Publications
2023
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(2023) BioRxiv. Abstract
Horizontal gene transfer (HGT) is a pivotal mechanism driving bacterial evolution, conferring adaptability within dynamic marine ecosystems. Among HGT mechanisms, conjugation mediated by Type IV secretion systems (T4SSs) plays a central role in the ecological success of marine bacteria. However, the triggers initiating conjugation events in the marine environment are not well understood. Roseobacters, abundant marine bacteria commonly associated with algae, possess a multitude of plasmids encoding T4SSs. Many Roseobacters are heterotrophic bacteria that rely on algal secreted compounds for supporting bacterial growth. Algal compounds therefore attract bacteria and promote colonization, including attachment to algal cells. Bacterial proximity, cell-to-cell contact, and attachment, can all foster HGT. Hence, we hypothesized that algal exudates, acting as chemoattractants for bacteria, may function as cues promoting bacterial HGT. Examination of various Roseobacters demonstrated that the genomic location of the T4SS impacts its functionality; a bacterial strain harboring a chromosomal-encoded T4SS does not perform conjugation, while a strain of the same species carrying a plasmid-encoded T4SS exhibits functional conjugation capability. Subsequently, we probed the influence of algal exudates on bacterial conjugation dynamics. Our findings revealed that algal exudates enhance plasmid transfer through conjugation but do not modulate the transcription of the conjugative machinery genes. These observations suggest that the bacterial responses to algal hosts evolved to correlate with an increased likelihood of encountering compatible partners for successful conjugation. Furthermore, since specific algae attract distinct bacterial populations, algae influence potential partners for genetic exchange and may shape the trajectory of bacterial evolution in the marine environment.Competing Interest StatementThe authors have declared no competing interest.
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(2023) BioRxiv. Abstract
An essential interaction between sunlight and eukaryotes involves the production of vitamin D through exposure to ultraviolet-B (UV-B) radiation. While extensively studied in vertebrates, the role of vitamin D in non-animal eukaryotes like microalgae remains unclear. Emiliania huxleyi, a microalga inhabiting shallow ocean depths exposed to UV-B radiation, is well-suited for this research. Our results show that E. huxleyi can produce vitamin D2 and D3, pointing to their potential role in the algal physiology. We further show that E. huxleyi algae respond to vitamin D at the transcriptional level, regulating the expression of protective mechanisms such as the light-harvesting complex stress-related protein (LHCSR) and heme oxygenase, and that vitamin D enhances the algal photosynthetic performance while reducing harmful reactive oxygen species buildup. Understanding the function of vitamin D in E. huxleyi has broader implications, shedding light on its role in non-animal eukaryotes and its potential importance in marine ecosystems. This research sets the stage for further investigations into the complex relationship between sunlight, vitamin D, and microalgal physiology, which contributes to our understanding of how eukaryotes adapt to diverse environmental conditions.
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(2023) ISME Communications. 3, 100. Abstract
Emiliania huxleyi is a unicellular micro-alga that forms massive oceanic blooms and plays key roles in global biogeochemical cycles. Mounting studies demonstrate various stimulatory and inhibitory influences that bacteria have on the E. huxleyi physiology. To investigate these algal-bacterial interactions, laboratory co-cultures have been established by us and by others. Owing to these co-cultures, various mechanisms of algal-bacterial interactions have been revealed, many involving bacterial pathogenicity towards algae. However, co-cultures represent a significantly simplified system, lacking the complexity of bacterial communities. In order to investigate bacterial pathogenicity within an ecologically relevant context, it becomes imperative to enhance the microbial complexity of co-culture setups. Phaeobacter inhibens bacteria are known pathogens that cause the death of E. huxleyi algae in laboratory co-culture systems. The bacteria depend on algal exudates for growth, but when algae senesce, bacteria switch to a pathogenic state and induce algal death. Here we investigate whether P. inhibens bacteria can induce algal death in the presence of a complex bacterial community. We show that an E. huxleyi-associated bacterial community protects the alga from the pathogen, although the pathogen occurs within the community. To study how the bacterial community regulates pathogenicity, we reduced the complex bacterial community to a five-member synthetic community (syncom). The syncom is comprised of a single algal host and five isolated bacterial species, which represent major bacterial groups that are naturally associated with E. huxleyi. We discovered that a single bacterial species in the reduced community, Sulfitobacter pontiacus, protects the alga from the pathogen. We further found that algal protection from P. inhibens pathogenicity is a shared trait among several Sulfitobacter species. Algal protection by bacteria might be a common phenomenon with ecological significance, which is overlooked in reduced co-culture systems.
