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Bacterioplankton of the marine Roseobacter clade have genomes that reflect a dynamic environment and diverse interactions with marine plankton. Comparative genome sequence analysis of three cultured representatives suggests that cellular requirements for nitrogen are largely provided by regenerated ammonium and organic compounds (polyamines, allophanate, and urea), while typical sources of carbon include amino acids, glyoxylate, and aromatic metabolites. An unexpectedly large number of genes are predicted to encode proteins involved in the production, degradation, and efflux of toxins and metabolites. A mechanism likely involved in cell-to-cell DNA or protein transfer was also discovered: vir -related genes encoding a type IV secretion system typical of bacterial pathogens. These suggest a potential for interacting with neighboring cells and impacting the routing of organic matter into the microbial loop. Genes shared among the three roseobacters and also common in nine draft Roseobacter genomes include those for carbon monoxide oxidation, dimethylsulfoniopropionate demethylation, and aromatic compound degradation. Genes shared with other cultured marine bacteria include those for utilizing sodium gradients, transport and metabolism of sulfate, and osmoregulation.

Microbial and photochemical processes affect chromophoric dissolved organic matter (CDOM) dynamics in the ocean. Some evidence suggests that dissolved nitrogen plays a role in CDOM formation, although this has received little systematic attention in marine ecosystems. Coastal seawater incubations were carried out in the presence of model dissolved organic nitrogen (DON: amino sugars and amino acids) and dissolved inorganic nitrogen (DIN) compounds to assess their role in biological and photochemical production of CDOM. For several of the dissolved N compounds, microbial processing resulted in a pulse of CDOM that was mainly labile, appearing and disappearing within 7 days. In contrast, a net loss of CDOM occurred when no N was added to the microbial incubations. The greatest net biological CDOM formation was found upon addition of amino sugars (formation of fluorescent, mostly labile CDOM) and tryptophan (formation of non-fluorescent, refractory CDOM). Photochemical formation of CDOM was only found with tryptophan, the one aromatic compound tested. This CDOM was highly fluorescent, with excitation–emission matrices (EEMs) resembling those of terrestrial, humic-like fluorophores. The heterogeneity in CDOM formation from this collection of labile N-containing compounds was surprising. These compounds are common components of biopolymers and humic substances in natural waters and likely to contribute to microbially- and photochemically-produced CDOM in coastal seawater.

In oligotrophic ocean waters where bacteria are often subjected to chronic nutrient limitation, community transcriptome sequencing has pointed to the presence of highly abundant small RNAs (sRNAs). The role of sRNAs in regulating response to nutrient stress was investigated in a model heterotrophic marine bacterium Ruegeria pomeroyi grown in continuous culture under carbon (C) and nitrogen (N) limitation. RNAseq analysis identified 99 putative sRNAs. Sixty-nine were cis-encoded and located antisense to a presumed target gene. Thirty were trans-encoded and initial target prediction was performed computationally. The most prevalent functional roles of genes anti-sense to the cis-sRNAs were transport, cell-cell interactions, signal transduction, and transcriptional regulation. Most sRNAs were transcribed equally under both C and N limitation, and may be involved in a general stress response. However, 14 were regulated differentially between the C and N treatments and may respond to specific nutrient limitations. A network analysis of the predicted target genes of the R. pomeroyi cis-sRNAs indicated that they average fewer connections than typical protein-encoding genes, and appear to be more important in peripheral or niche-defining functions encoded in the pan genome.

After publication of this study [1], the authors noticed that the reference numbers given in the original version of the Additional file 1: Table S1 were incorrect. The corrected Additional file 1: Table S1 can be found below

This protocol is from: Satinsky, Brandon M., et al. "Use of internal standards for quantitative metatranscriptome and metagenome analysis." Methods in enzymology 531 (2012): 237-250. Please see the full chapter for additional details.

Bourlyashchy is the largest and hottest pool in the Uzon Caldera, located in the territory of Kronotsky Nature Reserve, Kamchatka, Russia, with sediment surface temperatures at the margins ranging from 86 to 97 °C, and pH from 6.0 to 7.0. The microbial communities of the pool water and sediments were studied comprehensively from 2005 to 2014. Radioisotopic tracer studies revealed the processes of inorganic carbon assimilation, sulfate reduction, lithotrophic methanogenesis and potentially very active process of acetate oxidation to CO2. The total number of microbial cells in water was different in different years ranging from 5.2 to 7.0 × 10(6); in sediments, it changed from year to year between 6.3 × 10(6) and 1.75 × 10(8), increasing with a decrease in temperature. FISH with Archaea- and Bacteria-specific probes showed that the share of Bacteria differed with year, changing from 34 to 71%. According to 16S rRNA gene pyrosequencing data, lithoautotrophs (Aquificales and Thermoproteales) predominated in water samples, while in sediments they shared the niche with organotrophic Crenarchaeota, Korarchaeota, and bacteria of the genus Caldimicrobium (phylum Thermodesulfobacteria). The majority of organisms in water belonged to cultivated orders of prokaryotes; the only large uncultured group was that representing a novel order in class Thermoprotei. In sediments, unclassified Aquificeae comprised a significant part of the bacterial population. Thus, we showed that the hottest of the terrestrial hot pools studied contains numerous and active microbial populations where Bacteria represent a significant part of the microbial community, and planktonic and sediment populations differ in both composition and function.

Members of Thaumarchaeota are responsible for much of the ammonia oxidation occurring in the ocean. Recent studies showed that marine Thaumarchaeota have versatile metabolic capabilities, but sequencing additional genomes has not significantly increased the gene content ascribed to this group. We used the assembly-free dN pipeline software in combination with phylogenetic analyses to interrogate shotgun metagenomic data sets to gain a better understanding of the genomic diversity of Thaumarchaeota populations. The program confidently assigned ∼3,000 paired-end reads to Thaumarchaeota, independent of homologies to any known Thaumarchaeota genome sequence. Only 2% of these reads potentially harbor new genes that were absent from the genome of 'Candidatus Nitrosopumilus maritimus' str. SCM1, even though this strain was isolated from a marine aquarium rather than directly from the ocean. One of these novel genes encode proteins associated with the CRISPR/Cas system, Cas1, suggesting that phage defense through CRISPR may be also present in planktonic Thaumarchaeota lineages. Our results suggest that marine Thaumarchaeota populations have very low diversity in genome content, which is corroborated using computer simulation analyses of two bacterial lineages with known genome content diversity.

The optical properties and geochemical cycling of chromophoric dissolved organic matter (CDOM) are altered by its sorption to freshwater and estuarine sediments. Measured partition coefficients ( K p ) of Satilla River (Georgia) and Cape Fear River estuary (North Carolina) CDOM ranged from 19 to 233 L kg −1 when model freshwater and organic-rich estuarine sediments were added to solution (concentrations of 0.1, 1, or 10 g L −1 ), with the largest K p values measured in solutions with the lowest sediment concentrations. Sorption of Satilla River CDOM was augmented upon raising the ionic strength of solution by mixing with natural seawater, likely due to the ‘salting out effect’ of Ca +2 and Mg +2 ions. For turbid estuarine systems with particle loads of ∼100 mg L −1 , we estimate that ∼1–2% of the CDOM pool sorbs to settling particles, facilitating the transfer to a potentially large colored particulate organic matter (CPOM) reservoir within sediments. Our results also indicate that >30% of the colored organic matter pool within sediment pore waters (sediment concentration > 10 g L −1 ) may exist as CPOM. Spectral slope coefficients (300–700 nm) of initial CDOM samples increased as much as 20% after mixing with 10 g L −1 sediment and 5% after mixing with 1 g L −1 sediment indicating that sorption to particles has the potential to significantly alter the optical properties of CDOM in the water column of turbid shallow environments or in areas of high benthic exchange.

