3' untranslated region isoforms for >30% of the genes. These alternative isoforms often present marked length differences, implying that archaea can employ regulation via alternative 3' untranslated regions, similar to eukaryotes. Although most of the terminators are intergenic, we discover numerous cases in which termination of one gene occurs within the coding region of a downstream gene, implying that leaky termination may tune inter-transcript stoichiometry in multi-gene operons. These results provide the first high-throughput maps of transcriptional terminators in archaea and point to an evolutionary path linking bacterial and eukaryal non-coding regulatory strategies.

CRISPRCas: Spacer Diversity Determines the Efficiency of Defense

Lopatina A. & Sorek R. (2016) Current Biology. 26, 14, p. R683-R685

Transcriptome dynamics of a broad host-range cyanophage and its hosts

Doron S., Fedida A., Hernndez-Prieto M. A., Sabehi G., Karunker I., Stazic D., Feingersch R., Steglich C., Futschik M., Lindell D. & Sorek R. (2016) ISME Journal. 10, 6, p. 1437-1455

Cyanobacteria are highly abundant in the oceans and are constantly exposed to lytic viruses. The T4-like cyanomyoviruses are abundant in the marine environment and have broad host-ranges relative to other cyanophages. It is currently unknown whether broad host-range phages specifically tailor their infection program for each host, or employ the same program irrespective of the host infected. Also unknown is how different hosts respond to infection by the same phage. Here we used microarray and RNA-seq analyses to investigate the interaction between the Syn9 T4-like cyanophage and three phylogenetically, ecologically and genomically distinct marine Synechococcus strains: WH7803, WH8102 and WH8109. Strikingly, Syn9 led a nearly identical infection and transcriptional program in all three hosts. Different to previous assumptions for T4-like cyanophages, three temporally regulated gene expression classes were observed. Furthermore, a novel regulatory element controlled early-gene transcription, and host-like promoters drove middle gene transcription, different to the regulatory paradigm for T4. Similar results were found for the P-TIM40 phage during infection of Prochlorococcus NATL2A. Moreover, genomic and metagenomic analyses indicate that these regulatory elements are abundant and conserved among T4-like cyanophages. In contrast to the near-identical transcriptional program employed by Syn9, host responses to infection involved host-specific genes primarily located in hypervariable genomic islands, substantiating islands as a major axis of phage-cyanobacteria interactions. Our findings suggest that the ability of broad host-range phages to infect multiple hosts is more likely dependent on the effectiveness of host defense strategies than on differential tailoring of the infection process by the phage.

Term-seq reveals abundant ribo-regulation of antibiotics resistance in bacteria

Dar D., Shamir M., Mellin J. R., Koutero M., Stern-Ginossar N., Cossart P. & Sorek R. (2016) Science. 352, 6282, aad9822.

Accepted version

Dar et al 2016.pdf

Riboswitches and attenuators are cis-regulatory RNA elements, most of which control bacterial gene expression via metabolite-mediated, premature transcription termination. We developed an unbiased experimental approach for genome-wide discovery of such ribo-regulators in bacteria. We also devised an experimental platform that quantitatively measures the in vivo activity of all such regulators in parallel and enables rapid screening for ribo-regulators that respond to metabolites of choice. Using this approach, we detected numerous antibiotic-responsive ribo-regulators that control antibiotic resistance genes in pathogens and in the human microbiome. Studying one such regulator in Listeria monocytogenes revealed an attenuation mechanism mediated by antibiotic-stalled ribosomes. Our results expose broad roles for conditional termination in regulating antibiotic resistance and provide a tool for discovering riboswitches and attenuators that respond to previously unknown ligands.

Natural selection underlies apparent stress-induced mutagenesis in a bacteriophage infection model

Yosef I., Edgar R., Levy A., Amitai G., Sorek R., Munitz A., Qimron U., Mueller B., Tarkowski P. & Weiss D. (2016) Nature Microbiology. 1, 6, 16047.

The emergence of mutations following growth-limiting conditions underlies bacterial drug resistance, viral escape from the immune system and fundamental evolution-driven events. Intriguingly, whether mutations are induced by growth limitation conditions or are randomly generated during growth and then selected by growth limitation conditions remains an open question¹. Here, we show that bacteriophage T7 undergoes apparent stress-induced mutagenesis when selected for improved recognition of its host's receptor. In our unique experimental set-up, the growth limitation condition is physically and temporally separated from mutagenesis: growth limitation occurs while phage DNA is outside the host, and spontaneous mutations occur during phage DNA replication inside the host. We show that the selected beneficial mutations are not pre-existing and that the initial slow phage growth is enabled by the phage particle's low-efficiency DNA injection into the host. Thus, the phage particle allows phage populations to initially extend their host range without mutagenesis by virtue of residual recognition of the host receptor. Mutations appear during non-selective intracellular replication, and the frequency of mutant phages increases by natural selection acting on free phages, which are not capable of mutagenesis.

CRISPR-Cas adaptation: Insights into the mechanism of action

Amitai G. & Sorek R. (2016) Nature Reviews Microbiology. 14, 2, p. 67-76

Since the first demonstration that CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against phages and plasmids, numerous studies have yielded key insights into the molecular mechanisms governing how these systems attack and degrade foreign DNA. However, the molecular mechanisms underlying the adaptation stage, in which new immunological memory is formed, have until recently represented a major unresolved question. In this Progress article, we discuss recent discoveries that have shown both how foreign DNA is identified by the CRISPR-Cas adaptation machinery and the molecular basis for its integration into the chromosome to form an immunological memory. Furthermore, we describe the roles of each of the specific CRISPR-Cas components that are involved in memory formation, and consider current models for their evolutionary origin.

Comparative transcriptomics across the prokaryotic tree of life

Cohen O., Doron S., Wurtzel O., Dar D., Edelheit S., Karunker I., Mick E. & Sorek R. (2016) Nucleic Acids Research. 44, W1, p. W46-W53

Whole-transcriptome sequencing studies from recent years revealed an unexpected complexity in transcriptomes of bacteria and archaea, including abundant non-coding RNAs, cis-antisense transcription and regulatory untranslated regions (UTRs). Understanding the functional relevance of the plethora of non-coding RNAs in a given organism is challenging, especially since some of these RNAs were attributed to 'transcriptional noise'. To allow the search for conserved transcriptomic elements we produced comparative transcriptome maps for multiple species across the microbial tree of life. These transcriptome maps are detailed in annotations, comparable by gene families, and BLAST-searchable by user provided sequences. Our transcriptome collection includes 18 model organisms spanning 10 phyla/subphyla of bacteria and archaea that were sequenced using standardized RNA-seq methods. The utility of the comparative approach, as implemented in our web server, is demonstrated by highlighting genes with exceptionally long 5UTRs across species, which correspond to many known riboswitches and further suggest novel putative regulatory elements. Our study provides a standardized reference transcriptome to major clinically and environmentally important microbial phyla.

2015

In grateful recognition of our Editorial Board

Bolhuis J., Botelho R., Caetano-Anolles G., Carninci P., Cheah K., Dagan T., Frye M., Goodson H., Gough J., Groth A., Huang S., Isalan M., Kaeberlein M., López-García P., Pei D., Schierwater B., Schumacher B., Shen M., Sorek R., Thomas D., Tian X. L., Veitia R., Whitelaw E. & Wong R. (2015) BioEssays. 37, 12, p. 1254-1255

Growth dynamics of gut microbiota in health and disease inferred from single metagenomic samples

Korem T., Zeevi D., Suez J., Weinberger A., Avnit Sagi S. T., Pompan-Lotan M., Matot E., Jona G., Harmelin A., Cohen N., Sirota-Madi A., Thaiss C. A., Pevsner-Fischer M., Sorek R., Xavier R. J., Elinav E. & Segal E. (2015) Science (New York, N.Y.). 349, 6252, p. 1101-1106

Accepted version

Metagenomic sequencing increased our understanding of the role of the microbiome in health and disease, yet it only provides a snapshot of a highly dynamic ecosystem. Here, we show that the pattern of metagenomic sequencing read coverage for different microbial genomes contains a single trough and a single peak, the latter coinciding with the bacterial origin of replication. Furthermore, the ratio of sequencing coverage between the peak and trough provides a quantitative measure of a species' growth rate. We demonstrate this in vitro and in vivo, under different growth conditions, and in complex bacterial communities. For several bacterial species, peak-to-trough coverage ratios, but not relative abundances, correlated with the manifestation of inflammatory bowel disease and type II diabetes.

CRISPR adaptation biases explain preference for acquisition of foreign DNA

Levy A., Goren M. G., Yosef I., Auster O., Manor M., Amitai G., Edgar R., Qimron U. & Sorek R. (2015) Nature. 520, 7548, p. 505-510

Levy et al 2015.pdf

CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.

