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Gifford, S. M., S. Sharma, M. Booth, M. A. Moran. 2013. Expression patterns reveal niche diversification in a marine microbial assemblage. ISME Journal 7:281-298.
Resolving the ecological niches of coexisting marine microbial taxa is challenging due to the high species richness of microbial communities and the apparent functional redundancy in bacterial genomes and metagenomes. Here, we generated over 11 million Illumina reads of protein-encoding transcripts collected from well-mixed southeastern US coastal waters to characterize gene expression patterns distinguishing the ecological roles of hundreds of microbial taxa sharing the same environment. The taxa with highest in situ growth rates (based on relative abundance of ribosomal protein transcripts) were typically not the greatest contributors to community transcription, suggesting strong top-down ecological control, and their diverse transcriptomes indicated roles as metabolic generalists. The taxa with low in situ growth rates typically had low diversity transcriptomes dominated by specialized metabolisms. By identifying protein-encoding genes with atypically high expression for their level of conservation, unique functional roles of community members emerged related to substrate use (such as complex carbohydrates, fatty acids, methanesulfonate, taurine, tartrate, ectoine), alternative energy-conservation strategies (proteorhodopsin, AAnP, V-type pyrophosphatases, sulfur oxidation, hydrogen oxidation) and mechanisms for negotiating a heterogeneous environment (flagellar motility, gliding motility, adhesion strategies). On average, the heterotrophic bacterioplankton dedicated 7% of their transcriptomes to obtaining energy by non-heterotrophic means. This deep sequencing of a coastal bacterioplankton transcriptome provides the most highly resolved view of bacterioplankton niche dimensions yet available, uncovering a spectrum of unrecognized ecological strategies.
** Luo, H., M. Csürös, A. L. Hughes, and M. A. Moran. 2013. Evolution of divergent life history strategies in marine alphaproteobacteria. mBio 4:4.
Luo, H. and M. A. Moran. 2013. Assembly-free metagenomic analysis reveals new metabolic capabilities in surface ocean bacterioplankton. Environmental Microbiology. In press.
Uncovering the metabolic capabilities of microbes is key to understanding global energy flux and nutrient transformations. Since the vast majority of environmental microorganisms are uncultured, metagenomics has become an important tool to genotype the microbial community. This study uses a recently developed computational method to confidently assign metagenomic reads to microbial clades without the requirement of metagenome assembly by comparing the evolutionary pattern of nucleotide sequences at non-synonymous sites between metagenomic and orthologous reference genes. We found evidence for new, ecologically relevant metabolic pathways in several lineages of surface ocean bacterioplankton using the Global Ocean Survey (GOS) metagenomic data, including assimilatory sulfate reduction and alkaline phosphatase capabilities in the alphaproteobacterial SAR11 clade, and proteorhodopsin-like genes in the cyanobacterial genus Prochlorococcus. These findings raise new hypotheses about microbial roles in energy flux and organic matter transformation in the ocean.
Moran, M.A., B. Satinsky, S. M. Gifford, H. Luo, A. Rivers, L.-K. Chan, J. Meng, B. P. Durham, Chen Shen, V. A. Varaljay, C. B. Smith, P. L. Yager, and B. M. Hopkinson. 2013. Sizing up metatranscriptomics. ISME Journal 7:237-243.
A typical marine bacterial cell in coastal seawater contains only ~200 molecules of mRNA, each of which lasts only a few minutes before being degraded. Such a surprisingly small and dynamic cellular mRNA reservoir has important implications for understanding the bacterium’s responses to environmental signals, as well as for our ability to measure those responses. In this perspective, we review the available data on transcript dynamics in environmental bacteria, and then consider the consequences of a small and transient mRNA inventory for functional metagenomic studies of microbial communities.
Rivers, A. R., S. Sharma, S. G. Tringe , J. Martin, S. Joye, and M. A. Moran. 2013. Transcriptional response of bathypelagic marine bacterioplankton to the Deepwater Horizon oil spill. ISME Journal doi:10.1038/ismej.2013.129.