We analyzed bioavailability, photoreactivity, fluorescence, and isotopic composition of dissolved organic carbon (DOC) collected at 13 stations in the Sacramento-San Joaquin River Delta during various seasons to estimate the persistence of DOC from diverse shallow water habitat sources. Prospective large-scale wetland restorations in the Delta may change the amount of DOC available to the food web as well as change the quality of Delta water exported for municipal use.Our study indicates that DOC contributed by Delta sources is relatively refractory and likely mostly the dissolved remnants of vascular plant material from degrading soils and tidal marshes rather than phytoplankton production. Therefore, the prospective conversion of agricultural land into submerged, phytoplankton-dominated habitats may reduce the undesired export of DOC from the Delta to municipal users.A median of 10% of Delta DOC was rapidly utilizable by bacterio- plankton. A moderate dose of simulated solar radiation (286 W m � 2 for 4 h) decreased the DOC bioavailability by an average of 40%, with a larger relative decrease in samples with higher initial DOC bioavailability. Potentially, a DOC-based microbial food web could support £ 0.6 · 10 9 gC of protist production in the Delta annually, compared to � 17 · 10 9 g C phytoplankton primary production. Thus, DOC utilization via the microbial food web is unlikely to play an important role in the nutrition of Delta zooplankton and fish, and the possible decrease in DOC concentration due to wetland restoration is unlikely to have a direct effect on Delta fish productivity.

Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 μM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella , Oceanicola , and Sulfitobacter ( Roseobacter lineage, α -Proteobacteria ); Caulobacter (α -Proteobacteria ); and Brachymonas and Xenophilus (β -Proteobacteria ) in the May-02 sample and to Ketogulonicigenium ( Roseobacter lineage, α -Proteobacteria ) and novel γ -Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.

Since the recognition of prokaryotes as essential components of the oceanic food web, bacterioplankton have been acknowledged as catalysts of most major biogeochemical processes in the sea. Studying heterotrophic bacterioplankton has been challenging, however, as most major clades have never been cultured or have only been grown to low densities in sea water. Here we describe the genome sequence of Silicibacter pomeroyi, a member of the marine Roseobacter clade (Fig. 1), the relatives of which comprise approximately 10-20% of coastal and oceanic mixed-layer bacterioplankton. This first genome sequence from any major heterotrophic clade consists of a chromosome (4,109,442 base pairs) and megaplasmid (491,611 base pairs). Genome analysis indicates that this organism relies upon a lithoheterotrophic strategy that uses inorganic compounds (carbon monoxide and sulphide) to supplement heterotrophy. Silicibacter pomeroyi also has genes advantageous for associations with plankton and suspended particles, including genes for uptake of algal-derived compounds, use of metabolites from reducing microzones, rapid growth and cell-density-dependent regulation. This bacterium has a physiology distinct from that of marine oligotrophs, adding a new strategy to the recognized repertoire for coping with a nutrient-poor ocean.

Fluorescence-based observations provide useful, sensitive information concerning the nature and distribution of colored dissolved organic matter (CDOM) in coastal and freshwater environments. The excitation–emission matrix (EEM) technique has become widely used for evaluating sources and sinks of CDOM. Water scattering peaks, however, can create problems for quantitative analysis and display of the EEMs, especially for samples with low CDOM concentrations. Here we report a new method for eliminating Rayleigh and Raman scatter peaks from EEMs during post-processing of the data in MATLAB®. An algorithm was developed to excise scatter peaks (i.e. peak emission±10–15 nm at each excitation wavelength) from the scan data and replace the excised values using three-dimensional interpolation of the remaining data (Delaunay triangulation method). The interpolated surface was constrained to pass through the non-excised values so that only data in excised portions were replaced. Tests of the algorithm in non-scatter regions indicate expected deviations of 0–4% for interpolated regions of DOM fluorescence peaks (i.e. difference between measured and interpolated intensity after removal/interpolation), which is within machine error for the primary observations. This new scattering correction method is shown to provide much improved results in the quantitative analysis of EEMs compared to the conventional blank-subtraction procedure. The method is used to process EEMs and fluorescence quantum yields for water samples obtained along a salinity transect in a river located on the coast of the southeastern United States. Results of this analysis demonstrate observed shifts in EEM peak positions along most of the transect cannot be accounted for by a simple model that computes EEMs as a function of salinity assuming that the dominant driver of EEM spectral change is mixing between riverine and marine waters within the estuary. Other results show that fluorescence apparent quantum yields (AQYs) increased with increasing salinity and pH in the estuary and that the highest quantum yields are observed on excitation by 350–380-nm light. Modeling results and the observed EEM spectral changes indicate that photoreactions had an important effect on the fluorescent dissolved organic matter (FDOM) optical properties in the estuary. The increase in fluorescence quantum yields with increasing salinity and pH in the estuary likely were caused by reactions involving magnesium or hydroxide ions that reduced fluorescence quenching by chelated iron and possibly other paramagnetic ions. The results of the study indicate that EEM analysis with careful scatter correction can provide a powerful tool for evaluating pathways for carbon cycling in estuaries.

Chromophoric dissolved organic matter (CDOM) in the Mississippi plume region may have several distinct sources: riverine (terrestrial soils), wetland (terrestrial plants), biological production (phytoplankton, zooplankton, microbial), and sediments. Complex mixing, photodegradation, and biological processes make differentiation of the specific sources of CDOM difficult. Using a combination of high resolution in situ observations on an undulating vehicle, the ECOShuttle, a pumping system mounted on the vehicle, and detailed chemical and biological analyses of discrete samples allowed us to characterize two specific sources of CDOM in the Louisiana Bight: the river water constrained in the upper 12 m of the Mississippi River plume and several subsurface layers of CDOM below the plume. The subsurface CDOM maxima were coincident with steep pycnoclines and sometimes with maxima in chlorophyll a fluorescence. Both sources were actively supplying CDOM to the same location by entirely different processes. The subsurface CDOM was more biologically labile and photochemically refractory than the surface CDOM. Optical properties were also different with a relatively higher protein fluorescence and a lower spectral slope coefficient in the subsurface CDOM. The geographical extent of the two sources was determined by threedimensional mapping of the area, and due to the relatively calm conditions in the summer of 2000, thin layers of CDOM produced in the subsurface were observed throughout the region. While riverine inputs dominated the distribution of CDOM in surface waters