BREX is a novel phage resistance system widespread in microbial genomes

Goldfarb T., Sberro H., Weinstock E., Cohen O., Doron S., Charpak-Amikam Y., Afik S., Ofir G. & Sorek R. (2015) EMBO Journal. 34, 2, p. 169-183

The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include the CRISPR-Cas and restriction-modification systems, have proven to be invaluable in the biotechnology and dairy industries. Here, we report on a six-gene cassette in Bacillus cereus which, when integrated into the Bacillus subtilis genome, confers resistance to a broad range of phages, including both virulent and temperate ones. This cassette includes a putative Lon-like protease, an alkaline phosphatase domain protein, a putative RNA-binding protein, a DNA methylase, an ATPase-domain protein, and a protein of unknown function. We denote this novel defense system BREX (Bacteriophage Exclusion) and show that it allows phage adsorption but blocks phage DNA replication. Furthermore, our results suggest that methylation on non-palindromic TAGGAG motifs in the bacterial genome guides self/non-self discrimination and is essential for the defensive function of the BREX system. However, unlike restriction-modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense. Pan genomic analysis revealed that BREX and BREX-like systems, including the distantly related Pgl system described in Streptomyces coelicolor, are widely distributed in ∼10% of all sequenced microbial genomes and can be divided into six coherent subtypes in which the gene composition and order is conserved. Finally, we detected a phage family that evades the BREX defense, implying that anti-BREX mechanisms may have evolved in some phages as part of their arms race with bacteria.

2014

Pregnancy-associated listeriosis: Clinical characteristics and geospatial analysis of a 10-year period in Israel

Elinav H., Hershko-Klement A., Valinsky L., Jaffe J., Wiseman A., Shimon H., Braun E., Paitan Y., Block C., Sorek R. & Nir-Paz R. (2014) Clinical Infectious Diseases. 59, 7, p. 953-961

Background. Listeria monocytogenes is a foodborne pathogen that causes life-threatening infections in elderly, immunocompromised, and pregnant women. In pregnancy it may cause fetal loss or a preterm delivery, and the neonate is prone to neonatal sepsis and death. Methods. We created a cohort of all L. monocytogenes cases during 10 years (1998-2007) in Israel, by a comprehensive review of cases in hospitals throughout the country and cases reported to the Ministry of Health. Results. One hundred sixty-six pregnancy-related listeriosis cases were identified, resulting in a yearly incidence of 5-25 cases per 100 000 births. Presentation associated with fetal demise was more common in the second trimester (55.3%), and preterm labor (52.3%) and abnormal fetal heart rate monitoring (22.2%) were more common in the third trimester (P = .001). Fetal viability was low in the second trimester (29.2%) and much higher (95.3%) in the third trimester. Each additional week of pregnancy increased the survival chance by 33% (odds ratio, 1.331 [95% confidence interval, 1.189-1.489]). A single case of maternal mortality was identified. Listeria monocytogenes serotype 4b was more common in pregnancy-related than in non-pregnancy-related cases (79.5% vs 61.3%, P = .011). Pulsed-field gel electrophoresis analysis suggested that 1 pulsotype is responsible for 35.7% of the pregnancy cases between 2001 and 2007. This clone is closely related to the Italian gastroenteritis-associated HPB2262 and the invasive US Scott A L. monocytogenes strains. Conclusions. Our survey emphasizes the high rate of pregnancy-related listeriosis in Israel and shows that specific clones might account for this.

Sequestration of a two-component response regulator by a riboswitch-regulated noncoding RNA

Mellin J. R., Koutero M., Dar D., Nahori M. A., Sorek R. & Cossart P. (2014) Science. 345, 6199, p. 940-943

Riboswitches are ligand-binding elements contained within the 5 untranslated regions of bacterial transcripts, which generally regulate expression of downstream open reading frames. Here, we show that in Listeria monocytogenes, a riboswitch that binds vitamin B₁₂ controls expression of a noncoding regulatory RNA, Rli55. Rli55, in turn, controls expression of the eut genes, whose products enable ethanolamine utilization and require B₁₂ as a cofactor. Defects in ethanolamine utilization, or in its regulation by Rli55, significantly attenuate Listeria virulence in mice. Rli55 functions by sequestering the two-component response regulator EutV by means of a EutV-binding site contained within the RNA. Thus, Rli55 is a riboswitch-regulated member of the small group of regulatory RNAs that function by sequestering a protein and reveals a distinctive mechanism of signal integration in bacterial gene regulation.

Fur is the master regulator of the extraintestinal pathogenic Escherichia coli response to serum

Huja S., Oren Y., Biran D., Meyer S., Dobrindt U., Bernhard J., Becher D., Hecker M., Sorek R. & Ron E. Z. (2014) mBio. 5, 4, e01460-14.

Drug-resistant extraintestinal pathogenic Escherichia coli (ExPEC) strains are the major cause of colisepticemia (colibacillosis), a condition that has become an increasing public health problem in recent years. ExPEC strains are characterized by high resistance to serum, which is otherwise highly toxic to most bacteria. To understand how these bacteria survive and grow in serum, we performed system-wide analyses of their response to serum, making a clear distinction between the responses to nutritional immunity and innate immunity. Thus, mild heat inactivation of serum destroys the immune complement and abolishes the bactericidal effect of serum (inactive serum), making it possible to examine nutritional immunity. We used a combination of deep RNA sequencing and proteomics in order to characterize ExPEC genes whose expression is affected by the nutritional stress of serum and by the immune complement. The major change in gene expression induced by serumactive and inactiveinvolved metabolic genes. In particular, the serum metabolic response is coordinated by three transcriptional regulators, Fur, BasR, and CysB. Fur alone was responsible for more than 80% of the serum-induced transcriptional response. Consistent with its role as a major serum response regulator, deletion of Fur renders the bacteria completely serum sensitive. These results highlight the role of metabolic adaptation in colisepticemia and virulence.IMPORTANCE Drug-resistant extraintestinal pathogenic Escherichia coli (ExPEC) strains have emerged as major pathogens, especially in community- and hospital-acquired infections. These bacteria cause a large spectrum of syndromes, the most serious of which is septicemia, a condition with a high mortality rate. These bacterial strains are characterized by high resistance to serum, otherwise highly toxic to most bacteria. To understand the basis of this resistance, we carried out system-wide analyses of the response of ExPEC strains to serum by using proteomics and deep RNA sequencing. The major changes in gene expression induced by exposure to serum involved metabolic genes, not necessarily implicated in relation to virulence. One metabolic regulator- Fur-involved in iron metabolism was responsible for more than 80% of the serum-induced response, and its deletion renders the bacteria completely serum sensitive. These results highlight the role of metabolic adaptation in virulence.

High-resolution metagenomics

Mick E. & Sorek R. (2014) Nature biotechnology. 32, 8, p. 750-751

Rearrangement of a large novel Pseudomonas aeruginosa gene island in strains isolated from a patient developing ventilator-associated pneumonia

Singh G., Srinivasan R., Cheng J., Peng Z., Fujimura K., Baek M. S., Panzer A. R., Tringe S. G., Chen F., Sorek R., Weng L., Bristow J., Wiener-Kronish J. P. & Lynch S. V. (2014) Journal of Clinical Microbiology. 52, 7, p. 2430-2438

Bacterial gene islands add to the genetic repertoire of opportunistic pathogens. Here, we perform comparative analyses of three Pseudomonas aeruginosa strains isolated sequentially over a 3-week period from a patient with ventilator-associated pneumonia (VAP) who received clindamycin and piperacillin-tazobactam as part of their treatment regime. While all three strains appeared to be clonal by standard pulsed-field gel electrophoresis, whole-genome sequencing revealed subtle alterations in the chromosomal organization of the last two strains; specifically, an inversion event within a novel 124-kb gene island (PAGI 12) composed of 137 open reading frames [ORFs]. Predicted ORFs in the island included metabolism and virulence genes. Overexpression of a gene island-borne putative β-lactamase gene was observed following piperacillin-tazobactam exposure and only in those strains that had undergone the inversion event, indicating altered gene regulation following genomic remodeling. Examination of a separate cohort of 76 patients with VAP for integration at this tRNA^lys recombination site demonstrated that patients exhibiting evidence of integration at this site had significantly higher 28-day mortality. These findings provide evidence that P. aeruginosa can integrate, rapidly remodel, and express exogenous genes, which likely contributes to its fitness in a clinical setting.

2013

Computational evaluation of cellular metabolic costssuccessfully predicts genes whose expression is deleterious

Wagner A., Zarecki R., Reshef L., Gochev C., Sorek R., Gophna U. & Ruppin E. (2013) Proceedings of the National Academy of Sciences of the United States of America. 110, 47, p. 19166-19171

Gene suppression and overexpression are both fundamental tools in linking genotype to phenotype in model organisms. Computational methods have proven invaluable in studying and predicting the deleterious effects of gene deletions, and yet parallel computational methods for overexpression are still lacking. Here, we present Expression-Dependent Gene Effects (EDGE), an in silico method that can predict the deleterious effects resulting from overexpression of either native or foreign metabolic genes. We first test and validate EDGE's predictive power in bacteria through a combination of small-scale growth experiments that we performed and analysis of extant large-scale datasets. Second, a broad cross-species analysis, ranging from microorganisms to multiple plant and human tissues, shows that genes that EDGE predicts to be deleterious when overexpressed are indeed typically downregulated. This reflects a universal selection force keeping the expression of potentially deleterious genes in check. Third, EDGEbased analysis shows that cancer genetic reprogramming specifically suppresses genes whose overexpression impedes proliferation. The magnitude of this suppression is large enough to enable an almost perfect distinction between normal and cancerous tissues based solely on EDGE results. We expect EDGE to advance our understanding of human pathologies associated with up-regulation of particular transcripts and to facilitate the utilization of gene overexpression in metabolic engineering.