The Deepwater Horizon blowout released a massive amount of oil and gas into the deep ocean between April and July 2010, stimulating microbial blooms of petroleum-degrading bacteria. To understand the metabolic response of marine microorganisms, we sequenced ∼66 million community transcripts that revealed the identity of metabolically active microbes and their roles in petroleum consumption. Reads were assigned to reference genes from ∼2700 bacterial and archaeal taxa, but most assignments (39%) were to just six genomes representing predominantly methane- and petroleum-degrading Gammaproteobacteria. Specific pathways for the degradation of alkanes, aromatic compounds and methane emerged from the metatranscriptomes, with some transcripts assigned to methane monooxygenases representing highly divergent homologs that may degrade either methane or short alkanes. The microbial community in the plume was less taxonomically and functionally diverse than the unexposed community below the plume; this was due primarily to decreased species evenness resulting from Gammaproteobacteria blooms. Surprisingly, a number of taxa (related to SAR11, Nitrosopumilus and Bacteroides, among others) contributed equal numbers of transcripts per liter in both the unexposed and plume samples, suggesting that some groups were unaffected by the petroleum inputs and blooms of degrader taxa, and may be important for re-establishing the pre-spill microbial community structure.
Swan, B. K., B. Tupper, A. Sczyrba, F. M. Lauro, M. Martinez-Garcia, J. M. González, H. Luo, J. J. Wright, Z. C. Landry, N. W. Hanson, B. P. Thompson, N. J. Poulton, P. Schwientek, S. G. Acinas, S. J. Giovannoni, M. A. Moran, S. J. Hallam, R. Cavicchioli, T. Woyke, and R. Stepanauskas. 2013. Prevalent genome streamlining and latitudinal divergence of marine bacteria in the surface ocean. Proceedings of the National Academy of Sciences 110: 11463-11468.
Planktonic bacteria dominate surface ocean biomass and influence global biogeochemical processes, but remain poorly characterized owing to difficulties in cultivation. Using large-scale single cell genomics, we obtained insight into the genome content and biogeography of many bacterial lineages inhabiting the surface ocean. We found that, compared with existing cultures, natural bacterioplankton have smaller genomes, fewer gene duplications, and are depleted in guanine and cytosine, noncoding nucleotides, and genes encoding transcription, signal transduction, and noncytoplasmic proteins. These findings provide strong evidence that genome streamlining and oligotrophy are prevalent features among diverse, free-living bacterioplankton, whereas existing laboratory cultures consist primarily of copiotrophs. The apparent ubiquity of metabolic specialization and mixotrophy, as predicted from single cell genomes, also may contribute to the difficulty in bacterioplankton cultivation. Using metagenome fragment recruitment against single cell genomes, we show that the global distribution of surface ocean bacterioplankton correlates with temperature and latitude and is not limited by dispersal at the time scales required for nucleotide substitution to exceed the current operational definition of bacterial species. Single cell genomes with highly similar small subunit rRNA gene sequences exhibited significant genomic and biogeographic variability, highlighting challenges in the interpretation of individual gene surveys and metagenome assemblies in environmental microbiology. Our study demonstrates the utility of single cell genomics for gaining an improved understanding of the composition and dynamics of natural microbial assemblages.
** Chan, L K, R J Newton, S Sharma, C B Smith, P Rayapati, A J Limardo, et al. 2012. Transcriptional Changes Underlying Elemental Stoichiometry Shifts in a Marine Heterotrophic Bacterium. Frontiers in Aquatic Microbiology 3:159.