Members of the Roseobacter lineage, an ecologically important marine clade within the class α- Proteobacteria , harbor genes for the protocatechuate branch of the β-ketoadipate pathway, a major catabolic route for lignin-related aromatic compounds. The genes of this pathway are typically clustered, although gene order varies among organisms. Here we characterize genes linked to pcaH and -G , which encode protocatechuate 3,4-dioxygenase, in eight closely related members of the Roseobacter lineage (pairwise 16S rRNA gene sequence identities, 92 to 99%). Sequence analysis of genomic fragments revealed five unique pca gene arrangements. Identical gene organization was found for isolates demonstrating species-level identity (i.e., >99% 16S rRNA gene similarity). In one isolate, six functionally related genes were clustered: pcaQ , pobA , pcaD , pcaC , pcaH , and pcaG . The remaining seven isolates lacked at least one of these genes in their clusters, although the relative order of the remaining genes was preserved. Three genes ( pcaC , - H , and - G ) were physically linked in all isolates. A highly conserved open reading frame (ORF) was found immediately downstream of pcaG in all eight isolates. Reverse transcription-PCR analysis of RNA from one isolate, Silicibacter pomeroyi DSS-3, provides evidence that this ORF is coexpressed with upstream pca genes. The absence of this ORF in similar bacterial pca gene clusters from diverse microbes suggests a niche-specific role for its protein product in Roseobacter group members. Collectively, these comparisons of bacterial pca gene organization illuminate a complex evolutionary history and underscore the widespread ecological importance of the encoded β-ketoadipate pathway.

Both bacteria and fungi play critical roles in decomposition processes in many natural environments, yet only rarely have they been studied as an integrated microbial community. Here we describe the bacterial and fungal assemblages associated with two decomposition stages of Spartina alterniflora detritus in a productive southeastern U.S. salt marsh. 16S rRNA genes and 18S-to-28S internal transcribed spacer (ITS) regions were used to target the bacterial and ascomycete fungal communities, respectively, based on DNA sequence analysis of isolates and environmental clones and by using community fingerprinting based on terminal restriction fragment length polymorphism (T-RFLP) analysis. Seven major bacterial taxa (six affiliated with the α- Proteobacteria and one with the Cytophagales ) and four major fungal taxa were identified over five sample dates spanning 13 months. Fungal terminal restriction fragments (T-RFs) were informative at the species level; however, bacterial T-RFs frequently comprised a number of related genera. Amplicon abundances indicated that the salt marsh saprophyte communities have little-to-moderate variability spatially or with decomposition stage, but considerable variability temporally. However, the temporal variability could not be readily explained by either successional shifts or simple relationships with environmental factors. Significant correlations in abundance (both positive and negative) were found among dominant fungal and bacterial taxa that possibly indicate ecological interactions between decomposer organisms. Most associations involved one of four microbial taxa: two groups of bacteria affiliated with the α- Proteobacteria and two ascomycete fungi ( Phaeosphaeria spartinicola and environmental isolate “4clt”).

Three Gram-negative, rod-shaped, aerobic bacteria that were capable of degrading dimethylsulfoniopropionate (DMSP) were isolated from marine waters. These isolates (DSS-3(T), DSS-10 and ISM(T)) exhibited the ability to demethylate and cleave DMSP, as well as to degrade other sulfur compounds related to DMSP that are cycled in marine environments. Intracellular poly-beta-hydroxybutyrate inclusions, surface blebs and one polar, complex flagellum that rotated exclusively in the clockwise direction were observed for DSS-3(T). The outer membrane of ISM(T) was separated from the cytoplasm at the poles in a toga-like morphology. The primary fatty acid in both strains was C(18 : 1)omega7c. DNA G+C contents for the isolates were 68.0+/-0.1, 68.1+/-0.1 and 66.0+/-0.2 mol% for DSS-3(T), DSS-10 and ISM(T), respectively. 16S rRNA gene sequence analyses placed these organisms within the Roseobacter lineage of the alpha-PROTEOBACTERIA: Closely related species were Silicibacter lacuscaerulensis and Ruegeria atlantica (DSS-3(T) and DSS-10) and Roseovarius tolerans (ISM(T)). Neither DSS-3(T) nor ISM(T) exhibited 16S rRNA similarity97 % or DNA-DNA hybridization values45 % to their nearest described relatives. Genotypic and phenotypic analyses support the creation of two novel species: Silicibacter pomeroyi sp. nov. with strain DSS-3(T) (=ATCC 700808(T)=DSM 15171(T)) as the type strain, and Roseovarius nubinhibens sp. nov. with strain ISM(T) (=ATCC BAA-591(T)=DSM 15170(T)) as the type strain.

We examined seasonal and spatial variation in bacterioplankton composition in the Sacramento-San Joaquin River Delta (CA) using terminal restriction fragment length polymorphism (T-RFLP) analysis. Cloned 16S rRNA genes from this system were used for putative identification of taxa dominating the T-RFLP profiles. Both cloning and T-RFLP analysis indicated that Actinobacte- ria, Verrucomicrobia, Cytophaga-Flavobacterium and Proteobacteria were the most abundant bacte- rioplankton groups in the Delta. Despite the broad variety of sampled habitats (deep water channels, lakes, marshes, agricultural drains, freshwater and brackish areas), and the spatial and temporal dif- ferences in hydrology, temperature and water chemistry among the sampling campaigns, T-RFLP electropherograms from all samples were similar, indicating that the same bacterioplankton phylo- types dominated in the various habitats of the Delta throughout the year. However, principal compo- nent analysis (PCA) and partial least-squares regression (PLS) of T-RFLP profiles revealed consistent grouping of samples on a seasonal, but not a spatial, basis. β-Proteobacteria related to Ralstonia, Acti- nobacteria related to Microthrix, and β-Proteobacteria identical to the environmental Clone LD12 had the highest relative abundance in summer/fall T-RFLP profiles and were associated with low river flow, high pH, and a number of optical and chemical characteristics of dissolved organic carbon (DOC) indicative of an increased proportion of phytoplankton-produced organic material as opposed to allochthonous, terrestrially derived organic material. On the other hand, Geobacter-related β-Pro- teobacteria showed a relative increase in abundance in T-RFLP analysis during winter/spring, and probably were washed out from watershed soils or sediment. Various phylotypes associated with the same phylogenetic division, based on tentative identification of T-RFLP fragments, exhibited diverse seasonal patterns, suggesting that ecological roles of Delta bacterioplankton were partitioned at the genus or species level.

Temporal dynamics of three marine γ-Proteobacteria taxa withculturable members were followed in sediment communities in southeastern USsaltmarshes using a whole-genome hybridization approach. Labeled DNA from threebacterial isolates was used to probe sediment community DNA from two saltmarshes on Sapelo Island, GA at multiple time points over a three-year period.The relative abundance of the three isolates (and their close relatives)accounted for up to 28% of the total sediment community DNA. Hybridizationsignals for the taxon represented by isolate SIGA28M (with a 16S rRNA sequencesimilarity of 97% to Vibrio proteolyticus) showedsignificant temporal variation in both marshes, varying from 1% to 28% over thethree-year period. The taxa represented by isolates SIGA172a and SIGA198 (16SrRNA sequence similarities of 90% and 92% to Shewanellafrigidimarina and Vibrio nigripulchritudo,respectively) accounted for less than 6% of the sediment community at any timepoint. The variation in relative abundance of these three groups did not appearto follow clear seasonal trends, and could not be readily correlated withenvironmental variables.