Correlated Occurrence and Bypass of Frame-Shifting Insertion-Deletions (InDels) to Give Functional Proteins

Rockah-Shmuel L., Tóth-Petróczy Á., Sela A., Wurtzel O., Sorek R. & Tawfik D. S. (2013) PLoS Genetics. 9, 10, e1003882.

Short insertions and deletions (InDels) comprise an important part of the natural mutational repertoire. InDels are, however, highly deleterious, primarily because two-thirds result in frame-shifts. Bypass through slippage over homonucleotide repeats by transcriptional and/or translational infidelity is known to occur sporadically. However, the overall frequency of bypass and its relation to sequence composition remain unclear. Intriguingly, the occurrence of InDels and the bypass of frame-shifts are mechanistically related - occurring through slippage over repeats by DNA or RNA polymerases, or by the ribosome, respectively. Here, we show that the frequency of frame-shifting InDels, and the frequency by which they are bypassed to give full-length, functional proteins, are indeed highly correlated. Using a laboratory genetic drift, we have exhaustively mapped all InDels that occurred within a single gene. We thus compared the naive InDel repertoire that results from DNA polymerase slippage to the frame-shifting InDels tolerated following selection to maintain protein function. We found that InDels repeatedly occurred, and were bypassed, within homonucleotide repeats of 3-8 bases. The longer the repeat, the higher was the frequency of InDels formation, and the more frequent was their bypass. Besides an expected 8A repeat, other types of repeats, including short ones, and G and C repeats, were bypassed. Although obtained in vitro, our results indicate a direct link between the genetic occurrence of InDels and their phenotypic rescue, thus suggesting a potential role for frame-shifting InDels as bridging evolutionary intermediates.

Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m⁵C within Archaeal mRNAs

Edelheit S., Schwartz S., Mumbach M. R., Wurtzel O. & Sorek R. (2013) PLoS Genetics. 9, 6, e1003602.

The presence of 5-methylcytidine (m⁵C) in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m⁵C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis) and gram negative (E. coli) bacteria, an archaeon (S. solfataricus) and a eukaryote (S. cerevisiae), followed by massively parallel sequencing. We were able to recover most previously documented m⁵C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m⁵C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m⁵C was absent were also discovered. Intriguingly, we detected m⁵C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m⁵C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.

Differential translation tunes uneven production of operon-encoded proteins

Quax T. E. F., Wolf Y. I., Koehorst J. J., Wurtzel O., van der Oost d. O. R., Ran W., Blombach F., Makarova K. S., Brouns S. J. J., Forster A. C., Wagner E. G. H., Sorek R., Koonin E. V. & van der Oost d. O. J. (2013) Cell Reports. 4, 5, p. 938-944

Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in appropriate relative quantities. Using comparative genomic analysis, we show that differential translation is a key determinant of modulated expression of genes clustered in operons and that codon bias generally is the best insilico indicator of unequal protein production. Variable ribosome density profiles of polycistronic transcripts correlate strongly with differential translation patterns. In addition, we provide experimental evidence that de novo initiation of translation can occur at intercistronic sites, allowing for differential translation of any gene irrespective of its position on a polycistronic messenger. Thus, modulation of translation efficiency appears to be auniversal mode of control in bacteria and archaea that allows for differential production of operon-encodedproteins

Holding a grudge: Persisting anti-phage CRISPR immunity in multiple human gut microbiomes

Mick E., Stern A. & Sorek R. (2013) RNA Biology. 10, 5, p. 900-906

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system of bacteria and archaea constitutes a mechanism of acquired adaptive immunity against phages, which is based on genome-encoded markers of previously infecting phage sequences ("spacers"). As a repository of phage sequences, these spacers make the system particularly suitable for elucidating phage-bacteria interactions in metagenomic studies. Recent metagenomic analyses of CRISPRs associated with the human microbiome intriguingly revealed conserved "memory spacers" shared by bacteria in multiple unrelated, geographically separated individuals. Here, we discuss possible avenues for explaining this phenomenon by integrating insights from CRISPR biology and phage-bacteria ecology, with a special focus on the human gut. We further explore the growing body of evidence for the role of CRISPR/Cas in regulating the interplay between bacteria and lysogenic phages, which may be intimately related to the presence of memory spacers and sheds new light on the multifaceted biological and ecological modes of action of CRISPR/Cas.

A Global Transcriptional Switch between the Attack and Growth Forms of Bdellovibrio bacteriovorus

Karunker I., Rotem O., Dori-Bachash M., Jurkevitch E. & Sorek R. (2013) PLoS ONE. 8, 4, e61850.

Bdellovibrio bacteriovorus is an obligate predator of bacteria ubiquitously found in the environment. Its life cycle is composed of two essential phases: a free-living, non-replicative, fast swimming attack phase (AP) wherein the predator searches for prey; and a non-motile, actively dividing growth phase (GP) in which it consumes the prey. The molecular regulatory mechanisms governing the switch between AP and GP are largely unknown. We used RNA-seq to generate a single-base-resolution map of the Bdellovibrio transcriptome in AP and GP, revealing a specific "AP" transcriptional program, which is largely mutually exclusive of the GP program. Based on the expression map, most genes in the Bdellovibrio genome are classified as "AP only" or "GP only". We experimentally generated a genome-wide map of 140 AP promoters, controlling the majority of AP-specific genes. This revealed a common sigma-like DNA binding site highly similar to the E. coli flagellar genes regulator sigma28 (FliA). Further analyses suggest that FliA has evolved to become a global AP regulator in Bdellovibrio. Our results also reveal a non-coding RNA that is massively expressed in AP. This ncRNA contains a c-di-GMP riboswitch. We suggest it functions as an intracellular reservoir for c-di-GMP, playing a role in the rapid switch from AP to GP.

Discovery of Functional Toxin/Antitoxin Systems in Bacteria by Shotgun Cloning

Sberro H., Leavitt A., Kiro R., Koh E., Peleg Y., Qimron U. & Sorek R. (2013) Molecular Cell. 50, 1, p. 136-148

Toxin-antitoxin (TA) modules, composed of a toxic protein and a counteracting antitoxin, play important roles in bacterial physiology. We examined the experimental insertion of 1.5 million genes from 388 microbial genomes into an Escherichia coli host using more than 8.5 million random clones. This revealed hundreds of genes (toxins) that could only be cloned when the neighboring gene (antitoxin) was present on the same clone. Clustering of these genes revealed TA families widespread in bacterial genomes, some of which deviate from the classical characteristics previously described for such modules. Introduction of these genes into E. coli validated that the toxin toxicity is mitigated by the antitoxin. Infection experiments with T7 phage showed that two of the new modules can provide resistance against phage. Moreover, our experiments revealed an " antidefense" protein in phage T7 that neutralizes phage resistance. Our results expose active fronts in the arms race between bacteria and phage.

The excludon: A new concept in bacterial antisense RNA-mediated gene regulation

Sesto N., Wurtzel O., Archambaud C., Sorek R. & Cossart P. (2013) Nature Reviews Microbiology. 11, 2, p. 75-82

In recent years, non-coding RNAs have emerged as key regulators of gene expression. Among these RNAs, the antisense RNAs (asRNAs) are particularly abundant, but in most cases the function and mechanism of action for a particular asRNA remains elusive. Here, we highlight a recently discovered paradigm termed the excludon, which defines a genomic locus encoding an unusually long asRNA that spans divergent genes or operons with related or opposing functions. Because these asRNAs can inhibit the expression of one operon while functioning as an mRNA for the adjacent operon, they act as fine-tuning regulatory switches in bacteria.

CRISPR-Mediated Adaptive Immune Systems in Bacteria and Archaea

Sorek R., Lawrence C. M. & Wiedenheft B. (2013) Annual Review of Biochemistry. 82, p. 237-266

Effective clearance of an infection requires that the immune system rapidly detects and neutralizes invading parasites while strictly avoiding self-antigens that would result in autoimmunity. The cellular machinery and complex signaling pathways that coordinate an effective immune response have generally been considered properties of the eukaryotic immune system. However, a surprisingly sophisticated adaptive immune system that relies on small RNAs for sequence-specific targeting of foreign nucleic acids was recently discovered in bacteria and archaea. Molecular vaccination in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into a repetitive locus in the host chromosome known as a CRISPR (clustered regularly interspaced short palindromic repeat). Here we review the mechanisms of CRISPR-mediated immunity and discuss the ecological and evolutionary implications of these adaptive defense systems.