Levine, N M, V A Varaljay, D A Toole, J W H Dacey, S C Doney, M A Moran. 2012. Environmental, biochemical, and genetic drivers of DMSP degradation and DMS production in the Sargasso Sea. Environ. Microbio. 14(5):1210-23
Dimethylsulfide (DMS) is a climatically relevant trace gas produced and cycled by the surface ocean food web. Mechanisms driving intraannual variability in DMS production and dimethylsulfoniopropionate (DMSP) degradation in open-ocean, oligotrophic regions were investigated during a 10-month time-series at the Bermuda Atlantic Time-series Study site in the Sargasso Sea. Abundance and transcription of bacterial DMSP degradation genes, DMSP lyase enzyme activity, and DMS and DMSP concentrations, consumption rates and production rates were quantified over time and depth. This interdisciplinary data set was used to test current hypotheses of the role of light and carbon supply in regulating upper-ocean sulfur cycling. Findings supported UV-A-dependent phytoplankton DMS production. Bacterial DMSP degraders may also contribute significantly to DMS production when temperatures are elevated and UV-A dose is moderate, but may favour DMSP demethylation under low UV-A doses. Three groups of bacterial DMSP degraders with distinct intraannual variability were identified and niche differentiation was indicated. The combination of genetic and biochemical data suggest a modified 'bacterial switch' hypothesis where the prevalence of different bacterial DMSP degradation pathways is regulated by a complex set of factors including carbon supply, temperature and UV-A dose.
Luo, H, A Löytynoja, M Moran. 2012. Genome content of uncultivated marine Roseobacters in the surface ocean. Environ. Microbio. 14(1):41-51.
Understanding of the ecological roles and evolutionary histories of marine bacterial taxa can be complicated by mismatches in genome content between wild populations and their better-studied cultured relatives. We used computed patterns of non-synonymous (amino acid-altering) nucleotide diversity in marine metagenomic data to provide high-confidence identification of DNA fragments from uncultivated members of the Roseobacter clade, an abundant taxon of heterotrophic marine bacterioplankton in the world's oceans. Differences in gene stoichiometry in the Global Ocean Survey metagenomic data set compared with 39 sequenced isolates indicated that natural Roseobacter populations differ systematically in several genomic attributes from their cultured representatives, including fewer genes for signal transduction and cell surface modifications but more genes for Sec-like protein secretion systems, anaplerotic CO(2) incorporation, and phosphorus and sulfate uptake. Several of these trends match well with characteristics previously identified as distinguishing r- versus K-selected ecological strategies in bacteria, suggesting that the r-strategist model assigned to cultured roseobacters may be less applicable to their free-living oceanic counterparts. The metagenomic Roseobacter DNA fragments revealed several traits with evolutionary histories suggestive of horizontal gene transfer from other marine bacterioplankton taxa or viruses, including pyrophosphatases and glycosylation proteins.
Moran, M, C R Reisch, R P Kiene, W B Whitman. 2012. Genomic insights into bacterial DMSP transformations. Ann. Rev. Mar. Sci. 4:523-542.
Genomic and functional genomic methods applied to both model organisms and natural communities have rapidly advanced understanding of bacterial dimethylsulfoniopropionate (DMSP) degradation in the ocean. The genes for the two main pathways in bacterial degradation, routing DMSP to distinctly different biogeochemical fates, have recently been identified. The genes dmdA, -B, -C, and -D mediate the demethylation of DMSP and facilitate retention of carbon and sulfur in the marine microbial food web. The genes dddD, -L, -P, -Q, -W, and -Y mediate the cleavage of DMSP to dimethylsulfide (DMS), with important consequences for ocean-atmosphere sulfur flux. In ocean metagenomes, sufficient copies of these genes are present for approximately 60% of surface ocean bacterial cells to directly participate in DMSP degradation. The factors that regulate these two competing pathways remain elusive, but gene transcription analyses of natural bacterioplankton communities are making headway in unraveling the intricacies of bacterial DMSP processing in the ocean.
Rinta-Kanto, J, S Sun, S Sharma, R P Kiene, M Moran. 2012. Bacterial Community Transcription Patterns During a Marine Phytoplankton Bloom. Environ. Microbiol. 14(1):228-239.