Quantum yield spectra for the photochemical formation of biologically labile photoproducts from dissolved organic matter (DOM) have not been available previously, although they would greatly facilitate attempts to model photoproduct formation rates across latitudinal, seasonal, and depth-related changes in spectral irradiance. Apparent quantum yield spectra were calculated for two coastal environments from the southeastern United States using postirradiation bacterial respiration as a measure of total labile photoproduct formation and a cutoff filter method to model spectral dependence. As has been the case for previously studied classes of DOM photoproducts (i.e., dissolved inorganic carbon, CO, and H2O2), ultraviolet (UV)-B irradiance was significantly more efficient at forming labile photoproducts (i.e., compounds readily assimilated by marine bacterioplankton) than UV-A and visible irradiance. Calculations of DOM photoproduct formation in southeastern U.S. coastal surface waters indicate a formation ratio for biologically labile photoproducts : CO of 13 : 1. The slope of a natural log plot of the apparent quantum yield spectrum obtained for biologically labile photoproducts was similar to that for CO (0.028 nm 21 vs. 0.034 nm 21 ). Modeled kinetic rates therefore indicate that the production ratio of these photoproduct classes is approximately maintained despite variations in the solar spectrum that occur with depth in a water column or distance from shore. Application of the apparent quantum yield to coastal regions worldwide predicts an annual formation rate of biologically labile photoproducts in coastal waters of 206 3 10 12 gC .

Omics for understanding microbial functional dynamics Janet K. Jansson 1 *, Josh D. Neufeld 2 , Mary Ann Moran 3 , Jack A. Gilbert 4,5 Lawrence Berkeley National Laboratory, Earth Sciences Division Berkeley, CA, USA. Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. Department of Marine Sciences, University of Georgia, Athens, GA 30602, USA. Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439, USA. Department of Ecology and Evolution, University of Chicago, 5640 South Ellis Avenue, Chicago, IL 60637, USA. As a discipline, the field of microbial ecology has generally been limited by access to tools that have sufficient depth of resolution to enable exploration of complex microbial communities to determine ‘who is there?’ and ‘what they are doing?’. Until the mid 1980s most of our knowledge was based on the study of cultured microbial isolates or net enzymatic activities measured from laboratory enrich- ment experiments or environmental samples, without knowledge of the particular members of the community that were contributing to those functions. Since then, microbiologists have added a variety of molecular tools to our toolbox, allowing us to directly assess the identities and functions of microbial communities in a variety of environments without the necessity of prior cultivation. Most of the advances in molecular tools for assessing microbial community function have focused on the sequencing of nucleic acids. Sequencing of genomic or metagenomic DNA provides phylogenetic and functional gene information about specific organisms or communi- E-mail jrjansson@lbl.gov; Tel. (+1) 510 486 7487; Fax (+1) 510 486 7152. The submitted manuscript has been created in part by U. Chicago Argonne, LLC, Operator of Argonne National Laboratory (‘Argonne’). Argonne, a US Department of Energy Office of Science laboratory, is operated under Contract No. DE-AC02- 06CH11357. The US Government retains for itself, and others acting on its behalf, a paid-up nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the Government. ties respectively. These types of data have proven to be highly valuable for characterizing microbes that inhabit the various habitats on Earth, and importantly, their pos- sible roles in the function of that ecosystem. However, one should keep in mind that DNA sequence data only repre- sents the potential metabolic capacity of a system. In many environments, DNA that is sequenced may origi- nate from dead or dormant cells. Further, even actively growing microbes express only a fraction of their genes at any given time. For example, Escherichia coli genes are tightly regulated depending on, for example, the concen- tration of nutrients in their media (Tao et al., 1999). Only a third to a half of the genes in a given organism are expressed at any given time (Passalacqua et al., 2009). Methodology to address the longstanding dilemma of ‘which organisms are active?’ include the use of viability stains to first sort (e.g. with fluorescently activated cell sorting; FACS) and then sequence genomes from active populations, using stable isotopes to detect community members that are metabolically active (e.g. stable-isotope probing; SIP; Neufeld et al., 2007) and the use of bro- modeoxyuridine (BrdU), a thymidine analogue, to specifi- cally label DNA from growing cells that can then be isolated and sequenced (Edlund and Jansson, 2008; Mou et al., 2008). Alternatively, one can directly extract and sequence RNA to determine which microbes are active and which genes are transcribed. When applied to an entire microbial community, this analysis is referred to as ‘metatranscriptomics’. The metatranscriptomic approach has proven to be more challenging than metagenomics because RNA is less stable than DNA and because most of the RNA that is extracted is ribosomal RNA, usually representing over 90% of the total RNA (Urich et al., 2008). Ribosomal RNA sequences can inform about active community members due to a correlation between metabolic activity and ribosome abundance (Rehman et al., 2010), but do not reveal the genes and pathways expressed under a given set of conditions. In order to specifically focus on mRNA transcripts, several methods have been developed to enrich the mRNA fraction of the total RNA pool, usually by subtraction of the ribosomal RNA prior to sequencing (Fig. 1). The problem with loss of mRNA during the multiple processing steps is particularly

SUMMARY Members of the Roseobacter clade are equipped with a tremendous diversity of metabolic capabilities, which in part explains their success in so many different marine habitats. Ideas on how this diversity evolved and is maintained are reviewed, focusing on recent evolutionary studies exploring the timing and mechanisms of Roseobacter ecological diversification.

© 2015 International Society for Microbial Ecology. The 'bacterial switch' is a proposed regulatory point in the global sulfur cycle that routes dimethylsulfoniopropionate (DMSP) to two fundamentally different fates in seawater through genes encoding either the cleavage or demethylation pathway, and affects the flux of volatile sulfur from ocean surface waters to the atmosphere. Yet which ecological or physiological factors might control the bacterial switch remains a topic of considerable debate. Here we report the first field observations of dynamic changes in expression of DMSP pathway genes by a single marine bacterial species in its natural environment. Detection of taxon-specific gene expression in Roseobacter species HTCC2255 during a month-long deployment of an autonomous ocean sensor in Monterey Bay, CA captured in situ regulation of the first gene in each DMSP pathway (dddP and dmdA) that corresponded with shifts in the taxonomy of the phytoplankton community. Expression of the cleavage pathway was relatively greater during a high-DMSP-producing dinoflagellate bloom, and expression of the demethylation pathway was greater in the presence of a mixed diatom and dinoflagellate community. These field data fit the prevailing hypothesis for bacterial DMSP gene regulation based on bacterial sulfur demand, but also suggest a modification involving oxidative stress response, evidenced as upregulation of catalase via katG, when DMSP is demethylated.