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(2023) BioRxiv. Abstract
Marine ecosystems are influenced by phytoplankton aggregation, which affects processes like marine snow formation and harmful events such as marine mucilage outbreaks. Phytoplankton secrete exopolymers, creating an extracellular matrix (ECM) that promotes particle aggregation. This ECM attracts heterotrophic bacteria, providing a nutrient-rich and protective environment. In terrestrial environments, bacterial colonization near primary producers relies on attachment and the formation of multidimensional structures like biofilms. Bacteria were observed attaching and aggregating within algal-derived exopolymers, but it is unclear if bacteria produce an ECM that contributes to this colonization. This study, using Emiliania huxleyi algae and Phaeobacter inhibens bacteria in an environmentally relevant model system, reveals a shared algal-bacterial ECM scaffold that promotes algal-bacterial aggregation. Algal exudates play a pivotal role in promoting bacterial colonization, stimulating bacterial exopolysaccharide (EPS) production, and facilitating a joint ECM formation. A bacterial biosynthetic pathway responsible for producing a succinoglycan-like compound contributing to bacterial ECM formation is identified. Genes from this pathway show increased expression in algal-rich environments. These findings highlight the underestimated role of bacteria in aggregate-mediated processes in marine environments, offering insights into algal-bacterial interactions and ECM formation, with implications for understanding and managing disturbances like marine mucilage events.
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(2023) The ISME Journal. 17, 8, p. 1167-1183 Abstract[All authors]
Microbial interactions govern marine biogeochemistry. These interactions are generally considered to rely on exchange of organic molecules. Here we report on a novel inorganic route of microbial communication, showing that algal-bacterial interactions between Phaeobacter inhibens bacteria and Gephyrocapsa huxleyi algae are mediated through inorganic nitrogen exchange. Under oxygen-rich conditions, aerobic bacteria reduce algal-secreted nitrite to nitric oxide (NO) through denitrification, a well-studied anaerobic respiratory mechanism. The bacterial NO is involved in triggering a cascade in algae akin to programmed cell death. During death, algae further generate NO, thereby propagating the signal in the algal population. Eventually, the algal population collapses, similar to the sudden demise of oceanic algal blooms. Our study suggests that the exchange of inorganic nitrogen species in oxygenated environments is a potentially significant route of microbial communication within and across kingdoms.
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(2023) BioRxiv. Abstract
Short lag times are beneficial for heterotrophic bacteria that compete for resources in environments with fluctuating organic carbon levels. We found that the marine model bacterium Phaeobacter inhibens achieves shorter lag times in the presence of nano-to micromolar concentrations of N-or S-methylated compounds, which are abundantly produced by microalgae. To understand the underlying mechanism, we studied algal-bacterial co-cultures and bacterial pure cultures during their lag phase using transcriptomics analyses, 13C-labeled metabolomics, gene knock-out experiments and enzymatic characterizations. Our findings highlight methyl group synthesis as a bottleneck during the bacterial lag phase, which can be overcome by assimilating methyl groups from external sources. Our study reveals a fundamental aspect of the bacterial lag phase, emphasizing the importance of studying bacterial physiology within an ecological framework, particularly in the context of microbial interactions.
2022
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(2022) Proceedings of the National Academy of Sciences. 119, 32, e220360411. Abstract[All authors]
Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.