Bacterioplankton consume a large proportion of photosynthetically fixed carbon in the ocean and control its biogeochemical fate. We used an experimental metatranscriptomics approach to compare bacterial activities that route energy and nutrients during a phytoplankton bloom compared with non-bloom conditions. mRNAs were sequenced from duplicate bloom and control microcosms 1 day after a phytoplankton biomass peak, and transcript copies per litre of seawater were calculated using an internal mRNA standard. Transcriptome analysis revealed a potential novel mechanism for enhanced efficiency during carbon-limited growth, mediated through membrane-bound pyrophosphatases [V-type H(+)-translocating; hppA]; bloom bacterioplankton participated less in this metabolic energy scavenging than non-bloom bacterioplankton, with possible implications for differences in growth yields on organic substrates. Bloom bacterioplankton transcribed more copies of genes predicted to increase cell surface adhesiveness, mediated by changes in bacterial signalling molecules related to biofilm formation and motility; these may be important in microbial aggregate formation. Bloom bacterioplankton also transcribed more copies of genes for organic acid utilization, suggesting an increased importance of this compound class in the bioreactive organic matter released during phytoplankton blooms. Transcription patterns were surprisingly faithful within a taxon regardless of treatment, suggesting that phylogeny broadly predicts the ecological roles of bacterial groups across 'boom' and 'bust' environmental backgrounds.
** Schuller, D. J., C. R. Reisch, M. A. Moran, W. B. Whitman, and W. N. Lanzilotta. 2012. Structures of dimethylsulfoniopropionate-dependent demethylase from the marine organism Pelegabacter ubique. Protein Science 21: 289-298.
** Varaljay, V A, S M Gifford, S T Wilson, S Sharma, D M Karl, M. A. Moran. 2012. Bacterial dimethylsulfoniopropionate-degrading genes in the oligotrophic North Pacific Subtropical Gyre.Appl. Environ. Microbiol. Applied and Environmental Microbiology. 78:2775-2782.
Vila-Costa, M, J M Gasol, S Sharma, M Moran. 2012. Community analysis of high-and low-nucleic
acid-containing bacteria in NW Mediterranean coastal waters using 16S rDNA pyrosequencing. Environ. Microbiol. 14(6):1390-402.
The ecological significance of the marine bacterial populations distinguishable by flow cytometry on the basis of the fluorescence (FL) of their nucleic acid (NA) content and proxies of cell size (such as side scatter, SSC) remains largely unknown. Some studies have suggested that cells with high NA (HNA) content and high SSC (HS) represent the active members of the community, while the low NA (LNA) cells are inactive members of the same phylogenetic groups. But group-specific activity measurements and phylogenetic assignment after cell sorting have suggested this is not be the case, particularly in open-ocean communities. To test the extent to which the different NA subgroups are similar, and consequently the extent to which they likely have similar ecological and biogeochemical roles in the environment, we analysed the phylogenetic composition of three populations after cell sorting [high NA-high SC (HNA-HS), high NA-low SC (HNA-LS), low NA (LNA)] by 454 pyrosequencing in two contrasting periods of the year in NW Mediterranean coastal waters (BBMO, Blanes Bay Microbial Observatory) where these three populations have recurrent seasonal patterns. Statistical analyses showed that summer and winter samples were significantly different and, importantly, the sorted populations within a sample were composed of different taxa. The majority of taxa were associated with one NA fraction only, and the degree of overlap (i.e. OTUs present simultaneously in 2 fractions) between HNA and LNA and between summer and winter communities was very small. Rhodobacterales, SAR116 and Bacteroidetes contributed primarily to the HNA fraction, whereas other groups such as SAR11 and SAR86 contributed largely to the LNA fractions. Gammaproteobacteria other than SAR86 showed less preference for one particular NA fraction. An increase in diversity was observed from the LNA to the HNA-HS fraction for both sample dates. Our results suggest that, in Blanes Bay, flow cytometric signatures of natural communities track their phylogenetic composition.
Gifford, S M, S Sharma, J M Rinta-Kanto, M Moran. 2011. Quantitative analysis of a deeply-sequenced marine microbial metatranscriptome. ISME J. 5:461-472.