We investigated expression of genes mediating elemental cycling at the microspatial scale in the ocean's largest river plume using, to our knowledge, the first fully quantitative inventory of genes and transcripts. The bacterial and archaeal communities associated with a phytoplankton bloom in Amazon River Plume waters at the outer continental shelf in June 2010 harbored ∼ 1.0 × 10(13) genes and 4.7 × 10(11) transcripts per liter that mapped to several thousand microbial genomes. Genomes from free-living cells were more abundant than those from particle-associated cells, and they generated more transcripts per liter for carbon fixation, heterotrophy, nitrogen and phosphorus uptake, and iron acquisition, although they had lower expression ratios (transcripts ⋅ gene(-1)) overall. Genomes from particle-associated cells contributed more transcripts for sulfur cycling, aromatic compound degradation, and the synthesis of biologically essential vitamins, with an overall twofold up-regulation of expression compared with free-living cells. Quantitatively, gene regulation differences were more important than genome abundance differences in explaining why microenvironment transcriptomes differed. Taxa contributing genomes to both free-living and particle-associated communities had up to 65% of their expressed genes regulated differently between the two, quantifying the extent of transcriptional plasticity in marine microbes in situ. In response to patchiness in carbon, nutrients, and light at the micrometer scale, Amazon Plume microbes regulated the expression of genes relevant to biogeochemical processes at the ecosystem scale.

Microbial ecology is plagued by problems of an abstract nature. Cell sizes are so small and population sizes so large that both are virtually incomprehensible. Niches are so far from our everyday experience as to make their very definition elusive. Organisms that may be abundant and critical to our survival are little understood, seldom described and/or cultured, and sometimes yet to be even seen. One way to confront these problems is to use data of an even more abstract nature: molecular sequence data. Massive environmental nucleic acid sequencing, such as metagenomics or metatranscriptomics, promises functional analysis of microbial communities as a whole, without prior knowledge of which organisms are in the environment or exactly how they are interacting. But sequence-based ecological studies nearly always use a comparative approach, and that requires relevant reference sequences, which are an extremely limited resource when it comes to microbial eukaryotes [1]. In practice, this means sequence databases need to be populated with enormous quantities of data for which we have some certainties about the source. Most important is the taxonomic identity of the organism from which a sequence is derived and as much functional identification of the encoded proteins as possible. In an ideal world, such information would be available as a large set of complete, well-curated, and annotated genomes for all the major organisms from the environment in question. Reality substantially diverges from this ideal, but at least for bacterial molecular ecology, there is a database consisting of thousands of complete genomes from a wide range of taxa, supplemented by a phylogeny-driven approach to diversifying genomics [2]. For eukaryotes, the number of available genomes is far, far fewer, and we have relied much more heavily on random growth of sequence databases [3],[4], raising the question as to whether this is fit for purpose.

Phenotypic plasticity (PP) is the development of alternate phenotypes of a given taxon as an adaptation to environmental conditions. Methodological limitations have restricted the quantification of PP to the measurement of a few traits in single organisms. We used metatranscriptomic libraries to overcome these challenges and estimate PP using the expressed genes of multiple heterotrophic organisms as a proxy for traits in a microbial community. The metatranscriptomes captured the expression response of natural marine bacterial communities grown on differing carbon resource regimes in continuous cultures. We found that taxa with different magnitudes of PP coexisted in the same cultures, and that members of the order Rhodobacterales had the highest levels of PP. In agreement with previous studies, our results suggest that continuous culturing may have specifically selected for taxa featuring a rather high range of PP. On average, PP and abundance changes within a taxon contributed equally to the organism's change in functional gene abundance, implying that both PP and abundance mediated observed differences in community function. However, not all functional changes due to PP were directly reflected in the bulk community functional response: gene expression changes in individual taxa due to PP were partly masked by counterbalanced expression of the same gene in other taxa. This observation demonstrates that PP had a stabilizing effect on a community's functional response to environmental change.

The direct retrieval and sequencing of environmental RNA is emerging as a powerful technique to elucidate the in situ activities of microbial communities. Here we provide a metatranscriptomic protocol describing environmental sample collection, rRNA depletion, mRNA amplification, cDNA synthesis, and bioinformatic analysis. In addition, the preparation of internal RNA standards and their addition to the sample are discussed, providing a method by which transcript numbers can be expressed as absolute abundances in the environment and more readily compared to other biogeochemical and ecological measurements.

The composition of bacterial communities growing at the expense of high-molecular weight (HMW; >1000 Da) and low-molecular weight (LMW

Terrestrially derived dissolved organic matter (DOM) impacts the optical properties of coastal seawater and affects carbon cycling on a global scale. We studied sequential long-term photochemical and biological degradation of estuarine dissolved organic matter from the Satilla River, an estuary in the southeastern United States that is dominated by vascular plant-derived organic matter. During photodegradation, dissolved organic carbon (DOC) loss (amounting to 31% of the initial DOC) was much less extensive than colored dissolved organic matter (CDOM) or fluorescent dissolved organic matter (FDOM) loss (50% and 56% of the initial CDOM and FDOM), and analysis of kinetics suggested a reservoir of DOC that was resistant to photodegradation. In contrast, CDOM photodegradation closely followed first-order kinetics over two half-lives with no indication of a nondegradable component. FDOM loss was slightly biased toward fluorophores considered representative of terrestrial humic substances. Additional changes in optical properties included increases in spectral slope and shifts in fluorescence excitation/ emission maxima that were generally consistent with previous observations from field studies of photobleached DOM. Biological degradation of photobleached DOM was more rapid than that of unbleached material, and this net positive effect was evident even for extensively photodegraded material. Bacterial degradation caused shifts in the opposite direction from photochemical degradation for both spectral slope and excitation/emission maxima and thus dampened but did not eliminate changes in optical properties caused by photobleaching.

Organic sulfur compounds are present in all aquatic systems, but their use as sources of sulfur for bacteria is generally not considered important because of the high sulfate concentrations in natural waters. This study investigated whether dimethylsulfoniopropionate (DMSP), an algal osmolyte that is abundant and rapidly cycled in seawater, is used as a source of sulfur by bacterioplankton. Natural populations of bacterioplankton from subtropical and temperate marine waters rapidly incorporated 15 to 40% of the sulfur from tracer-level additions of [35S]DMSP into a macromolecule fraction. Tests with proteinase K and chloramphenicol showed that the sulfur from DMSP was incorporated into proteins, and analysis of protein hydrolysis products by high-pressure liquid chromatography showed that methionine was the major labeled amino acid produced from [35S]DMSP. Bacterial strains isolated from coastal seawater and belonging to the α-subdivision of the divisionProteobacteriaincorporated DMSP sulfur into protein only if they were capable of degrading DMSP to methanethiol (MeSH), whereas MeSH was rapidly incorporated into macromolecules by all tested strains and by natural bacterioplankton. These findings indicate that the demethylation/demethiolation pathway of DMSP degradation is important for sulfur assimilation and that MeSH is a key intermediate in the pathway leading to protein sulfur. Incorporation of sulfur from DMSP and MeSH by natural populations was inhibited by nanomolar levels of other reduced sulfur compounds including sulfide, methionine, homocysteine, cysteine, and cystathionine. In addition, propargylglycine and vinylglycine were potent inhibitors of incorporation of sulfur from DMSP and MeSH, suggesting involvement of the enzyme cystathionine γ-synthetase in sulfur assimilation by natural populations. Experiments with [methyl-3H]MeSH and [35S]MeSH showed that the entire methiol group of MeSH was efficiently incorporated into methionine, a reaction consistent with activity of cystathionine γ-synthetase. Field data from the Gulf of Mexico indicated that natural turnover of DMSP supplied a major fraction of the sulfur required for bacterial growth in surface waters. Our study highlights a remarkable adaptation by marine bacteria: they exploit nanomolar levels of reduced sulfur in apparent preference to sulfate, which is present at 106- to 107-fold higher concentrations.