2012

Roles of CRISPR in regulation of physiological processes

Amitai G. & Sorek R. (2012) CRISPR-Cas Systems : RNA-Mediated Adaptive Immunity in Bacteria and Archaea . van der Oost J. & Barrangou R.(eds.). p. 251-266

It is now well established, following numerous independent studies, that the CRISPR-Cas system is an adaptive immune system widespread in bacteria and archaea. The primary role of CRISPR-Cas in protection against foreign DNA is nowadays undisputed. However, there is also strong evidence suggesting that, at least in some organisms, CRISPR-Cas systems have adapted to take nonimmune-related regulatory roles that are hardwired into the core regulatory programs of bacterial physiology. In at least two cases, CRISPR was shown to regulate bacterial social behavior; in other cases, CRISPRs were suggested to directly regulate endogenous gene expression. Curious cases of CRISPR-derived autoimmunity were also described. This chapter discusses major examples of such nonimmune roles of CRISPR-Cas that were described to date, their putative mechanisms of action, and their functional and evolutionary implications.

CRISPR targeting reveals a reservoir of common phages associated with the human gut microbiome

Stern A., Mick E., Tirosh I., Sagy O. & Sorek R. (2012) Genome Research. 22, 10, p. 1985-1994

The bacterial community in the human gut has crucial health roles both in metabolic functions and in protection against pathogens. Phages, which are known to significantly affect microbial community composition in many ecological niches, have the potential to impact the gut microbiota, yet thorough characterization of this relationship remains elusive. We have reconstructed the content of the CRISPR bacterial immune system in the human gut microbiomes of 124 European individuals and used it to identify a catalog of 991 phages targeted by CRISPR across all individuals. Our results show that 78% of these phages are shared among two or more individuals. Moreover, a significant fraction of phages found in our study are shown to exist in fecal samples previously derived from American and Japanese individuals, identifying a common reservoir of phages frequently associated with the human gut microbiome. We further inferred the bacterial hosts for more than 130 such phages, enabling a detailed analysis of phage-bacteria interactions across the 124 individuals by correlating patterns of phage abundance with bacterial abundance and resistance. A subset of phages demonstrated preferred association with host genomes as lysogenized prophages, with highly increased abundance in specific individuals. Overall, our results imply that phage-bacterial attack-resistance interactions occur within the human gut microbiome, possibly affecting microbiota composition and human health. Our finding of global sharing of gut phages is surprising in light of the extreme genetic diversity of phages found in other ecological niches.

The Single-Nucleotide Resolution Transcriptome of Pseudomonas aeruginosa Grown in Body Temperature

Wurtzel O., Yoder-Himes D. R., Han K., Dandekar A. A., Edelheit S., Greenberg E. P., Sorek R. & Lory S. (2012) PLoS Pathogens. 8, 9, e1002945.

One of the hallmarks of opportunistic pathogens is their ability to adjust and respond to a wide range of environmental and host-associated conditions. The human pathogen Pseudomonas aeruginosa has an ability to thrive in a variety of hosts and cause a range of acute and chronic infections in individuals with impaired host defenses or cystic fibrosis. Here we report an in-depth transcriptional profiling of this organism when grown at host-related temperatures. Using RNA-seq of samples from P. aeruginosa grown at 28°C and 37°C we detected genes preferentially expressed at the body temperature of mammalian hosts, suggesting that they play a role during infection. These temperature-induced genes included the type III secretion system (T3SS) genes and effectors, as well as the genes responsible for phenazines biosynthesis. Using genome-wide transcription start site (TSS) mapping by RNA-seq we were able to accurately define the promoters and cis-acting RNA elements of many genes, and uncovered new genes and previously unrecognized non-coding RNAs directly controlled by the LasR quorum sensing regulator. Overall we identified 165 small RNAs and over 380 cis-antisense RNAs, some of which predicted to perform regulatory functions, and found that non-coding RNAs are preferentially localized in pathogenicity islands and horizontally transferred regions. Our work identifies regulatory features of P. aeruginosa genes whose products play a role in environmental adaption during infection and provides a reference transcriptional landscape for this pathogen.

PanDaTox: A tool for accelerated metabolic engineering

Amitai G. & Sorek R. (2012) Bioengineered. 3, 4, p. 218-221

Metabolic engineering is often facilitated by cloning of genes encoding enzymes from various heterologous organisms into E. coli. Such engineering efforts are frequently hampered by foreign genes that are toxic to the E. coli host. We have developed PanDaTox (www.weizmann.ac.il/pandatox), a web-based resource that provides experimental toxicity information for more than 1.5 million genes from hundreds of different microbial genomes. The toxicity predictions, which were extensively experimentally verified, are based on serial cloning of genes into E. coli as part of the Sanger whole genome shotgun sequencing process. PanDaTox can accelerate metabolic engineering projects by allowing researchers to exclude toxic genes from the engineering plan and verify the clonability of selected genes before the actual metabolic engineering experiments are conducted.

Topology of the human and mouse m⁶A RNA methylomes revealed by m⁶A-seq

Dominissini D., Moshitch-Moshkovitz S., Schwartz S., Salmon-Divon M., Ungar L., Osenberg S., Cesarkas K., Jacob-Hirsch J., Amariglio N., Kupiec M., Sorek R. & Rechavi G. (2012) Nature. 485, 7397, p. 201-206

An extensive repertoire of modifications is known to underlie the versatile coding, structural and catalytic functions of RNA, but it remains largely uncharted territory. Although biochemical studies indicate that N⁶-methyladenosine (m⁶A) is the most prevalent internal modification in messenger RNA, an in-depth study of its distribution and functions has been impeded by a lack of robust analytical methods. Here we present the human and mouse m⁶A modification landscape in a transcriptome-wide manner, using a novel approach, m⁶A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12, 000 m⁶A sites characterized by a typical consensus in the transcripts of more than 7, 000 human genes. Sites preferentially appear in two distinct landmarks-around stop codons and within long internal exons-and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m⁶A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signalling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m⁶A has a fundamental role in regulation of gene expression.

A vast collection of microbial genes that are toxic to bacteria

Kimelman A., Levy A., Sberro H., Kidron S., Leavitt A., Amitai G., Yoder-Himes D. R., Wurtzel O., Zhu Y., Rubin E. M. & Sorek R. (2012) Genome Research. 22, 4, p. 802-809

In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes, we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes, our assays revealed novel toxins and restriction enzymes, and new classes of small, non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short, toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator DnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.

Transcriptome-wide discovery of circular RNAs in Archaea

Danan M., Schwartz S., Edelheit S. & Sorek R. (2012) Nucleic Acids Research. 40, 7, p. 3131-3142

Circular RNA forms had been described in all domains of life. Such RNAs were shown to have diverse biological functions, including roles in the life cycle of viral and viroid genomes, and in maturation of permuted tRNA genes. Despite their potentially important biological roles, discovery of circular RNAs has so far been mostly serendipitous. We have developed circRNA-seq, a combined experimental/computational approach that enriches for circular RNAs and allows profiling their prevalence in a whole-genome, unbiased manner. Application of this approach to the archaeon Sulfolobus solfataricus P2 revealed multiple circular transcripts, a subset of which was further validated independently. The identified circular RNAs included expected forms, such as excised tRNA introns and rRNA processing intermediates, but were also enriched with non-coding RNAs, including C/D box RNAs and RNase P, as well as circular RNAs of unknown function. Many of the identified circles were conserved in Sulfolobus acidocaldarius, further supporting their functional significance. Our results suggest that circular RNAs, and particularly circular non-coding RNAs, are more prevalent in archaea than previously recognized, and might have yet unidentified biological roles. Our study establishes a specific and sensitive approach for identification of circular RNAs using RNA-seq, and can readily be applied to other organisms.

RNA-seq analysis of small RNPs in Trypanosoma brucei reveals a rich repertoire of non-coding RNAs

Michaeli S., Doniger T., Gupta S. K., Wurtzel O., Romano M., Visnovezky D., Sorek R., Unger R. & Ullu E. (2012) Nucleic Acids Research. 40, 3, p. 1282-1298

The discovery of a plethora of small non-coding RNAs (ncRNAs) has fundamentally changed our understanding of how genes are regulated. In this study, we employed the power of deep sequencing of RNA (RNA-seq) to examine the repertoire of ncRNAs present in small ribonucleoprotein particles (RNPs) of Trypanosoma brucei, an important protozoan parasite. We identified new C/D and H/ACA small nucleolar RNAs (snoRNAs), as well as tens of putative novel non-coding RNAs; several of these are processed from trans-spliced and polyadenylated transcripts. The RNA-seq analysis provided information on the relative abundance of the RNAs, and their 5-and 3-termini. The study demonstrated that three highly abundant snoRNAs are involved in rRNA processing and highlight the unique trypanosome-specific repertoire of these RNAs. Novel RNAs were studied using in situ hybridization, association in RNP complexes, and 'RNA walk' to detect interaction with their target RNAs. Finally, we showed that the abundance of certain ncRNAs varies between the two stages of the parasite, suggesting that ncRNAs may contribute to gene regulation during the complex parasite's life cycle. This is the first study to provide a whole-genome analysis of the large repertoire of small RNPs in trypanosomes.

