Project description:Extracellular vesicles are small (50-200 nm diameter) membrane-bound structures released by cells from all domains of life. Vesicles have been implicated in numerous microbial processes, including gene transfer, signaling, pathogenesis, and defense. While vesicles are abundant in the oceans, little is known about their contents or whether they interact with cells in this dilute environment. Here we show that extracellular vesicles contain a remarkable diversity of organic carbon compounds and likely mediate a complex network of export, transport and exchange within marine microbial communities. We report that vesicles from selected strains of Prochlorococcus, the most abundant cyanobacterium in the sea, and other bacteria can interact with cells of various marine microbes, including the numerically abundant heterotroph Pelagibacter. Through a combination of lipidomic, metabolomic and proteomic analyses, we show that two strains of Prochlorococcus export an enormous array of organic and inorganic compounds – including active, biogeochemically relevant enzymes – within vesicles. Vesicles from both strains contained some materials in common as well as numerous strain-specific differences, reflecting an even greater degree of functional complexity within natural vesicle populations. These data suggest additional roles for vesicles in carrying out extracellular biogeochemical reactions, mitigating toxicity from reactive oxygen species, and mediating energy and nutrient transfers in the oceans.
Project description:Carbon fixation plays a central role in determining cellular redox poise, increasingly understood to be a key parameter in cyanobacterial physiology. In the cyanobacterium Prochlorococcus--—the most abundant phototroph in the oligotrophic oceans--—the carbon-concentrating mechanism (CCM) is reduced to the bare essentials. Given the ability of Prochlorococcus populations to grow under a wide range of oxygen concentrations in the ocean, we wondered how carbon and oxygen physiology intersect in this minimal phototroph. We monitored genome-wide transcription in cells shocked with acute limitation of CO2, O2, or both. O2 limitation produced much smaller transcriptional changes than the broad suppression seen under CO2 limitation and CO2/O2 co-limitation. Strikingly, the transcriptional responses evoked by both CO2 limitation conditions were initially similar to that previously seen in high light stress, but at later timepoints we observed O2-dependent recovery of photosynthesis-related transcripts. These results suggest that oxygen plays a protective role in Prochlorococcus when carbon fixation is not a sufficient sink for light energy.
Project description:Carbon fixation plays a central role in determining cellular redox poise, increasingly understood to be a key parameter in cyanobacterial physiology. In the cyanobacterium Prochlorococcus--—the most abundant phototroph in the oligotrophic oceans--—the carbon-concentrating mechanism (CCM) is reduced to the bare essentials. Given the ability of Prochlorococcus populations to grow under a wide range of oxygen concentrations in the ocean, we wondered how carbon and oxygen physiology intersect in this minimal phototroph. We monitored genome-wide transcription in cells shocked with acute limitation of CO2, O2, or both. O2 limitation produced much smaller transcriptional changes than the broad suppression seen under CO2 limitation and CO2/O2 co-limitation. Strikingly, the transcriptional responses evoked by both CO2 limitation conditions were initially similar to that previously seen in high light stress, but at later timepoints we observed O2-dependent recovery of photosynthesis-related transcripts. These results suggest that oxygen plays a protective role in Prochlorococcus when carbon fixation is not a sufficient sink for light energy. Two biological replicates of timecourses under four conditions: medium bubbled with air (control) or three experimental gases (low CO2; low O2; or low CO2 and low O2)
Project description:Cyanobacteria Prochlorococcus marinus subsp. pastoris str. CCMP1986 (MED4) and Prochlorococcus marinus str. MIT 9313 (MIT9313) are oceanic oxygenic phototrophs, where MED4 is abundant in surface waters (~0-50 meters) and MIT9313 is abundant at depths of ~100 meters. To explore nitrogen-regulated changes in gene expression in these Prochlorococcus ecotypes, log phase cultures of MED4 and MIT9313 were transferred to either nitrogen-replete (800 uM ammonium) or medium lacking supplemental nitrogen. Samples were taken over a time series in order to characterize changes in physiology and gene expression during increasing nitrogen starvation. The two ecotypes' molecular responses to different nitrogen sources were also assessed by comparing gene expression of log phase cultures growing in ammonium vs. urea and cyanate (MED4), and vs. urea and nitrite (MIT9313).
Project description:Prochlorococcus contributes significantly to ocean primary productivity. The link between primary productivity and iron in specific ocean regions is well established and iron limitation of Prochlorococcus cell division rates in these regions has been shown. However, the extent of ecotypic variation in iron metabolism among Prochlorococcus and the molecular basis for differences is not understood. Here, we examine the growth and transcriptional response of Prochlorococcus strains, MED4 and MIT9313, to changing iron concentrations. During steady state, MIT9313 sustains growth at an order-of-magnitude lower iron concentration than MED4. To explore this difference, we measured the whole-genome transcriptional response of each strain to abrupt iron starvation and rescue. Only four of the 1159 orthologs of MED4 and MIT9313 were differentially expressed in response to iron in both strains. However, in each strain, the expression of over a hundred additional genes changed, many of which are in labile genomic regions, suggesting a role for lateral gene transfer in establishing diversity of iron metabolism among Prochlorococcus. Furthermore, we found that MED4 lacks three genes near the iron-deficiency-induced gene (idiA) that are present and induced by iron stress in MIT9313. These genes are interesting targets for studying the adaptation of natural Prochlorococcus assemblages to local iron conditions as they show more diversity than other genomic regions in environmental metagenomic databases.