We measured dissolved O2 concentrations, pCO2 values, and respiratory rates in five estuaries of the southeastern U.S. in October 1995 and July 1996. In the low-salinity sections of the coastal plain rivers, dissolved O 2 saturation states were typically only 50%, while pCO2 values were over 4,000 matm. Respiratory rates measured concurrently in estuarine water averaged 8 and 23 mmol m 23 d 21 in October 1995 and July 1996, but they showed little variability either within or among the five estuaries. Benthic chamber incubations in the adjacent intertidal marshes indicated fluxes of 30‐40 mmol m 22 d 21 and 50‐120 mmol m 22 d 21 for O2 and total dissolved inorganic carbon (DIC), respectively. For the Satilla River estuary, simple calculations revealed that neither respiratory activity in estuarine waters and sediments nor any other within-estuary process (not including the intertidal marsh system) was sufficient to account for the observed O2 concentrations and pCO2 values. Dissolved oxygen concentrations in four other southeastern U.S. estuaries fit the same general pattern as the Satilla, and likewise, within-estuary processes could not explain observed gas concentrations. Measured O2 concentrations, pCO2 values, pelagic respiratory rates, and benthic fluxes were used to construct a mass-balance model, focusing on the influence of the extensive intertidal marshes on O2 and CO2 mass balance and water‐atmosphere gas exchange in the five estuaries. Results indicate that respiratory activity in the sediments and overlying water of the marshes during high tide leaves a signal that is funneled back to the estuary during ebb tide and can account for the estuarine gas concentrations and fluxes. Both experimental and modeling approaches argue that the intertidal marshes of the southeastern U.S. export considerable amounts of inorganic respiratory products to the estuaries and that ‘‘outwelling’’ of organic matter to the estuaries is a minor process by comparison. The DIC exported to the coastal ocean, however, is only a small fraction of the total gas mass flow between the marsh, estuary, and atmosphere.

We studied the photoproduction of biologically labile compounds from 2 chemically defined fractions of dissolved organic matter (DOM) and whole (unfractionated) DOM from an acidic freshwater wetland (Okefenokee Swamp, Georgia, USA). Water samples were collected in summer (July 1995) and winter (February 1996), and for the winter sample, DOM was separated into humic substances and 'non-humic' compounds by fractionation on an XAD-8 resin. Following exposure in quartz flasks to either natural or s~rnulated sunlight, bacteria were found to utilize as much as 300% more organic matter in irradiated whole DOM and chemical fractions compared to non-irradiated counterparts. Previous studies have shown that humic substances, which have strong absorption in the UV region of sunlight, are important sources of biologically labile photoproducts. We now demonstrate that compounds outside this operational definition are also sources for labile photoproducts, and that production rates on an absorbance-normalized basis can be equivalent for the humic and non-humic components of DOM. Hurnic substances were nonetheless calculated to be the primary source of biologically lablle photoproducts in these samples, based on the fact that they absorbed most (>85%) of the sunlight. Exposure of DOM fractions to sunlight for the equivalent of 7 h of noontime sun (at 34" N latitude) resulted in the bacterial utilization (over a subsequent 4 d period) of up to 6 % of the humic substances pool (compared to 1.5 % for non-irradiated controls), and 11 % of the non-humic compounds (compared to 3 % for non-irradiated controls).

Dissolved and particulate organic matter (DOM and POM) collected from rivers or groundwater feeding five estuaries along the east and west coasts of the USA were characterized with a variety of biogeochemical techniques and related to bioavailability to estuarine microbes. Surface water was sampled from the Columbia, Satilla, Susquehanna and Parker Rivers and groundwater was sampled from the Childs River. Several geochemical descriptors (percent organic matter of suspended particulate matter, C/N, lignin phenol content, ratio of vanillic acid to vanillin) suggested an ordering of the systems with respect to POM lability: Satilla 2000 pM for the Satilla River. Elemental analysis of DOM concentrates (>I000 D) was used to predict organic matter composition and to calculate degree of substrate reduction using two different modeling approaches. Models predicted aliphatic carbon ranging between 43 and 60% and aromatic carbon between 26 and 36%, with aliphatic content lowest in the Satilla and highest in the Columbia River. The degree of substrate reduction of the organic matter concentrates followed a pattern similar to that for aliphatic C, being lowest in the Satilla (3.5) and highest in the Columbia (4.0). Extracellular enzyme activity varied broadly across the systems, but again ordered sites in the same way as did aliphatic content and degree of substrate reduction. Bacterial growth rates ranged from 1.3 ug mg-' d-' DOC in the Satilla to 1.7 ug mg-' d-' DOC in the Parker River. Bioassays confirmed patterns of dissolved organic matter lability predicted by the chemical models. Between 67% to 75% of the variation in bacterial growth could be explained by differences in organic matter composition.

The organosulfur compound dimethylsulfoniopropionate (DMSP) is produced by phytoplankton and is ubiquitous in the surface ocean. Once released from phytoplankton, marine bacteria degrade DMSP by either the cleavage pathway to form the volatile gas dimethylsulfide (DMS) or the demethylation pathway, yielding methanethiol (MeSH), which is readily assimilated or oxidized. The enzyme DmdB, a methylmercaptopropionate (MMPA)-coenzyme A (CoA) ligase, catalyzes the second step in the demethylation pathway and is a major regulatory point. The two forms of DmdB present in the marine roseobacter Ruegeria pomeroyi DSS-3, RPO_DmdB1 and RPO_DmdB2, and the single form in the SAR11 clade bacterium “ Candidatus Pelagibacter ubique” HTCC1062, PU_DmdB1, were characterized in detail. DmdB enzymes were also examined from Ruegeria lacuscaerulensis ITI-1157, Pseudomonas aeruginosa PAO1, and Burkholderia thailandensis E264. The DmdB enzymes separated into two phylogenetic clades. All enzymes had activity with MMPA and were sensitive to inhibition by salts, but there was no correlation between the clades and substrate specificity or salt sensitivity. All Ruegeria species enzymes were inhibited by physiological concentrations (70 mM) of DMSP. However, ADP reversed the inhibition of RPO_DmdB1, suggesting that this enzyme was responsive to cellular energy charge. MMPA reversed the inhibition of RPO_DmdB2 as well as both R. lacuscaerulensis ITI-1157 DmdB enzymes, suggesting that a complex regulatory system exists in marine bacteria. In contrast, the DmdBs of the non-DMSP-metabolizing P. aeruginosa PAO1 and B. thailandensis E264 were not inhibited by DMSP, suggesting that DMSP inhibition is a specific adaptation of DmdBs from marine bacteria.