Comparative transcriptomics of pathogenic and non-pathogenic Listeria species

Wurtzel O., Sesto N., Mellin J. R., Karunker I., Edelheit S., Bécavin C., Archambaud C., Cossart P. & Sorek R. (2012) Molecular Systems Biology. 8, 1, 583.

Listeria monocytogenes is a human, food-borne pathogen. Genomic comparisons between L. monocytogenes and Listeria innocua, a closely related non-pathogenic species, were pivotal in the identification of protein-coding genes essential for virulence. However, no comprehensive comparison has focused on the non-coding genome. We used strand-specific cDNA sequencing to produce genome-wide transcription start site maps for both organisms, and developed a publicly available integrative browser to visualize and analyze both transcriptomes in different growth conditions and genetic backgrounds. Our data revealed conservation across most transcripts, but significant divergence between the species in a subset of non-coding RNAs. In L. monocytogenes, we identified 113 small RNAs (33 novel) and 70 antisense RNAs (53 novel), significantly increasing the repertoire of ncRNAs in this species. Remarkably, we identified a class of long antisense transcripts (lasRNAs) that overlap one gene while also serving as the 5 UTR of the adjacent divergent gene. Experimental evidence suggests that lasRNAs transcription inhibits expression of one operon while activating the expression of another. Such a lasRNA/operon structure, that we named 'excludon', might represent a novel form of regulation in bacteria.

2011

Bacterial genomes: From regulatory complexity to engineering

Sorek R. & Serrano L. (2011) Current Opinion in Microbiology. 14, 5, p. 577-578

Genomic island variability facilitates Prochlorococcusvirus coexistence

Avrani S., Wurtzel O., Sharon I., Sorek R. & Lindell D. (2011) Nature. 474, 7353, p. 604-608

Prochlorococcus cyanobacteria are extremely abundant in the oceans, as are the viruses that infect them. How hosts and viruses coexist in nature remains unclear, although the presence of both susceptible and resistant cells may allow this coexistence. Combined whole-genome sequencing and PCR screening technology now enables us to investigate the effect of resistance on genome evolution and the genomic mechanisms behind the long-term coexistence of Prochlorococcus and their viruses. Here we present a genome analysis of 77 substrains selected for resistance to ten viruses, revealing mutations primarily in non-conserved, horizontally transferred genes that localize to a single hypervariable genomic island. Mutations affected viral attachment to the cell surface and imposed a fitness cost to the host, manifested by significantly lower growth rates or a previously unknown mechanism of more rapid infection by other viruses. The mutant genes are generally uncommon in nature yet some carry polymorphisms matching those found experimentally. These data are empirical evidence indicating that viral-attachment genes are preferentially located in genomic islands and that viruses are a selective pressure enhancing the diversity of both island genes and island gene content. This diversity emerges as a genomic mechanism that reduces the effective host population size for infection by a given virus, thus facilitating long-term coexistence between viruses and their hosts in nature.

The phage-host arms race: Shaping the evolution of microbes

Stern A. & Sorek R. (2011) BioEssays. 33, 1, p. 43-51

Bacteria, the most abundant organisms on the planet, are outnumbered by a factor of 10 to 1 by phages that infect them. Faced with the rapid evolution and turnover of phage particles, bacteria have evolved various mechanisms to evade phage infection and killing, leading to an evolutionary arms race. The extensive co-evolution of both phage and host has resulted in considerable diversity on the part of both bacterial and phage defensive and offensive strategies. Here, we discuss the unique and common features of phage resistance mechanisms and their role in global biodiversity. The commonalities between defense mechanisms suggest avenues for the discovery of novel forms of these mechanisms based on their evolutionary traits.

2010

Validation of two ribosomal RNA removal methods for microbial metatranscriptomics

He S., Wurtzel O., Singh K., Froula J. L., Yilmaz S., Tringe S. G., Wang Z., Chen F., Lindquist E. A., Sorek R. & Hugenholtz P. (2010) Nature Methods. 7, 10, p. 807-812

The predominance of rRNAs in the transcriptome is a major technical challenge in sequence-based analysis of cDNAs from microbial isolates and communities. Several approaches have been applied to deplete rRNAs from (meta)transcriptomes, but no systematic investigation of potential biases introduced by any of these approaches has been reported. Here we validated the effectiveness and fidelity of the two most commonly used approaches, subtractive hybridization and exonuclease digestion, as well as combinations of these treatments, on two synthetic five-microorganism metatranscriptomes using massively parallel sequencing. We found that the effectiveness of rRNA removal was a function of community composition and RNA integrity for these treatments. Subtractive hybridization alone introduced the least bias in relative transcript abundance, whereas exonuclease and in particular combined treatments greatly compromised mRNA abundance fidelity. Illumina sequencing itself also can compromise quantitative data analysis by introducing a G+C bias between runs.

Self-targeting by CRISPR: Gene regulation or autoimmunity?

Stern A., Keren L., Wurtzel O., Amitai G. & Sorek R. (2010) Trends in Genetics. 26, 8, p. 335-340

The recently discovered prokaryotic immune system known as CRISPR (clustered regularly interspaced short palindromic repeats) is based on small RNAs ('spacers') that restrict phage and plasmid infection. It has been hypothesized that CRISPRs can also regulate self gene expression by utilizing spacers that target self genes. By analyzing CRISPRs from 330 organisms we found that one in every 250 spacers is self-targeting, and that such self-targeting occurs in 18% of all CRISPR-bearing organisms. However, complete lack of conservation across species, combined with abundance of degraded repeats near self-targeting spacers, suggests that self-targeting is a form of autoimmunity rather than a regulatory mechanism. We propose that accidental incorporation of self nucleic acids by CRISPR can incur an autoimmune fitness cost, and this could explain the abundance of degraded CRISPR systems across prokaryotes.

A single-base resolution map of an archaeal transcriptome

Wurtzel O., Sapra R., Chen F., Zhu Y., Simmons B. A. & Sorek R. (2010) Genome Research. 20, 1, p. 133-141

Organisms of the third domain of life, the Archaea, share molecular characteristics both with Bacteria and Eukarya. These organisms attract scientific attention as research models for regulation and evolution of processes such as transcription, translation, and RNA processing. We have reconstructed the primary transcriptome of Sulfolobus solfataricus P2, one of the most widely studied model archaeal organisms. Analysis of 625 million bases of sequenced cDNAs yielded a single-basepair resolution map of transcription start sites and operon structures for more than 1000 transcriptional units. The analysis led to the discovery of 310 expressed noncoding RNAs, with an extensive expression of overlapping cis-antisense transcripts to a level unprecedented in any bacteria or archaea but resembling that of eukaryotes. As opposed to bacterial transcripts, most Sulfolobus transcripts completely lack 59-UTR sequences, suggesting that mRNA/ncRNA interactions differ between Bacteria and Archaea. The data also reveal internal hotspots for transcript cleavage linked to RNA degradation and predict sequence motifs that promote RNA destabilization. This study highlights transcriptome sequencing as a key tool for understanding the mechanisms and extent of RNA-based regulation in Bacteria and Archaea.

Prokaryotic transcriptomics: A new view on regulation, physiology and pathogenicity

Sorek R. & Cossart P. (2010) Nature Reviews Genetics. 11, 1, p. 9-16

Transcriptome-wide studies in eukaryotes have been instrumental in the characterization of fundamental regulatory mechanisms for more than a decade. By contrast, in prokaryotes (bacteria and archaea) whole-transcriptome studies have not been performed until recently owing to the general view that microbial gene structures are simple, as well as technical difficulties in enriching for mRNAs that lack poly(A) tails. Deep RNA sequencing and tiling array studies are now revolutionizing our understanding of the complexity, plasticity and regulation of microbial transcriptomes.

Mutation detection with next-generation resequencing through a mediator genome

Wurtzel O., Dori-Bachash M., Pietrokovski S., Jurkevitch E. & Sorek R. (2010) PLoS ONE. 5, 12, e15628.

The affordability of next generation sequencing (NGS) is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing itto the wild-type (WT) genome, thus identifying acquired mutations. A major limitation for this approach is the need for an apriori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WT and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.