The potential of metatranscriptomic sequencing to provide insights into the environmental factors that regulate microbial activities depends on how fully the sequence libraries capture community expression (that is, sample-sequencing depth and coverage depth), and the sensitivity with which expression differences between communities can be detected (that is, statistical power for hypothesis testing). In this study, we use an internal standard approach to make absolute (per liter) estimates of transcript numbers, a significant advantage over proportional estimates that can be biased by expression changes in unrelated genes. Coastal waters of the southeastern United States contain 1 × 10(12) bacterioplankton mRNA molecules per liter of seawater (~200 mRNA molecules per bacterial cell). Even for the large bacterioplankton libraries obtained in this study (~500,000 possible protein-encoding sequences in each of two libraries after discarding rRNAs and small RNAs from >1 million 454 FLX pyrosequencing reads), sample-sequencing depth was only 0.00001%. Expression levels of 82 genes diagnostic for transformations in the marine nitrogen, phosphorus and sulfur cycles ranged from below detection (<1 × 10(6) transcripts per liter) for 36 genes (for example, phosphonate metabolism gene phnH, dissimilatory nitrate reductase subunit napA) to >2.7 × 10(9) transcripts per liter (ammonia transporter amt and ammonia monooxygenase subunit amoC). Half of the categories for which expression was detected, however, had too few copy numbers for robust statistical resolution, as would be required for comparative (experimental or time-series) expression studies. By representing whole community gene abundance and expression in absolute units (per volume or mass of environment), 'omics' data can be better leveraged to improve understanding of microbially mediated processes in the ocean.
Hollibaugh, J T, S Gifford, S Sharma, N Bano, M Moran. 2011. Metatranscriptomic analysis of ammonia-oxidizing organisms in an estuarine bacterioplankton assemblage. ISME J. 5:866-878.
Quantitative PCR (qPCR) analysis revealed elevated relative abundance (1.8% of prokaryotes) of marine group 1 Crenarchaeota (MG1C) in two samples of southeastern US coastal bacterioplankton, collected in August 2008, compared with samples collected from the same site at different times (mean 0.026%). We analyzed the MG1C sequences in metatranscriptomes from these samples to gain an insight into the metabolism of MG1C population growing in the environment, and for comparison with ammonia-oxidizing bacteria (AOB) in the same samples. Assemblies revealed low diversity within sequences assigned to most individual MG1C open reading frames (ORFs) and high homology with 'Candidatus Nitrosopumilus maritimus' strain SCM1 genome sequences. Reads assigned to ORFs for ammonia uptake and oxidation accounted for 37% of all MG1C transcripts. We did not recover any reads for Nmar_1354-Nmar_1357, proposed to encode components of an alternative, nitroxyl-based ammonia oxidation pathway; however, reads from Nmar_1259 and Nmar_1667, annotated as encoding a multicopper oxidase with homology to nirK, were abundant. Reads assigned to two homologous ORFs (Nmar_1201 and Nmar_1547), annotated as hypothetical proteins were also abundant, suggesting that their unknown function is important to MG1C. Superoxide dismutase and peroxiredoxin-like transcripts were more abundant in the MG1C transcript pool than in the complete metatranscriptome, suggesting an enhanced response to oxidative stress by the MG1C population. qPCR indicated low AOB abundance (0.0010% of prokaryotes), and we found no transcripts related to ammonia oxidation and only one RuBisCO transcript among the transcripts assigned to AOB, suggesting they were not responding to the same environmental cues as the MG1C population.
Howard, E C, S Sun, C R Reisch, D A del Valle, H Bürgmann, R P Kiene, et al. 2011. Changes in DMSP demethylase gene assemblages in response to an induced phytoplankton bloom. Appl. Environ. Microb. 77:524-531.