The direct retrieval and sequencing of environmental RNA is emerging as a powerful technique to elucidate the in situ activities of microbial communities. Here we provide a metatranscriptomic protocol describing environmental sample collection, rRNA depletion, mRNA amplification, cDNA synthesis, and bioinformatic analysis. In addition, the preparation of internal RNA standards and their addition to the sample are discussed, providing a method by which transcript numbers can be expressed as absolute abundances in the environment and more readily compared to other biogeochemical and ecological measurements.

A cluster of marine bacteria within the alpha-3 subclass of the class Proteobacteria accounted for up to 28% of the 16S ribosomal DNA (rDNA) sequences in seawater samples from the coast of the southeastern United States. Two independent oligonucleotide probes targeting 16S rDNA of this "marine alpha" cluster indicate that the group dominates bacterioplankton communities in estuarine and nearshore regions of the southeastern U.S. coast. Marine alpha bacteria decline predictably in abundance with decreasing salinity along estuarine transsects and are not detectable in low-salinity (5%) or freshwater samples. Sequences of 16S rDNA obtained from seawater by PCR with one group-specific oligonucleotide as a primer confirm that the oligonucleotide targets only members of this phylogenetic cluster. Likewise, sequences of 16S rDNA obtained from seawater by PCR with several different pairs of nonspecific primers show an unusually high abundance of marine alpha sequences (52 to 84%) among the clones, which possibly indicates a PCR bias toward the group. Members of the marine alpha group were readily cultured from coastal seawater, accounting for 40% of the colonies isolated on low-nutrient marine agar, based on hybridizations with the group-specific 16S rDNA probe and on sequence analysis. This is the first description of a numerically dominant cluster of coastal bacteria, identified by molecular techniques, that can be readily cultured and studied in the laboratory.

Dissolved organic matter (DOM) can be degraded by sunlight into a variety of photoproducts that stimulate the growth and activity of microorganisms in aquatic environments. All biologically labile photoproducts identified to date fall into one of four categories: (1) low-molecular-weight (MW) organic compounds (carbonyl compounds with MW of 20% of the bacterial carbon demand. Likewise, 30% of the bacterial nitrogen demand can be met by photodegradation of the nitrogen components of DOM, a process likely to be of particular importance in nitrogen-limited systems. When considered on a depth-integrated basis around the globe, at least 1.0 X 10 15 g C and 0.15 X 10 15 g N are estimated to be available annually for utilization by planktonic microorganisms through the conversion of light-absorbing fractions of DOM to more biologically labile compounds. By comparison, direct photochemical mineralization of DOM is estimated to convert 12-16 X 10 15 g C to CO 2 annually.

The interaction between photochemical and biological processes in the degradation of marine dissolved organic matter (DOM) was investigated with seawater from a coastal southeastern U.S. salt marsh. Seawater supplemented with humic substances was exposed to alternating cycles of sunlight (equivalent to 8 h of midday sun) and dark incubations with natural bacterial populations (l-2 weeks in length). Photochemical degradation of the DOM was monitored during sunlight exposure by direct measurements of dissolved inorganic carbon (DIC) and carbon monoxide (CO) formation in 0.2-Frn filtered seawater. Bacterial degradation was monitored during dark incubations by tritiated leucine uptake and changes in bacterial numbers in bacterivore-free incubations and by direct measurements of DOM loss. The alternating cycles of sunlight and microbial activity resulted in more complete degradation of bulk DOM and marine humic substances than was found for nonirradiated controls (i.e. with microbial activity alone) by a factor of up to 3-fold. Increased decomposition was due both to direct losses of carbon gas photoproducts (DIC and CO in a 15 : 1 ratio) and to enhanced microbial degradation of photodegraded DOM, with approximately equal contributions from each pathway. Mass balance calculations indicated that low-molecular-weight carbon photoproducts, currently considered to be the compounds responsible for stimulating bacterial activity following photodegradation of DOM, were insufficient to account for the enhanced bacterial production observed. Thus, higher molecular weight, chemically uncharacterized fractions of DOM may also be modified to more biologically available forms during exposure to natural sunlight. Photochemical processes play a number of important roles in the biogeochemical cycling of dissolved organic matter (DOM) in natural waters. Water optical properties, biological processes, and trace element distributions have all been demonstrated to be affected, either directly or indirectly, by

Two numerically important bacteria in marine pulp mill effluent enrichment cultures were isolated. These organisms were gram-negative, rod-shaped, strictly aerobic bacteria. Isolate IRE-31T (T = type strain) produced hydrolytic enzymes for the breakdown of cellulose, xylan, chitin, gelatin, and Tween 80. It also utilized a variety of monosaccharides, disaccharides, amino acids, and volatile fatty acids for growth. Isolate KW-40T did not utilize natural polymers, but it could grow on a variety of monosaccharides, disaccharides, alcohols, and amino acids. It also utilized methanol and aromatic compounds. The surfaces of both organisms were covered by blebs and vesicles. 16S rRNA analyses placed both organisms in the gamma-3 subclass of the phylum Proteobacteria. They were related to Oceanospirillum linum, Marinomonas vaga, Pseudomonas putida, and Halomonas elongata, although a close association with any of these bacteria was not found. The guanine-plus-cytosine contents of strain IRE-31T and KW-40T were 57.6 and 54.9 mol%, respectively. On the basis of 16S rRNA sequence and phenotypic characterizations, these isolates were different enough so that they could be considered members of new genera. Thus, the following two new genera and species are proposed: Microbulbifer hydrolyticus, with type strain IRE-31 (= ATCC 700072), and Marinobacterium georgiense, with type strain KW-40 (= ATCC 700074).

To better understand the functional responses in prokaryotes to dissolved organic matter (DOM), we compared the transcriptional pattern of natural prokaryotic communities grown in continuous cultures on seawater amended with phytoplankton-derived DOM. Metatranscriptomic reads were classified taxonomically (by genomic binning) and functionally (using Kyoto Encyclopedia of Genes and Genomes), and the relative gene expression of individual taxa (genome bins) was compared with the total community response. In the first experiment comparing seawater and seawater amended with diatom-derived DOM, metatranscriptomes revealed pronounced differences in pathways involved in carbohydrate and lipid metabolism. In the second experiment comparing seawater amended with cyanobacteria- and diatom-derived DOM, metatranscriptomes had similar functional profiles, likely reflecting more similar DOM regimes in this experimental setup. Among the five most abundant taxa investigated in more detail, two featured pronounced differences in transcript abundance between treatments suggesting that they were specialized in the use of only one of the two DOM regimes. However, these two taxa were less involved in carbohydrate metabolism than others and had few genes that were significantly regulated in response to the DOM source. Our results indicate that both substrate composition and the competitive interplay of community members were decisive for the functional response of a microbial system.

Previous studies based on analysis of amoA, 16S ribosomal RNA or accA gene sequences have established that marine Thaumarchaeota fall into two phylogenetically distinct groups corresponding to shallow- and deep-water clades, but it is not clear how water depth interacts with other environmental factors, including light, temperature and location, to affect this pattern of diversification. Earlier studies focused on single-gene distributions were not able to link phylogenetic structure to other aspects of functional adaptation. Here, we analyzed the genome content of 46 uncultivated single Thaumarchaeota cells sampled from epi- and mesopelagic waters of subtropical, temperate and polar oceans. Phylogenomic analysis showed that populations diverged by depth, as expected, and that mesopelagic populations from different locations were well mixed. Functional analysis showed that some traits, including putative DNA photolyase and catalase genes that may be related to adaptive mechanisms to reduce light-induced damage, were found exclusively in members of the epipelagic clade. Our analysis of partial genomes has thus confirmed the depth differentiation of Thaumarchaeota populations observed previously, consistent with the distribution of putative mechanisms to reduce light-induced damage in shallow- and deep-water populations.

