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Microbial biofilms play critical roles in marine ecosystems and are hot spots for microbial interactions that play a role in the development and function of these communities. Roseobacteraceae are an abundant and active family of marine heterotrophic bacteria forming close associations with phytoplankton and carrying out key transformations in biogeochemical cycles. Group members are aggressive primary colonizers of surfaces, where they set the stage for the development of multispecies biofilm communities.

Viruses that infect microbial hosts have traditionally been studied in laboratory settings with a focus on either obligate lysis or persistent lysogeny. In the environment, these infection archetypes are part of a continuum that spans antagonistic to beneficial modes. In this Review, we advance a framework to accommodate the context-dependent nature of virus–microorganism interactions in ecological communities by synthesizing knowledge from decades of virology research, eco-evolutionary theory and recent technological advances. We discuss that nuanced outcomes, rather than the extremes of the continuum, are particularly likely in natural communities given variability in abiotic factors, the availability of suboptimal hosts and the relevance of multitrophic partnerships. We revisit the ‘rules of life’ in terms of how long-term infections shape the fate of viruses and microbial cells, populations and ecosystems. In this Review, Correa and colleagues revisit the rules of life for viruses of microorganisms by advancing a conceptual framework that recognizes virus–host interactions across a continuum of infection modalities and by examining the influence of these modalities on viruses, their hosts and ecosystems.

In the oceans, viruses that infect bacteria (phages) influence a variety of microbially mediated processes that drive global biogeochemical cycles. The nature of their influence is dependent upon infection mode, be it lytic or lysogenic. Temperate phages are predicted to be prevalent in marine systems where they are expected to execute both types of infection modes. Understanding the range and outcomes of temperate phage-host interactions is fundamental for evaluating their ecological impact. Here, we (i) review phage-mediated rewiring of host metabolism, with a focus on marine systems, (ii) consider the range and nature of temperate phage-host interactions, and (iii) draw on studies of cultivated model systems to examine the consequences of lysogeny among several dominant marine bacterial lineages. We also readdress the prevalence of lysogeny among marine bacteria by probing a collection of 1239 publicly available bacterial genomes, representing cultured and uncultivated strains, for evidence of complete prophages. Our conservative analysis, anticipated to underestimate true prevalence, predicts 18% of the genomes examined contain at least one prophage, the majority (97%) were found within genomes of cultured isolates. These results highlight the need for cultivation of additional model systems to better capture the diversity of temperate phage-host interactions in the oceans.

Microbial sulfur metabolism contributes to biogeochemical cycling on global scales. Sulfur metabolizing microbes are infected by phages that can encode auxiliary metabolic genes (AMGs) to alter sulfur metabolism within host cells but remain poorly characterized. Here we identified 191 phages derived from twelve environments that encoded 227 AMGs for oxidation of sulfur and thiosulfate (dsrA, dsrC/tusE, soxC, soxD and soxYZ). Evidence for retention of AMGs during niche-differentiation of diverse phage populations provided evidence that auxiliary metabolism imparts measurable fitness benefits to phages with ramifications for ecosystem biogeochemistry. Gene abundance and expression profiles of AMGs suggested significant contributions by phages to sulfur and thiosulfate oxidation in freshwater lakes and oceans, and a sensitive response to changing sulfur concentrations in hydrothermal environments. Overall, our study provides fundamental insights on the distribution, diversity, and ecology of phage auxiliary metabolism associated with sulfur and reinforces the necessity of incorporating viral contributions into biogeochemical configurations., Some bacteriophage encode auxiliary metabolic genes (AMGs) that impact host metabolism and biogeochemical cycling during infection. Here the authors identify hundreds of AMGs in environmental phage encoding sulfur oxidation genes and use their global distribution to infer phage-mediated biogeochemical impacts.