2009

When new exons are born

Sorek R. (2009) Heredity. 103, 4, p. 279-280

Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing

Yoder-Himes D. R., Chain P. S., Zhu Y., Wurtzel O., Rubin E. M., Tiedje J. M. & Sorek R. (2009) Proceedings of the National Academy of Sciences of the United States of America. 106, 10, p. 3976-3981

Determining how an organism responds to its environment by altering gene expression is key to understanding its ecology. Here, we used RNA-seq to comprehensively and quantitatively assess the transcriptional response of the bacterial opportunistic cystic fibrosis (CF) pathogen and endemic soil dweller, Burkholderia cenocepacia, in conditions mimicking these 2 environments. By sequencing 762 million bases of cDNA from 2 closely related B. cenocepacia strains (one isolated from a CF patient and one from soil), we identified a number of potential virulence factors expressed under CF-like conditions, whereas genes whose protein products are involved in nitrogen scavenging and 2-component sensing were among those induced under soil-like conditions. Interestingly, 13 new putative noncoding RNAs were discovered using this technique, 12 of which are preferentially induced in the soil environment, suggesting that ncRNAs play an important role in survival in the soil. In addition, we detected a surprisingly large number of regulatory differences between the 2 strains, which may represent specific adaptations to the niches from which each strain was isolated, despite their high degree of DNA sequence similarity. Compared with the CF strain, the soil strain shows a stronger global gene expression response to its environment, which is consistent with the need for a more dynamic reaction to the heterogeneous conditions of soil.

The dynamics and time scale of ongoing genomic erosion in symbiotic bacteria

Moran N. A., McLaughlin H. J. & Sorek R. (2009) Science. 323, 5912, p. 379-382

Among cellular organisms, symbiotic bacteria provide the extreme examples of genome degradation and reduction. However, only isolated snapshots of eroding symbiont genomes have previously been available. We documented the dynamics of symbiont genome evolution by sequencing seven strains of Buchnera aphidicola from pea aphid hosts. We estimated a spontaneous mutation rate of at least 4 × 10^-9 substitutions per site per replication, which is more than 10 times as high as the rates previously estimated for any bacteria. We observed a high rate of small insertions and deletions associated with abundant DNA homopolymers, and occasional larger deletions. Although purifying selection eliminates many mutations, some persist, resulting in ongoing loss of genes and DNA from this already tiny genome. Our results provide a general model for the stepwise process leading to genome reduction.

2008

CRISPR - A widespread system that provides acquired resistance against phages in bacteria and archaea

Sorek R., Kunin V. & Hugenholtz P. (2008) Nature Reviews Microbiology. 6, 3, p. 181-186

Arrays of clustered, regularly interspaced short palindromic repeats (CRISPRs) are widespread in the genomes of many bacteria and almost all archaea. These arrays are composed of direct repeats that are separated by similarly sized non-repetitive spacers. CRISPR arrays, together with a group of associated proteins, confer resistance to phages, possibly by an RNA-interference-like mechanism. This Progress discusses the structure and function of this newly recognized antiviral mechanism.

2007

Genome-wide experimental determination of barriers to horizontal gene transfer

Sorek R., Zhu Y., Creevey C. J., Francino M. P., Bork P. & Rubin E. M. (2007) Science. 318, 5855, p. 1449-1452

Horizontal gene transfer, in which genetic material is transferred from the genome of one organism to that of another, has been investigated in microbial species mainly through computational sequence analyses. To address the lack of experimental data, we studied the attempted movement of 246,045 genes from 79 prokaryotic genomes into Escherichia coli and identified genes that consistently fail to transfer. We studied the mechanisms underlying transfer inhibition by placing coding regions from different species under the control of inducible promoters. Our data suggest that toxicity to the host inhibited transfer regardless of the species of origin and that increased gene dosage and associated increased expression may be a predominant cause for transfer failure. Although these experimental studies examined transfer solely into E. coli, a computational analysis of gene-transfer rates across available bacterial and archaeal genomes supports that the barriers observed in our study are general across the tree of life.

Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite

Warnecke F., Luginbühl P., Ivanova N., Ghassemian M., Richardson T. H., Stege J. T., Cayouette M., McHardy A. C., Djordjevic G., Aboushadi N., Sorek R., Tringe S. G., Podar M., Martin H. G., Kunin V., Dalevi D., Madejska J., Kirton E., Platt D., Szeto E., Salamov A., Barry K., Mikhailova N., Kyrpides N. C., Matson E. G., Ottesen E. A., Zhang X., Hernández M., Murillo C., Acosta L. G., Rigoutsos I., Tamayo G., Green B. D., Chang C., Rubin E. M., Mathur E. J., Robertson D. E., Hugenholtz P. & Leadbetter J. R. (2007) Nature. 450, 7169, p. 560-565

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms ¹ and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis². Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding 'higher' Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H₂ metabolism, CO₂-reductive acetogenesis and N₂ fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-μl environment can be.

The birth of new exons: Mechanisms and evolutionary consequences

Sorek R. (2007) RNA. 13, 10, p. 1603-1608

A significant amount of literature was dedicated to hypotheses concerning the origin of ancient introns and exons, but accumulating evidence indicates that new exons are also constantly being added to evolving genomes. Several mechanisms contribute to the creation of novel exons in metazoan genomes, including whole gene and single exon duplications, but perhaps the most intriguing are events of exonization, where intronic sequences become exons de novo. Exonizations of intronic sequences, particularly those originating from repetitive elements, are now widely documented in many genomes including human, mouse, dog, and fish. Such de novo appearance of exons is very frequently associated with alternative splicing, with the new exon-containing variant typically being the rare one. This allows the new variant to be evolutionarily tested without compromising the original one, and provides an evolutionary strategy for generation of novel functions with minimum damage to the existing functional repertoire. This review discusses the molecular mechanisms leading to exonization, its extent in vertebrate genomes, and its evolutionary implications. Published by Cold Spring Harbor Laboratory Press.

Evolutionary conservation of sequence and secondary structures in CRISPR repeats

Kunin V., Sorek R. & Hugenholtz P. (2007) GENOME BIOLOGY. 8, 4, R61.

Background: Clustered regularly interspaced short palindromic repeats (CRISPRs) are a novel class of direct repeats, separated by unique spacer sequences of similar length, that are present in approximately 40% of bacterial and most archaeal genomes analyzed to date. More than 40 gene families, called CRISPR-associated sequences (CASs), appear in conjunction with these repeats and are thought to be involved in the propagation and functioning of CRISPRs. It has been recently shown that CRISPR provides acquired resistance against viruses in prokaryotes. Results: Here we analyze CRISPR repeats identified in 195 microbial genomes and show that they can be organized into multiple clusters based on sequence similarity. Some of the clusters present stable, highly conserved RNA secondary structures, while others lack detectable structures. Stable secondary structures exhibit multiple compensatory base changes in the stem region, indicating evolutionary and functional conservation. Conclusion: We show that the repeat-based classification corresponds to, and expands upon, a previously reported CAS gene-based classification, including specific relationships between CRISPR and CAS subtypes.

RNA-editing-mediated exon evolution

Lev-Maor G., Sorek R., Levanon E. Y., Paz N., Eisenberg E. & Ast G. (2007) GENOME BIOLOGY. 8, 2, R29.

Background: Alu retroelements are specific to primates and abundant in the human genome. Through mutations that create functional splice sites within intronic Alus, these elements can become new exons in a process denoted exonization. It was recently shown that Alu elements are also heavily changed by RNA editing in the human genome. Results: Here we show that the human nuclear prelamin A recognition factor contains a primate-specific Alu-exon that exclusively depends on RNA editing for its exonization. We demonstrate that RNA editing regulates the exonization in a tissue-dependent manner, through both the creation of a functional AG 3 splice site, and alteration of functional exonic splicing enhancers within the exon. Furthermore, a premature stop codon within the Alu-exon is eliminated by an exceptionally efficient RNA editing event. The sequence surrounding this editing site is important not only for editing of that site but also for editing in other neighboring sites as well. Conclusion: Our results show that the abundant RNA editing of Alu sequences can be recruited as a mechanism supporting the birth of new exons in the human genome.

2006

Assessing the number of ancestral alternatively spliced exons in the human genome

Sorek R., Dror G. & Shamir R. (2006) BMC Genomics. 7, 273.

Background: It is estimated that between 35% and 74% of all human genes undergo alternative splicing. However, as a gene that undergoes alternative splicing can have between one and dozens of alternative exons, the number of alternatively spliced genes by itself is not informative enough. An additional parameter, which was not addressed so far, is therefore the number of human exons that undergo alternative splicing. We have previously described an accurate machine-learning method allowing the detection of conserved alternatively spliced exons without using ESTs, which relies on specific features of the exon and its genomic vicinity that distinguish alternatively spliced exons from constitutive ones. Results: In this study we use the above-described approach to calculate that 7.2% (± 1.1%) of all human exons that are conserved in mouse are alternatively spliced in both species. Conclusion: This number is the first estimation for the extent of ancestral alternatively spliced exons in the human genome.