Next generation sequencing-enabled metatranscriptomic and metagenomic datasets are providing unprecedented insights into the functional diversity of microbial communities, allowing detection of the genes present in a community as well as differentiation of those being actively transcribed. An emerging challenge of meta-omics approaches is how to quantitatively compare metagenomes and metatranscriptomes collected across spatial and temporal scales, or among treatments in experimental manipulations. Here, we describe the use of internal DNA and mRNA standards in meta-omics methodologies, and highlight how data collected in an absolute framework (per L or per cell) provides increased comparative power and insight into underlying causes of differences between samples.

A fundamental question in marine microbial ecology is how microbes adapt to ocean environments. Although numerically dominant populations are typically considered more successful, higher census population sizes (Nc) do not equate directly to a greater capability for adaptation. Instead, effective population size (Ne) determines the fate of deleterious and favourable mutations, and thus is a key parameter for determining the adaptive potential of a population. In the case of the SAR11 and Roseobacter lineages, two abundant heterotrophic bacteria in ocean surface waters with contrasting life history strategies, culture-independent population surveys suggest that SAR11s have greater Nc than Roseobacters. To determine relative Ne, we compared the ratio of nonsynonymous to synonymous substitution rates (ω) of recently diverged lineages of these taxa. Values of ω associated with several of the Roseobacter subclades were lower than for SAR11 subclades, suggesting greater Ne in these cases. Most Roseobacter lineages also had smaller ω values compared with an atypical basal Roseobacter lineage with a large Nc. This finding provides insight into variability in Ne across two important marine bacterial lineages, and provides an evolutionary context for considering how heterotrophic marine bacteria may differ in their ability to adapt to changing ocean habitats.

Marine bacteria in the Roseobacter and SAR11 lineages successfully exploit the ocean habitat, together accounting for ~40% of bacteria in surface waters, yet have divergent life histories that exemplify patch-adapted versus free-living ecological roles. Here, we use a phylogenetic birth-and-death model to understand how genome content supporting different life history strategies evolved in these related alphaproteobacterial taxa, showing that the streamlined genomes of free-living SAR11 were gradually downsized from a common ancestral genome only slightly larger than the extant members (~2,000 genes), while the larger and variably sized genomes of roseobacters evolved along dynamic pathways from a sizeable common ancestor (~8,000 genes). Genome changes in the SAR11 lineage occurred gradually over ~800 million years, whereas Roseobacter genomes underwent more substantial modifications, including major periods of expansion, over ~260 million years. The timing of the first Roseobacter genome expansion was coincident with the predicted radiation of modern marine eukaryotic phytoplankton of sufficient size to create nutrient-enriched microzones and is consistent with present-day ecological associations between these microbial groups. We suggest that diversification of red-lineage phytoplankton is an important driver of divergent life history strategies among the heterotrophic bacterioplankton taxa that dominate the present-day ocean., IMPORTANCE One-half of global primary production occurs in the oceans, and more than half of this is processed by heterotrophic bacterioplankton through the marine microbial food web. The diversity of life history strategies that characterize different bacterioplankton taxa is an important subject, since the locations and mechanisms whereby bacteria interact with seawater organic matter has effects on microbial growth rates, metabolic pathways, and growth efficiencies, and these in turn affect rates of carbon mineralization to the atmosphere and sequestration into the deep sea. Understanding the evolutionary origins of the ecological strategies that underlie biochemical interactions of bacteria with the ocean system, and which scale up to affect globally important biogeochemical processes, will improve understanding of how microbial diversity is maintained and enable useful predictions about microbial response in the future ocean.

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.

The vast majority of surface ocean bacteria are uncultivated. Compared with their cultured relatives, they frequently exhibit a streamlined genome, reduced G+C content and distinct gene repertoire. These genomic traits are relevant to environmental adaptation, and have generally been thought to become fixed in marine bacterial populations through selection. Using single-cell genomics, we sequenced four uncultivated cells affiliated with the ecologically relevant Roseobacter clade and used a composition-heterogeneous Bayesian phylogenomic model to resolve these single-cell genomes into a new clade. This lineage has no representatives in culture, yet accounts for ∼35% of Roseobacters in some surface ocean waters. Analyses of multiple genomic traits, including genome size, G+C content and percentage of noncoding DNA, suggest that these single cells are representative of oceanic Roseobacters but divergent from isolates. Population genetic analyses showed that substitution of physicochemically dissimilar amino acids and replacement of G+C-rich to G+C-poor codons are accelerated in the uncultivated clade, processes that are explained equally well by genetic drift as by the more frequently invoked explanation of natural selection. The relative importance of drift vs selection in this clade, and perhaps in other marine bacterial clades with streamlined G+C-poor genomes, remains unresolved until more evidence is accumulated.

Swan, Brandon K. ... et al.-- 6 pages, 4 figures.-- Data deposition: Whole-genome sequence data for single amplified genomes used for our analyses are available in the Joint Genome Institute’s Integrated Microbial Genome database, http://img.jgi.doe.gov/cgi-bin/w/main.cgi (accession nos. 643886079, 643886118, 2228664025-26, 2228664028-29, 2228664032, 2228664034, 2228664052-53, 2228664055-56, 2236347001, 2236347003, 2236347013, 2236347015, 2236347017-19, 2236347021-24, 2236347026-27, 2236347030-33, 2236347035-36, 2236347039, 2236347041, 2236347043, 2236661010, 2236661014, 2236661017-18, 2507262045, 2507262047, and 2517572139).-- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1304246110/-/DCSupplemental, 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, freeliving 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, This work was supported by National Science Foundation Grants EF-826924 (to R.S.), OCE-821374 (to R.S.), and OCE-1232982 (to R.S. and B.K.S.); US Department of Energy (DOE) JGI 2011 Microbes Program Grant CSP 387 (to R.S., B.K.S., S.G., M.A.M., F.M.L., R.C. and S.G.A.); the Gordon and Betty Moore Foundation (M.A.M.); Spanish Ministry of Science and Innovation Grant CGL2011-26848/BOS (to S.G.A) and CONSOLIDER-INGENIO2010 Program Grant CSD2008-00077 (to S.G.A. and J.M.G.); the Natural Sciences and Engineering Research Council of Canada (NSERC); the Canada Foundation for Innovation, and the Canadian Institute for Advanced Research (CIFAR; S.J.H.). J.J.W. was supported by NSERC. Research activities of R.C. and F.M.L. are supported by the Australian Research Council, and research activities of R.C. are supported by the Australian Antarctic Science program. Work conducted by the DOE Joint Genome Institute is supported by the DOE’s Office of Science under Contract DE-AC02-05CH11231. This is contribution no. 006 of the Tara Oceans Expedition 2009–2012