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(2022) Geobiology. 20, 3, Abstract
Coccolithophores are a diverse group of calcifying microalgae that have left a prominent fossil record on Earth. Various coccolithophore relics, both organic and inorganic, serve as proxies for reconstruction of past oceanic conditions. Emiliania huxleyi is the most widely distributed representative of the coccolithophores in modern oceans and is known to engage in dynamic interactions with bacteria. Algal–bacterial interactions influence various aspects of algal physiology and alter algal alkenone unsaturation (UK’37), a frequently used organic coccolithophore-derived paleo-temperature proxy. Whether algal–bacterial interactions influence inorganic coccolithophore-derived paleo-proxies is yet unknown. A commonly used inorganic proxy for past productivity and sea surface temperature is the Sr/Ca ratio of the coccolith calcite. Interestingly, during interactions between bacteria and a population of calcifying algae, bacteria were shown to physically attach only to non-calcified algal cells, suggesting an influence on algal calcification. In this study, we explore the effects of algal–bacterial interactions on calcification and coccolith Sr/Ca ratios. We find that while bacteria attach only to non-calcified algal cells, coccolith cell coverage and overall calcite production in algal populations with and without bacteria is similar. Furthermore, we find that Sr/Ca values are impacted only by water temperature and algal growth rate, regardless of bacterial influences on algal physiology. Our observations reinforce the robustness of coccolith Sr/Ca ratios as a paleo-proxy independent of microbial interactions and highlight a fundamental difference between organic and inorganic paleo-proxies.
2021
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(2021) Applied and environmental microbiology : AEM. 88, 2, Abstract
Microalgae are key ecological players with a complex evolutionary history. Genomic diversity, in addition to limited availability of high-quality genomes, challenge studies that aim to elucidate molecular mechanisms underlying microalgal ecophysiology. Here, we present a novel and comprehensive transcriptomic hybrid approach to generate a reference for genetic analyses, and resolve the microalgal gene landscape at the strain level. The approach is demonstrated for a strain of the coccolithophore microalga Emiliania huxleyi, which is a species complex with considerable genome variability. The investigated strain is commonly studied as a model for algal-bacterial interactions, and was therefore sequenced in the presence of bacteria to elicit the expression of interaction-relevant genes. We applied complementary PacBio Iso-Seq full-length cDNA, and poly(A)-independent Illumina total RNA sequencing, which resulted in a de novo assembled, near complete hybrid transcriptome. In particular, hybrid sequencing improved the reconstruction of long transcripts and increased the recovery of full-length transcript isoforms. To use the resulting hybrid transcriptome as a reference for genetic analyses, we demonstrate a method that collapses the transcriptome into a genome-like dataset, termed “synthetic genome” (sGenome). We used the sGenome as a reference to visually confirm the robustness of the CCMP3266 gene assembly, to conduct differential gene expression analysis, and to characterize novel E. huxleyi genes. The newly-identified genes contribute to our understanding of E. huxleyi genome diversification, and are predicted to play a role in microbial interactions. Our transcriptomic toolkit can be implemented in various microalgae to facilitate mechanistic studies on microalgal diversity and ecology.
2018
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(2018) Frontiers in Marine Science. 5, p. 144+ 144. Abstract
Microscopic marine phytoplankton drift freely in the ocean, harvesting sunlight through photosynthesis. These unicellular microorganisms account for half of the primary productivity on Earth and play pivotal roles in the biogeochemistry of our planet (Field et al., 1998). The major groups of microalgae that comprise the phytoplankton community are coccolithophores, diatoms and dinoflagellates. In present oceans, phytoplankton individuals and populations are forced to rapidly adjust, as key chemical and physical parameters defining marine habitats are changing globally. Here we propose that microalgal populations often display the characteristics of a multicellular-like community rather than a random collection of individuals. Evolution of multicellularity entails a continuum of events starting from single cells that go through aggregation or clonal divisions (Brunet and King, 2017). Phytoplankton may be an intermediate state between single cells and aggregates of physically attached cells that communicate and co-operate; perhaps an evolutionary snapshot toward multicellularity. In this opinion article, we journey through several studies conducted in two key phytoplankton groups, coccolithophores and diatoms, to demonstrate how observations in these studies could be interpreted in a multicellular context.
2016
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(2016) eLife. 5, NOVEMBER2016, e17473. Abstract
Emiliania huxleyi is a model coccolithophore micro-alga that generates vast blooms in the ocean. Bacteria are not considered among the major factors influencing coccolithophore physiology. Here we show through a laboratory model system that the bacterium Phaeobacter inhibens, a well-studied member of the Roseobacter group, intimately interacts with E. huxleyi. While attached to the algal cell, bacteria initially promote algal growth but ultimately kill their algal host. Both algal growth enhancement and algal death are driven by the bacterially-produced phytohormone indole-3-acetic acid. Bacterial production of indole-3-acetic acid and attachment to algae are significantly increased by tryptophan, which is exuded from the algal cell. Algal death triggered by bacteria involves activation of pathways unique to oxidative stress response and programmed cell death. Our observations suggest that bacteria greatly influence the physiology and metabolism of E. huxleyi. Coccolithophore-bacteria interactions should be further studied in the environment to determine whether they impact micro-algal population dynamics on a global scale.