The hydroxycinnamates (HCAs) ferulate and p-coumarate are among the most abundant constituents of lignin, and their degradation by bacteria is an essential step in the remineralization of vascular plant material. Here, we investigate the catabolism of these two HCAs by the marine bacterium Sagittula stellata E-37, a member of the roseobacter lineage with lignolytic potential. Bacterial degradation of HCAs is often initiated by the activity of a hydroxycinnamoyl-coenzyme A (hydroxycinnamoyl-CoA) synthase. Genome analysis of S. stellata revealed the presence of two feruloyl-CoA (fcs) synthase homologs, an unusual occurrence among characterized HCA degraders. In order to elucidate the role of these homologs in HCA catabolism, fcs-1 and fcs-2 were disrupted using insertional mutagenesis, yielding both single and double fcs mutants. Growth on p-coumarate was abolished in the fcs double mutant, whereas maximum cell yield on ferulate was only 2% of that of the wild type. Interestingly, the single mutants demonstrated opposing phenotypes, where the fcs-1 mutant showed impaired growth (extended lag and ∼60% of wild-type rate) on p-coumarate, and the fcs-2 mutant showed impaired growth (extended lag and ∼20% of wild-type rate) on ferulate, pointing to distinct but overlapping roles of the encoded fcs homologs, with fcs-1 primarily dedicated to p-coumarate utilization and fcs-2 playing a dominant role in ferulate utilization. Finally, a tripartite ATP-independent periplasmic (TRAP) family transporter was found to be required for growth on both HCAs. These findings provide evidence for functional redundancy in the degradation of HCAs in S. stellata E-37 and offer important insight into the genetic complexity of aromatic compound degradation in bacteria. IMPORTANCE Hydroxycinnamates (HCAs) are essential components of lignin and are involved in various plant functions, including defense. In nature, microbial degradation of HCAs is influential to global carbon cycling. HCA degradation pathways are also of industrial relevance, as microbial transformation of the HCA, ferulate, can generate vanillin, a valuable flavoring compound. Yet, surprisingly little is known of the genetics underlying bacterial HCA degradation. Here, we make comparisons to previously characterized bacterial HCA degraders and use a genetic approach to characterize genes involved in catabolism and uptake of HCAs in the environmentally relevant marine bacterium Sagittula stellata. We provide evidence of overlapping substrate specificity between HCA degradation pathways and uptake proteins. We conclude that S. stellata is uniquely poised to utilize HCAs found in the complex mixtures of plant-derived compounds in nature. This strategy may be common among marine bacteria residing in lignin-rich coastal waters and has potential relevance to biotechnology sectors.

The marine roseobacter Phaeobacter sp. strain Y4I synthesizes the blue antimicrobial secondary metabolite indigoidine when grown in a biofilm or on agar plates. Prior studies suggested that indigoidine production may be, in part, regulated by cell-to-cell communication systems. Phaeobacter sp. strain Y4I possesses two luxR and luxI homologous N -acyl- l -homoserine lactone (AHL)-mediated cell-to-cell communication systems, designated pgaRI and phaRI . We show here that Y4I produces two dominant AHLs, the novel monounsaturated N -(3-hydroxydodecenoyl)- l -homoserine lactone (3OHC 12:1 -HSL) and the relatively common N -octanoyl- l -homoserine lactone (C 8 -HSL), and provide evidence that they are synthesized by PhaI and PgaI, respectively. A Tn 5 insertional mutation in either genetic locus results in the abolishment ( pgaR ::Tn 5 ) or reduction ( phaR ::Tn 5 ) of pigment production. Motility defects and denser biofilms were also observed in these mutant backgrounds, suggesting an overlap in the functional roles of these systems. Production of the AHLs occurs at distinct points during growth on an agar surface and was determined by isotope dilution high-performance liquid chromatography–tandem mass spectrometry (ID-HPLC-MS/MS) analysis. Within 2 h of surface inoculation, only 3OHC 12:1 -HSL was detected in agar extracts. As surface-attached cells became established (at ∼10 h), the concentration of 3OHC 12:1 -HSL decreased, and the concentration of C 8 -HSL increased rapidly over 14 h. After longer (>24-h) establishment periods, the concentrations of the two AHLs increased to and stabilized at ∼15 nM and ∼600 nM for 3OHC 12:1 -HSL and C 8 -HSL, respectively. In contrast, the total amount of indigoidine increased steadily from undetectable to 642 μM by 48 h. Gene expression profiles of the AHL and indigoidine synthases ( pgaI , phaI , and igiD ) were consistent with their metabolite profiles. These data provide evidence that pgaRI and phaRI play overlapping roles in the regulation of indigoidine biosynthesis, and it is postulated that this allows Phaeobacter sp. strain Y4I to coordinate production of indigoidine with different growth-phase-dependent physiologies.