Over half of the bacterioplankton cells in ocean surface waters are capable of carrying out a demethylation of the phytoplankton metabolite dimethylsulfoniopropionate (DMSP) that routes the sulfur moiety away from the climatically active gas dimethylsulfide (DMS). In this study, we tracked changes in dmdA, the gene responsible for DMSP demethylation, over the course of an induced phytoplankton bloom in Gulf of Mexico seawater microcosms. Analysis of >91,000 amplicon sequences indicated 578 different dmdA sequence clusters at a conservative clustering criterion of ≥90% nucleotide sequence identity over the 6-day study. The representation of the major clades of dmdA, several of which are linked to specific taxa through genomes of cultured marine bacterioplankton, remained fairly constant. However, the representation of clusters within these major clades shifted significantly in response to the bloom, including two Roseobacter-like clusters and a SAR11-like cluster, and the best correlate with shifts of the dominant dmdA clades was chlorophyll a concentration. Concurrent 16S rRNA amplification and sequencing indicated the presence of Roseobacter, SAR11, OM60, and marine Rhodospirillales populations, all of which are known to harbor dmdA genes, although the largest taxonomic change was an increase in Flavobacteriaceae, a group not yet demonstrated to have DMSP-demethylating capabilities. Sequence heterogeneity in dmdA and other functional gene populations is becoming increasingly evident with the advent of high-throughput sequencing technologies, and understanding the ecological implications of this heterogeneity is a major challenge for marine microbial ecology.
Jansson, J K, J D Neufeld, M Moran, J A Gilbert. 2011. Omics for understanding microbial functional dynamics. Environ. Microb.14(1):1-3.
Mou, X, M Vila-Costa, S Sun, W Zhao, S Sharma, M Moran. 2011. Metatranscriptomic Signature of Exogenous Polyamine Utilization by Coastal Bacterioplankton. Environ. Microbiol. 3(6):798-806.
The polyamines putrescine (PUT) and spermidine (SPD) are ubiquitous in seawater, but mechanisms that drive the degradation of these important nitrogen sources by marine bacteria remain unclear. We employed a comparative metatranscriptomics approach to compare gene transcription patterns between coastal bacterioplankton communities with and without amendments of PUT or SPD, in an effort to understand how bacterial communities and their genes shape polyamine biogeochemistry in the ocean. Statistically different transcript categories in the PUT (25 COG groups) and SPD (23 COG groups) samples, relative to controls that received no amendment (CTRL), indicated that genes encoding the cellular translation machinery and the metabolism of organic nitrogen and carbon became enriched in the community transcriptome when polyamine availability increased. Of the three known pathways for bacterial polyamine degradation, only genes in the transamination pathway were enriched in the PUT and SPD libraries, suggesting that this route dominated polyamine degradation. Taxonomic affiliation of significantly enriched diagnostic genes in the PUT and SPD libraries pointed to roseobacter- and SAR11-affiliated bacteria as the predominant taxa driving transformation in this coastal ocean, although other diverse marine bacterioplankton groups (Gammaproteobacteria, Betaproteobacteria, Actinobacteria and Bacteroidetes) also contributed to polyamine-related gene transcription.
Reisch, C R, M J Stoudemayer, V A Varaljay, I J Amster, M Moran, W B Whitman. 2011. Novel pathway for assimilation of dimethylsulphoniopropionate widespread in marine bacteria. Nature 473:208–211.
Dimethylsulphoniopropionate (DMSP) accounts for up to 10% of carbon fixed by marine phytoplankton in ocean surface waters, producing an estimated 11.7-103 Tmol S per year, most of which is processed by marine bacteria through the demethylation/demethiolation pathway. This pathway releases methanethiol (MeSH) instead of the climatically active gas dimethylsulphide (DMS) and enables marine microorganisms to assimilate the reduced sulphur. Despite recognition of this critical microbial transformation for over two decades, the biochemical pathway and enzymes responsible have remained unidentified. Here we show that three new enzymes related to fatty acid β-oxidation constitute the pathway that assimilates methylmercaptopropionate (MMPA), the first product of DMSP demethylation/demethiolation, and that two previously unknown coenzyme A (CoA) derivatives, 3-methylmercaptopropionyl-CoA (MMPA-CoA) and methylthioacryloyl-CoA (MTA-CoA), are formed as novel intermediates. A member of the marine roseobacters, Ruegeria pomeroyi DSS-3, requires the MMPA-CoA pathway for MMPA assimilation and MeSH production. This pathway and the ability to produce MeSH from MMPA are present in diverse bacteria, and the ubiquitous SAR11 clade bacterium Pelagibacter ubique possesses enzymes for at least the first two steps. Analysis of marine metagenomic data indicates that the pathway is widespread among bacterioplankton in the ocean surface waters, making it one of the most important known routes for acquisition of reduced carbon and sulphur by surface ocean heterotrophs.