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(2016) Algal Research-Biomass Biofuels And Bioproducts. 19, p. 370-380 Abstract
Flow cytometry (FCM) is a well-established tool in the field of aquatic phytoplankton ecology and microalgal biotechnology, which allows for rapid assessment of the viability and physiological state of individual cells in algal populations. However, the autofluorescent spectra of different types of chlorophyll and other algal pigments may overlap with fluorescent dyes and affect the resolution of algae clusters, sensitivity, and signal-to-noise ratio. Dying algal cells continue to exhibit a strong autofluorescent signal, which may affect the evaluation of algal viability. Herein, we tested two different approaches to measure algal fluorescence in the presence of a strong autofluorescent signal: 1) by separating dyes between different excitation lasers in order to reach minimal spectral overlap with the autofluorescent signal using flow and imaging cytometry and 2) through full spectrum analysis, virtual filtering and spectral unmixing of dye combinations and algal pigments' autofluorescence via spectral flow cytometry. For this purpose, we used viability dyes from the SYTOX family and lipophilic dyes. Among the dyes tested, the SYTOX Blue (SB) dye had minimal overlap with chlorophyll fluorescence and can be combined with autofluorescence assessment and lipophilic dyes (validated with Emiliania huxleyi algal monocultures). Imaging cytometry provided a detailed characterization of algal subpopulations stained with a combination of fluorescent dyes. A spectral flow cytometer allowed us to analyze environmental phytoplankton samples stained with fluorescent dyes in the presence of strong and heterogeneous autofluorescence from intrinsic algal pigments. We concluded that the multi-color staining of algal samples can be achieved in the presence of strong and diverse algal autofluorescence using dyes with minimal spectral overlap, a multi-laser approach (flow and imaging cytometry) and/or virtual filter and spectral flow cytometry instrumentation. This can open a new page for analytical and cell sorting algal applications.
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(2016) Journal of Phycology. 52, 1, p. 125-130 Abstract
The microalga Emiliania huxleyi produces alkenone lipids that are important proxies for estimating past sea surface temperatures. Field calibrations of this proxy are robust but highly variable results are obtained in culture. Here, we present results suggesting that algal-bacterial interactions may be responsible for some of this variability. Co-cultures of E. huxleyi and the bacterium Phaeobacter inhibens resulted in a 2.5-fold decrease in algal alkenone-containing lipid bodies. In addition levels of unsaturated alkenones increase in co-cultures. These changes result in an increase in the reconstructed growth temperature of up to 2°C relative to axenic algal cultures.
2015
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(2015) PLoS ONE. 10, 11, e0141300. Abstract
The Roseobacterclade is a key group of bacteria in the ocean exhibiting diverse metabolic repertoires and a wide range of symbiotic life-styles. Many Roseobacters possess remarkable capabilities of attachment to both biotic and abiotic surfaces. When attached to each other, these bacteria form multi-cellular structures called rosettes. Phaeobacter inhibens, a well-studied Roseobacter, exhibits various cell sizes and morphologies that are either associated with rosettes or occur as single cells. Here we describe the distribution of P. inhibens morphologies and rosettes within a population. We detect an N-acetylglucosamine-containing polysaccharide on the poles of some cells and at the center of all rosettes. We demonstrate that rosettes are formed by the attachment of individual cells at the polysaccharide-containing pole rather than by cell division. Finally, we show that P. inhibens attachment to abiotic surfaces is hindered by the presence of DNA from itself, but not from other bacteria. Taken together, our findings demonstrate that cell adhesiveness is likely to play a significant role in the life cycle of P. inhibens as well as other Roseobacters.