Project description:RNA decay was measured in Prochlorococcus after inhibition of transcription by rifampicin using customized Affymetrix gene expression arrays. RNA turnover plays an important role in the gene regulation of microorganisms and influences their speed of acclimation to environmental changes. We investigated whole-genome RNA stability of Prochlorococcus, a relatively slow-growing marine cyanobacterium doubling approximately once a day, which is extremely abundant in the oceans. Using a combination of microarrays, quantitative RT-PCR and a new algorithm for determining RNA decay rates, we found a median half-life of 2.4 min and a median decay rate of 2.6 min for expressed genes â two-fold faster than that reported for any organism. The shortest transcript half-life (33 seconds) was for a gene of unknown function, while some of the longest (ca. 18 min) were for highly expressed genes. Genes organized in operons displayed intriguing mRNA decay patterns, such as increased stability, and delayed onset of decay with greater distance from the transcriptional start site. The same phenomenon was observed on a single probe resolution for genes greater than 2 kb. We hypothesize that the fast turnover relative to generation time in Prochlorococcus may enable a swift response to environmental changes through rapid recycling of nucleotides, which could be advantageous in nutrient poor oceans. Our growing understanding of RNA half-lives will inform on the modelling of cell processes and help interpret the growing bank of metatranscriptomic studies of wild populations of Prochlorococcus. The surprisingly complex decay patterns of large transcripts reported here, and the method developed to describe them, will open new avenues for the investigation and understanding of RNA decay for all organisms. Prochlorococcus cells were treated with rifampicin, which prevents initiation of new transcripts. Cells were harvested at 0 min (before rifampicin addition), 2.5 min, 5 min, 10 min, 20 min, 40 min and 60 min after rifampicin addition.
Project description:RNA decay was measured in Prochlorococcus after inhibition of transcription by rifampicin using customized Affymetrix gene expression arrays. RNA turnover plays an important role in the gene regulation of microorganisms and influences their speed of acclimation to environmental changes. We investigated whole-genome RNA stability of Prochlorococcus, a relatively slow-growing marine cyanobacterium doubling approximately once a day, which is extremely abundant in the oceans. Using a combination of microarrays, quantitative RT-PCR and a new algorithm for determining RNA decay rates, we found a median half-life of 2.4 min and a median decay rate of 2.6 min for expressed genes – two-fold faster than that reported for any organism. The shortest transcript half-life (33 seconds) was for a gene of unknown function, while some of the longest (ca. 18 min) were for highly expressed genes. Genes organized in operons displayed intriguing mRNA decay patterns, such as increased stability, and delayed onset of decay with greater distance from the transcriptional start site. The same phenomenon was observed on a single probe resolution for genes greater than 2 kb. We hypothesize that the fast turnover relative to generation time in Prochlorococcus may enable a swift response to environmental changes through rapid recycling of nucleotides, which could be advantageous in nutrient poor oceans. Our growing understanding of RNA half-lives will inform on the modelling of cell processes and help interpret the growing bank of metatranscriptomic studies of wild populations of Prochlorococcus. The surprisingly complex decay patterns of large transcripts reported here, and the method developed to describe them, will open new avenues for the investigation and understanding of RNA decay for all organisms.
Project description:Extracellular vesicles are small (~50–200 nm diameter) membrane-bound structures released by cells from all domains of life. While extremely abundant in the oceans, our understanding of their functions, both for cells and the emergent ecosystem, is in its infancy. To advance this understanding, we analyzed the lipid, metabolite, and protein content of vesicles produced by two strains of the most abundant phytoplankton cell in the ocean, the cyanobacterium Prochlorococcus. We show that Prochlorococcus exports an enormous array of cellular compounds into their surroundings via extracellular vesicles. The vesicles produced by the two different strains contained some materials in common, but also displayed numerous strain-specific differences, reflecting functional complexity within natural vesicle populations. Prochlorococcus vesicles contain active enzymes, indicating that they can mediate biogeochemically relevant extracellular reactions in the wild. Interaction assays demonstrate that vesicles from Prochlorococcus and multiple genera of heterotrophic bacteria can associate with other marine microbes, including Pelagibacter, the most abundant heterotrophic group in the oceans. Our observations suggest that vesicles may play diverse functional roles in the oceans, including but not limited to mediating energy and nutrient transfers, catalyzing extracellular biochemical reactions, and mitigating toxicity of reactive oxygen species. These findings further indicate that a portion of the ‘dissolved’ compounds in the oceans are not truly dissolved, but are instead packaged within locally structured, colloidal vesicles.