** Reisch, C R, M A Moran and W B Whitman. 2011. Bacterial catabolism of dimethylsulfoniopropionate (DMSP). Frontiers in Microbial Physiology and Metabolism.
Robbins, S, J Jacob, X Lu, M Moran, X Mou. 2011. Bromodeoxyuridine (BrdU) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton. J Visual Exp. 55 doi:10.3791/2855.
Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS). Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.
Vila-Costa, M, S Sharma, M Moran, E O Casamayor. 2011. Diel Gene Expression Profiles of a Phosphorus Limited Mountain Lake Using Metatranscriptomics. Environ Microbiol. 15(4):1190-203
The genetic basis of bacterial functionality in freshwater systems remains largely unexplored despite its relevance in biogeochemical cycles. In this study, we used metatranscriptomic sequencing to analyse day and night gene expression profiles of the bacterial planktonic assemblage from the phosphorus (P) limited Lake Llebreta (1620 m above sea level) in the Limnological Observatory of the Pyrenees (LOOP, Central Pyrenees). The goal of the study was to obtain clues about the ecological strategies of bacteria in a highly oligotrophic environment, particularly those related to processing P and energy capture. An average of 37 871 unique reads were obtained per treatment using 454 pyrosequencing of amplified messenger RNA (mRNA), of which ∼ 37% matched a protein function in BLASTx analysis against the NCBI RefSeq database. In general, an overabundance of transcripts for energy acquisition (e.g. photosynthesis, oxidative phosphorylation, proteorhodopsins and bacteriochlorophyll a) was observed in the day compared with the night. Several different forms of P were metabolized by the community, with the relative abundance of transcripts related to phosphonate and phosphate uptake pointing to a major role of organic P in controlling this ecosystem. Bacteroidetes and Betaproteobacteria were the most actively transcribing phyla in the community, but showed different strategies for supplemental sources of energy: Bacteroidetes appeared to rely on creating H+ gradients across the membrane by using proteorhodopsins during the day and pyrophosphatases at night, whereas Betaproteobacteria appeared to be oxidizing carbon monoxide (CO) that potentially was generated by photooxidation of dissolved organic matter. When these diel freshwater metatranscriptomes were compared with those from two pelagic marine systems, gene expression patterns distinguished freshwater versus marine samples but showed common differences between day and night transcriptomes related to energy production.
** Newton, R J, L Griffin, K Bowles, C Meile, S Gifford, C E Givens, et al. 2010. Genome characteristics of a generalist marine bacterial lineage. ISME J. 4:784-798.
Poretsky, R S, S Sun, X Mou, M Moran. 2010. Transporter genes expressed by coastal bacterioplankton in response to dissolved organic carbon. Environ. Microbiol. 12(3):616-627.
Coastal ocean bacterioplankton control the flow of dissolved organic carbon (DOC) from terrestrial and oceanic sources into the marine food web, and regulate the release of inorganic carbon to atmospheric and offshore reservoirs. While the fate of the chemically complex coastal DOC reservoir has long been recognized as a critical feature of the global carbon budget, it has been problematic to identify both the compounds that serve as major conduits for carbon flux and the roles of individual bacterioplankton taxa in mediating that flux. Here we analyse random libraries of expressed genes from a coastal bacterial community to identify sequences representing DOC-transporting proteins. Predicted substrates of expressed transporter genes indicated that carboxylic acids, compatible solutes, polyamines and lipids may be key components of the biologically labile DOC pool in coastal waters, in addition to canonical bacterial substrates such as amino acids, oligopeptides and carbohydrates. Half of the expressed DOC transporter sequences in this coastal ocean appeared to originate from just eight taxa: Roseobacter, SAR11, Flavobacteriales and five orders of gamma-Proteobacteria. While all major taxa expressed transporter genes for some DOC components (e.g. amino acids), there were indications of specialization within the bacterioplankton community for others (e.g. carbohydrates, carboxylic acids and polyamines). Experimental manipulations of the natural DOC pool that increased the concentration of phytoplankton- or vascular plant-derived compounds invoked a readily measured response in bacterial transporter gene expression. This highly resolved view of the potential for carbon flux into heterotrophic bacterioplankton cells identifies possible bioreactive components of the coastal DOC pool and highlights differing ecological roles in carbon turnover for the resident bacterial taxa..