Genomic fossils as a snapshot of the human transcriptome

Shemesh R., Novik A., Edelheit S. & Sorek R. (2006) Proceedings of the National Academy of Sciences of the United States of America. 103, 5, p. 1364-1369

Processed pseudogenes (PPGs) are cDNA sequences that were generated through reverse transcription of mature, spliced mRNAs and have subsequently been reinserted at a new genomic location. These cDNA sequences are usually no longer transcribed and are considered "dead on arrival." Here we show that PPGs can be used to generate a map of the transcriptome. By analyzing thousands of human PPGs, we were able to discover hundreds of transcript variants so far unidentified. An experimental verification of a subset of these variants by RT-PCR indicates that most of them are still active in the human transcriptome. Furthermore, we demonstrate that PPGs can enable the identification of ancient splice variants that were expressed ancestrally but are now extinct. Our results show that the genome itself carries a "virtual cDNA library" that can readily be used to analyze both present and ancestral transcripts. Our approach can be applied to sequenced metazoan genomes to computationally annotate splicing variation even when expressed sequences are unavailable.

Transcription-mediated gene fusion in the human genome

Akiva P., Toporik A., Edelheit S., Peretz Y., Diber A., Shemesh R., Novik A. & Sorek R. (2006) Genome Research. 16, 1, p. 30-36

Transcription of a gene usually ends at a regulated termination point, preventing the RNA-polymerase from reading through the next gene. However, sporadic reports suggest that chimeric transcripts, formed by transcription of two consecutive genes into one RNA, can occur in human. The splicing and translation of such RNAs can lead to a new, fused protein, having domains from both original proteins. Here, we systematically identified over 200 cases of intergenic splicing in the human genome (involving 421 genes), and experimentally demonstrated that at least half of these fusions exist in human tissues. We showed that unique splicing patterns dominate the functional and regulatory nature of the resulting transcripts, and found intergenic distance bias in fused compared with nonfused genes. We demonstrate that the hundreds of fused genes we identified are only a subset of the actual number of fused genes in human. We describe a novel evolutionary mechanism where transcription-induced chimerism followed by retroposition results in a new, active fused gene. Finally, we provide evidence that transcription-induced chimerism can be a mechanism contributing to the evolution of protein complexes.

2005

Is there any sense in antisense editing?

Neeman Y., Dahary D., Levanon E. Y., Sorek R. & Eisenberg E. (2005) Trends in Genetics. 21, 10, p. 544-547

Several recent studies have hypothesized that sense-antisense RNA-transcript pairs create dsRNA duplexes that undergo extensive A-to-I RNA editing. We studied human and mouse genomic antisense regions and found that the editing level in these areas is negligible. This observation questions the scope of sense-antisense duplexes formation in-vivo, which is the basis for several proposed regulatory mechanisms.

Accurate identification of alternatively spliced exons using support vector machine

Dror G., Sorek R. & Shamir R. (2005) Bioinformatics. 21, 7, p. 897-901

Motivation: Alternative splicing is a major component of the regulatory action on mammalian transcriptomes. It is estimated that over half of all human genes have more than one splice variant. Previous studies have shown that alternatively spliced exons possess several features that distinguish them from constitutively spliced ones. Recently, we have demonstrated that such features can be used to distinguish alternative from constitutive exons. In the current study, we used advanced machine learning methods to generate robust classifier of alternative exons. Results: We extracted several hundred local sequence features of constitutive as well as alternative exons. Using feature selection methods we find seven attributes that are dominant for the task of classification. Several less informative features help to slightly increase the performance of the classifier. The classifier achieves a true positive rate of 50% for a false positive rate of 0.5%. This result enables one to reliably identify alternatively spliced exons in exon databases that are believed to be dominated by constitutive exons.

Naturally occurring antisense: Transcriptional leakage or real overlap?

Dahary D., Elroy-Stein O. & Sorek R. (2005) Genome Research. 15, 3, p. 364-368

Naturally occurring antisense transcription is associated with the regulation of gene expression through a variety of biological mechanisms. Several recent genome-wide studies reported the identification of potential antisense transcripts for thousands of mammalian genes, many of them resulting from alternatively polyadenylated transcripts or heterogeneous transcription start sites. However, it is not clear whether this transcriptional plasticity is intentional, leading to regulated overlap between the transcripts, or, alternatively, represents a "leakage" of the RNA transcription machinery. To address this question through an evolutionary approach, we compared the genomic organization of genes, with or without antisense, between human, mouse, and the pufferfish Fugu rubripes. Our hypothesis was that if two neighboring genes overlap and have a sense-antisense relationship, we would expect negative selection acting on the evolutionary separation between them. We found that antisense gene pairs are twice as likely to preserve their genomic organization throughout vertebrates' evolution compared to nonantisense pairs, implying an overlap existence in the ancestral genome. In addition, we show that increasing the genomic distance between pairs of genes having a sense-antisense relationship is selected against. These findings indicate that, at least in part, the abundance of antisense transcripts observed in expressed data represents real overlap rather than transcriptional leakage. Moreover, our results imply that natural antisense transcription has considerably affected vertebrate genome evolution.

Is abundant A-to-I RNA editing primate-specific?

Eisenberg E., Nemzer S., Kinar Y., Sorek R., Rechavi G. & Levanon E. Y. (2005) Trends in Genetics. 21, 2, p. 77-81

A-to-I RNA editing is common in all eukaryotes, and is associated with various neurological functions. Recently, A-to-I editing was found to occur frequently in the human transcriptome. In this article, we show that the frequency of A-to-I editing in humans is at least an order of magnitude higher than in the mouse, rat, chicken or fly genomes. The extraordinary frequency of RNA editing in human is explained by the dominance of the primate-specific Alu element in the human transcriptome, which increases the number of double-stranded RNA substrates.

2004

A non-EST-based method for exon-skipping prediction

Sorek R., Shemesh R., Cohen Y., Basechess O., Ast G. & Shamir R. (2004) Genome Research. 14, 8, p. 1617-1623

It is estimated that between 35% and 74% of all human genes can undergo alternative splicing. Currently, the most efficient methods for large-scale detection of alternative splicing use expressed sequence tags (ESTs) or microarray analysis. As these methods merely sample the transcriptome, splice variants that do not appear in deeply sampled tissues have a low probability of being detected. We present a new method by which we can predict that an internal exon is skipped (namely whether it is a cassette-exon) merely based on its naked genomic sequence and on the sequence of its mouse ortholog. No other data, such as ESTs, are required for the prediction. Using our method, which was experimentally validated, we detected hundreds of novel splice variants that were not detectable using ESTs. We show that a substantial fraction of the splice variants in the human genome could not be identified through current human EST or cDNA data.

Minimal conditions for exonization of intronic sequences: 5 splice site formation in Alu exons

Sorek R., Lev-Maor G., Reznik M., Dagan T., Belinky F., Graur D. & Ast G. (2004) Molecular Cell. 14, 2, p. 221-231

Alu exonization, which is an evolutionary pathway that creates primate-specific transcriptomic diversity, is a powerful tool for studying alternative-splicing regulation. Through bioinformatic analyses combined with experimental methodology, we identified the mutational changes needed to create functional 5 splice sites in Alu. We revealed a complex mechanism by which the sequence composition of the 5 splice site and its base pairing with the small nuclear RNA U1 govern alternative splicing. We show that in Alu-derived GC introns the strength of the base pairing between U1 snRNA and the 5 splice site controls the skipping/inclusion ratio of alternative splicing. Based on these findings, we identified 7810 Alus within the human genome that are prone to exonization. Mutations in these Alus may cause genetic disorders or contribute to human-specific protein diversity.

How prevalent is functional alternative splicing in the human genome?

Sorek R., Shamir R. & Ast G. (2004) Trends in Genetics. 20, 2, p. 68-71

Comparative analyses of ESTs and cDNAs with genomic DNA predict a high frequency of alternative splicing in human genes. However, there is an ongoing debate as to how many of these predicted splice variants are functional and how many are the result of aberrant splicing (or 'noise'). To address this question, we compared alternatively spliced cassette exons that are conserved between human and mouse with EST-predicted cassette exons that are not conserved in the mouse genome. Presumably, conserved exon-skipping events represent functional alternative splicing. We show that conserved (functional) cassette exons possess unique characteristics in size, repeat content and in their influence on the protein. By contrast, most non-conserved cassette exons do not share these characteristics. We conclude that a significant portion of cassette exons evident in EST databases is not functional, and might result from aberrant rather than regulated splicing.

In search of antisense

Lavorgna G., Dahary D., Lehner B., Sorek R., Sanderson C. M. & Casari G. (2004) Trends in Biochemical Sciences. 29, 2, p. 88-94

In recent years, natural antisense transcripts (NATs) have been implicated in many aspects of eukaryotic gene expression including genomic imprinting, RNA interference, translational regulation, alternative splicing, X-inactivation and RNA editing. Moreover, there is growing evidence to suggest that antisense transcription might have a key role in a range of human diseases. Consequently, there have been several recent attempts to identify novel NATs. To date, ∼2500 mammalian NATs have been found, indicating that antisense transcription might be a common mechanism of regulating gene expression in human cells. There are increasingly diverse ways in which antisense transcription can regulate gene expression and evidence for the involvement of NATs in human disease is emerging. A range of bioinformatic resources could be used to assist future antisense research.