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(2015) Molecular Cell. 57, 4, p. 695-707 Abstract
The bacterial spore can rapidly convert from a dormant to a fully active cell. Here we study this remarkable cellular transition in Bacillus subtilis and reveal the identity of the newly synthesized proteins throughout spore revival. Our analysis uncovers a highly ordered developmental program that correlates with the spore morphological changes and reveals thespatial and temporal molecular events fundamental to reconstruct a cell. As opposed to current knowledge, we found that translation takes place during the earliest revival event, termed germination, a process hitherto considered to occur without the need for any macromolecule synthesis. Furthermore, we demonstrate that translation is required for execution of germination and relies on the bona fide translational factors RpmE and Tig. Our study sheds light on the spore revival process and on the vital building blocks underlying cellular awakening, thereby paving the way for designing new antimicrobial agents to eradicate spore-forming pathogens.
2013
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(2013) Journal of Bacteriology. 195, 9, p. 1875-1882 Abstract
Bacterial spores can remain dormant for years, yet they possess a remarkable potential to rapidly resume a vegetative life form. Here, we identified a distinct phase at the onset of spore outgrowth, designated the ripening period. This transition phase is exploited by the germinating spore for molecular reorganization toward elongation and subsequent cell division. We have previously shown that spores of different ages, kept under various temperatures, harbor dissimilar molecular reservoirs (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139 -149, 2012). Utilizing this phenomenon, we observed that the length of the ripening period can vary according to the spore molecular content. Importantly, the duration of the ripening period was found to correlate with the initial spore rRNA content and the kinetics of rRNA accumulation upon exiting dormancy. Further, the synthesis of the ribosomal protein RplA and the degradation of the spore-specific protein SspA also correlated with the duration of the ripening period. Our data suggest that the spore molecular cargo determines the extent of the ripening period, a potentially crucial phase for a germinating spore in obtaining limited resources during revival.
2012
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(2012) Cell. 148, 1-2, p. 139-149 Abstract
Upon starvation, the bacterium Bacillus subtilis enters the process of sporulation, lasting several hours and culminating in formation of a spore, the most resilient cell type known. We show that a few days following sporulation, the RNA profile of spores is highly dynamic. In aging spores incubated at high temperatures, RNA content is globally decreased by degradation over several days. This degradation might be a strategy utilized by the spore to facilitate its dormancy. However, spores kept at low temperature exhibit a different RNA profile with evidence supporting transcription. Further, we demonstrate that germination is affected by spore age, incubation temperature, and RNA state, implying that spores can acquire dissimilar characteristics at a time they are considered dormant. We propose that, in contrast to current thinking, entering dormancy lasts a few days, during which spores are affected by the environment and undergo corresponding molecular changes influencing their emergence from quiescence.
2006
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(2006) GEOCHEMISTRY GEOPHYSICS GEOSYSTEMS. 7, Q02P09. Abstract
[1] The ratio of magnesium to calcium (Mg/Ca) in CaCO3 shells of foraminifera is widely used to determine paleotemperatures. However, Mg/Ca is highly variable within and between species, suggesting a strong physiological influence on the incorporation of Mg2+ into the shells. While most field and laboratory calibrations have focused on the effect of temperature, we chose to study the effect of ambient Mg/Ca on the calcification process and the final shell composition. We cultured two species of symbiont-bearing benthic foraminifera, Amphistegina lobifera and Amphistegina lessonii, in seawater with different Mg/Ca ratios. Electron probe analysis of the shell Mg/Ca revealed a positive ( but not entirely linear) correlation with Mg/Ca in the culturing media with slightly different curves for each species. Partition coefficients of Mg2+ (D-Mg) in the calcite shells showed a decrease by a factor of roughly 2 between the lowest and highest Mg/Ca in the ambient water. This was previously demonstrated in inorganic calcite precipitation experiments. However, the biogenic DMg was significantly lower than the inorganic one, suggesting a physiological mechanism that reduces Mg/Ca at the calcification site. Unlike inorganic experiments that display a dependence of DMg on the kinetics of precipitation, the biogenic DMg is not correlated with the rate of calcification. Both DMg and calcification rates in our experiment were sensitive to the Mg/Ca ratio rather than the concentration of either Ca2+ or Mg2+. The largest addition of CaCO3 was obtained at Mg/Ca of 1, and not at present-day seawater ratio (Mg/Ca = 5). This may reflect the Mg/Ca that prevailed during the Eocene (Mg/Ca similar to 1.5), when this genus evolved.