Rinta-Kanto, J, H Bürgmann, S M Gifford, S Sun, S Sharma, D A del Valle, et al. 2010. Analysis of sulfur-related transcription by Roseobacter communities using a taxon-specific functional gene microarray. Environ. Microbiol. 13:453-467.
The fraction of dissolved dimethylsulfoniopropionate (DMSPd) converted by marine bacterioplankton into the climate-active gas dimethylsulfide (DMS) varies widely in the ocean, with the factors that determine this value still largely unknown. One current hypothesis is that the ratio of DMS formation: DMSP demethylation is determined by DMSP availability, with 'availability' in both an absolute sense (i.e. concentration in seawater) and in a relative sense (i.e. proportionally to other labile organic S compounds) proposed as the critical factor. We investigated these models during an experimentally induced phytoplankton bloom using a taxon-specific microarray targeting DMSP-related gene transcription in members of the Roseobacter clade, a group hypothesized to play an important role in the surface ocean sulfur cycle and well represented by genome sequences. The array consisted of 1578 probes to 431 genes and was designed to target diverse Roseobacter communities in natural seawater by using hierarchical probe design based on 13 genome sequences. The prevailing pattern of Roseobacter gene transcription showed relative depletion of DMSP-related transcripts during the peak of the bloom, despite increasing absolute concentrations and flux of DMSP-related compounds. DMSPd thus appeared to be assimilated by Roseobacter populations in proportion to its relative abundance in the organic matter pool (the 'relative sense' hypothesis), rather than assimilated in preference to other labile organic sulfur or carbon compounds produced during the bloom. The relative investment of the Roseobacter community in DMSP demethylation was not useful for predicting the formation of DMS, however, suggesting a complex regulatory process that may involve multiple taxa and alternative fates of DMSPd.
** Varaljay V A, Howard E C, Sun S, Moran M. 2010. Deep sequencing of a dimethylsulfoniopropionate-degrading gene (dmdA) by using PCR primer pairs designed on the basis of marine metagenomic data. Appl. Environ. Microbiol. 76(2):609-17.
Vila-Costa, M, J M Rinta-Kanto, S Sun, S Sharma, R Poretsky, M Moran. 2010. Transcriptomic analysis of a marine bacterial community enriched with dimethylsulfoniopropionate. ISME J. 4:1410-1420.
Dimethylsulfoniopropionate (DMSP) is an important source of reduced sulfur and carbon for marine microbial communities, as well as the precursor of the climate-active gas dimethylsulfide (DMS). In this study, we used metatranscriptomic sequencing to analyze gene expression profiles of a bacterial assemblage from surface waters at the Bermuda Atlantic Time-series Study (BATS) station with and without a short-term enrichment of DMSP (25 nM for 30 min). An average of 303 143 reads were obtained per treatment using 454 pyrosequencing technology, of which 51% were potential protein-encoding sequences. Transcripts from Gammaproteobacteria and Bacteroidetes increased in relative abundance on DMSP addition, yet there was little change in the contribution of two bacterioplankton groups whose cultured members harbor known DMSP degradation genes, Roseobacter and SAR11. The DMSP addition led to an enrichment of transcripts supporting heterotrophic activity, and a depletion of those encoding light-related energy generation. Genes for the degradation of C3 compounds were significantly overrepresented after DMSP addition, likely reflecting the metabolism of the C3 component of DMSP. Mapping these transcripts to known biochemical pathways indicated that both acetyl-CoA and succinyl-CoA may be common entry points of this moiety into the tricarboxylic acid cycle. In a short time frame (30 min) in the extremely oligotrophic Sargasso Sea, different gene expression patterns suggest the use of DMSP by a diversity of marine bacterioplankton as both carbon and sulfur sources.