AluGene: A database of Alu elements incorporated within protein-coding genes

Dagan T., Sorek R., Sharon E., Ast G. & Graur D. (2004) Nucleic Acids Research. 32, Suppl.1, p. D489-D492

Alu elements are short interspersed elements (SINEs) ∼300 nucleotides in length. More than 1 million Alus are found in the human genome. Despite their being genetically functionless, recent findings suggest that Alu elements may have a broad evolutionary impact by affecting gene structures, protein sequences, splicing motifs and expression patterns. Because of these effects, compiling a genomic database of Alu sequences that reside within protein-coding genes seemed a useful enterprise. Presently, such data are limited since the structural and positional information on genes and Alu sequences are scattered throughout incompatible and unconnected databases. AluGene (http://Alugene.tau.ac.il/) provides easy access to a complete Alu map of the human genome, as well as Alu-associated information. The Alu elements are annotated with respect to coding region and exon/intron location. This design facilitates queries on Alu sequences, locations, as well as motifs and compositional properties via a one-stop search page.

2003

Expressed Sequence Tags: Clean before Using. Correspondence re: Z. Wang et al., Computational Analysis and Experimental Validation of Tumor-associated Alternative RNA Splicing in Human Cancer. Cancer Res., 63: 655-657, 2003 [1] (multiple letters)

Sorek R., Basechess O., Safer H. M., Wang Z. & Lee M. P. (2003) Cancer Research. 63, 20, p. 6996-6997

Intronic sequences flanking alternatively spliced exons are conserved between human and mouse

Sorek R. & Ast G. (2003) Genome Research. 13, 7, p. 1631-1637

Comparison of the sequences of mouse and human genomes revealed a surprising number of nonexonic, nonexpressed conserved sequences, for which no function could be assigned. To study the possible correlation between these conserved intronic sequences and alternative splicing regulation, we developed a method to identify exons that are alternatively spliced in both human and mouse. We compiled two exon sets: one of alternatively spliced conserved exons and another of constitutively spliced conserved exons. We found that 77% of the conserved alternatively spliced exons were flanked on both sides by long conserved intronic sequences. In comparison, only 17% of the conserved constitutively spliced exons were flanked by such conserved intronic sequences. The average length of the conserved intronic sequences was 103 bases in the upstream intron and 94 bases in the downstream intron. The average identity levels in the immediately flanking intronic sequences were 88% and 80% for the upstream and downstream introns, respectively, higher than the conservation levels of 77% that were measured in promoter regions. Our results suggest that the function of many of the intronic sequence blocks that are conserved between human and mouse is the regulation of alternative splicing.

The birth of an alternatively spliced exon: 3 Splice-site selection in Alu exons

Lev-Maor G., Sorek R., Shomron N. & Ast G. (2003) Science. 300, 5623, p. 1288-1291

Alu repetitive elements can be inserted into mature messenger RNAs via a splicing-mediated process termed exonization. To understand the molecular basis and the regulation of the process of turning intronic Alus into new exons, we compiled and analyzed a data set of human exonized Alus. We revealed a mechanism that governs 3 splice-site selection in these exons during alternative splicing. On the basis of these findings, we identified mutations that activated the exonization of a silent intronic Alu.

Widespread occurrence of antisense transcription in the human genome

Yelin R., Dahary D., Sorek R., Levanon E. Y., Goldstein O., Shoshan A., Diber A., Biton S., Tamir Y., Khosravi R., Nemzer S., Pinner E., Walach S., Bernstein J., Savitsky K. & Rotman G. (2003) Nature biotechnology. 21, 4, p. 379-386

An increasing number of eukaryotic genes are being found to have naturally occurring antisense transcripts. Here we study the extent of antisense transcription in the human genome by analyzing the public databases of expressed sequences using a set of computational tools designed to identify sense-antisense transcriptional units on opposite DNA strands of the same genomic locus. The resulting data set of 2,667 sense-antisense pairs was evaluated by microarrays containing strand-specific oligonucleotide probes derived from the region of overlap. Verification of specific cases by northern blot analysis with strand-specific riboprobes proved transcription from both DNA strands. We conclude that ≥60% of this data set, or ∼1,600 predicted sense-antisense transcriptional units, are transcribed from both DNA strands. This indicates that the occurrence of antisense transcription, usually regarded as infrequent, is a very common phenomenon in the human genome. Therefore, antisense modulation of gene expression in human cells may be a common regulatory mechanism.

Evolutionary dynamics of large Numts in the human genome: Rarity of independent insertions and abundance of post-insertion duplications

Hazkani-Covo E., Sorek R. & Graur D. (2003) Journal of Molecular Evolution. 56, 2, p. 169-174

We determined the phylogenetic positions of 82 large nuclear pseudogenes of mitochondrial origin (numts) within the human genome. For each numt, two possibilities pertaining to its origin were considered: (1) independent insertion from the mitochondria into the nucleus, or (2) genomic duplication subsequent to the insertion. A significant increase in the rate of numt accumulation is seen after the divergence of Platyrrhini (New World monkeys) from the Catarrhini (Old World monkeys, apes and humans). By using pairwise phylogenetic analyses, we were able to demonstrate that this peak in numt accumulation is mostly the result of duplication of preexisting nuclear numts rather than the result of an increase in mitochondrial-sequence insertion. In fact, only about a third of all the numt repertoire in the human nuclear genome is due to insertions of mitochondrial sequences, the rest originated as duplications of preexisting numts. Hence, we conclude that numt insertion occurs at a much lower rate than previously reported. As expected under the assumption that genomic duplications occur at rates that are uninfluenced by content, older numts were found to be duplicated more times than recently inserted ones.

A novel algorithm for computational identification of contaminated EST libraries

Sorek R. & Safer H. M. (2003) Nucleic Acids Research. 31, 3, p. 1067-1074

A key goal of the Human Genome Project was to understand the complete set of human proteins, the proteome. Since the genome sequence by itself is not sufficient for predicting new genes and alternative splicing events that lead to new proteins, expressed sequence tags (ESTs) are used as the primary tool for these purposes. The high prevalence of artifacts in dbEST, however, often leads to invalid predictions. Here we describe a novel method for recognizing genomic DNA contamination and other artifacts that cannot be identified using current EST cleaning techniques. Our method uses the alignment of the entire set of ESTs to the human genome to identify highly contaminated EST libraries. We discovered 53 highly contaminated libraries and a subset of 24 766 ESTs from these libraries that probably represent contamination with genomic DNA, pre-mRNA, and ESTs that span non-canonical introns. Although this is only a small fraction of the entire EST dataset, each contaminating sequence could create a spurious transcript prediction. Indeed, in the clustering and assembly tool that we used, these sequences would have caused incorrect inference of 9575 new splice variants and 6370 new genes. Conclusions based on EST analysis, including prediction of alternative splicing, should be re-evaluated in light of these results. Our method, along with the identified set of contaminated sequences, will be essential for applications that depend on large EST datasets.

The importance of alternative splicing in the drug discovery process

Levanon E. Y. & Sorek R. (2003) Drug Discovery Today: TARGETS. 2, 3, p. 109-114

The publication of the sequence of the human genome revealed that the gene count in humans is much lower than previously estimated. Although textbooks usually place the number at 100,000, it is currently estimated that the human genome contains no more than 30,000 protein-coding genes. How can the great complexity of human life be explained by this number, which is less than twice the number of genes in the primitive worm C. elegans? The answer probably lies in the recent discovery that about half of all human genes undergo alternative splicing. This paper reviews the broad implications of alternative splicing for the drug-discovery process.

2002

Alu-containing exons are alternatively spliced

Sorek R., Ast G. & Graur D. (2002) Genome Research. 12, 7, p. 1060-1067

Alu repetitive elements are found in ∼1.4 million copies in the human genome, comprising more than one-tenth of it. Numerous studies describe exonizations of Alu elements, that is, splicing-mediated insertions of parts of Alu sequences into mature mRNAs. To study the connection between the exonization of Alu elements and alternative splicing, we used a database of ESTs and cDNAs aligned to the human genome. We compiled two exon sets, one of 1176 alternatively spliced internal exons, and another of 4151 constitutively spliced internal exons. Sixty one alternatively spliced internal exons (5.2%) had a significant BLAST hit to an Alu sequence, but none of the constitutively spliced internal exons had such a hit. The vast majority (84%) of the Alu-containing exons that appeared within the coding region of mRNAs caused a frame-shift or a premature termination codon. Alu-containing exons were included in transcripts at lower frequencies than alternatively spliced exons that do not contain an Alu sequence. These results indicate that internal exons that contain an Alu sequence are predominantly, if not exclusively, alternatively spliced. Presumably, evolutionary events that cause a constitutive insertion of an Alu sequence into an mRNA are deleterious and selected against.

2001

Piecing together the significance of splicing

Sorek R. & Amitai M. (2001) Nature biotechnology. 19, 3, p. 196