Marine phytoplankton blooms are annual spring events that sustain active and diverse bloom-associated bacterial populations. Blooms vary considerably in terms of eukaryotic species composition and environmental conditions, but a limited number of heterotrophic bacterial lineages - primarily members of the Flavobacteriia, Alphaproteobacteria and Gammaproteobacteria - dominate these communities. In this Review, we discuss the central role that these bacteria have in transforming phytoplankton-derived organic matter and thus in biogeochemical nutrient cycling. On the basis of selected field and laboratory-based studies of flavobacteria and roseobacters, distinct metabolic strategies are emerging for these archetypal phytoplankton-associated taxa, which provide insights into the underlying mechanisms that dictate their behaviours during blooms.

Dissolved organic matter (DOM) in the oceans is one of the largest pools of reduced carbon on Earth, comparable in size to the atmospheric CO 2 reservoir. A vast number of compounds are present in DOM, and they play important roles in all major element cycles, contribute to the storage of atmospheric CO 2 in the ocean, support marine ecosystems, and facilitate interactions between organisms. At the heart of the DOM cycle lie molecular-level relationships between the individual compounds in DOM and the members of the ocean microbiome that produce and consume them. In the past, these connections have eluded clear definition because of the sheer numerical complexity of both DOM molecules and microorganisms. Emerging tools in analytical chemistry, microbiology, and informatics are breaking down the barriers to a fuller appreciation of these connections. Here we highlight questions being addressed using recent methodological and technological developments in those fields and consider how these advances are transforming our understanding of some of the most important reactions of the marine carbon cycle.

Members of the Roseobacter lineage of marine bacteria are prolific surface colonizers in marine coastal environments, and antimicrobial secondary metabolite production has been hypothesized to provide a competitive advantage to colonizing roseobacters. Here, we report that the roseobacter Phaeobacter sp. strain Y4I produces the blue pigment indigoidine via a nonribosomal peptide synthase (NRPS)-based biosynthetic pathway encoded by a novel series of genetically linked genes: igiBCDFE . A Tn 5 -based random mutagenesis library of Y4I showed a perfect correlation between indigoidine production by the Phaeobacter strain and inhibition of Vibrio fischeri on agar plates, revealing a previously unrecognized bioactivity of this molecule. In addition, igiD null mutants (igiD encoding the indigoidine NRPS) were more resistant to hydrogen peroxide, less motile, and faster to colonize an artificial surface than the wild-type strain. Collectively, these data provide evidence for pleiotropic effects of indigoidine production in this strain. Gene expression assays support phenotypic observations and demonstrate that igiD gene expression is upregulated during growth on surfaces. Furthermore, competitive cocultures of V. fischeri and Y4I show that the production of indigoidine by Y4I significantly inhibits colonization of V. fischeri on surfaces. This study is the first to characterize a secondary metabolite produced by an NRPS in roseobacters.

Determining in situ growth rates for specific bacterioplankton is of critical importance to understanding their contributions to energy and matter flow in the Ocean. Quantifying expression of genes central to cell division is a plausible approach for obtaining these measurements. In order to test this approach's assumptions, a quantitative PCR assay targeting the cell division gene ftsZ in the ubiquitous NAC11-7 group of the Rhodobacterales order of marine bacteria was developed. ftsZ genes and their corresponding mRNAs were measured in diel in situ samples and in parallel on-deck incubations. Strong correlations between ftsZ expression and gene abundance (R-squared = 0.62) were observed in situ. Rapid changes in NAC11-7 ftsZ gene copies suggested that different populations from different water types were sampled with a significant positive correlation between ftsZ expression and water temperature (R-squared = 0.68, P < 0.001). An outlier to this trend occurred at a single time point (9:00), which was remarkably consistent with a concomitant peak in ftsZ expression in on-deck incubations, suggesting the possibility of synchronous population growth.

Microbes play critical roles in the biogeochemical cycling of nitrogen and sulfur in aquatic environments. Here we investigated the interaction between the naturally occurring organic sulfur compound methanethiol (MeSH) and the final step of the denitrification pathway, the reduction of nitrous oxide (N2 O) to dinitrogen (N2 ) gas, in sediment slurries from the temperate Douro and Ave estuaries (NW Portugal) and in pure cultures of the marine bacterium Ruegeria pomeroyi. Sediment slurries and cell suspensions were amended with a range of concentrations of either MeSH (0-120 µM) or methionine (0-5 mM), a known precursor of MeSH. MeSH or methionine additions caused N2 O to accumulate and this accumulation was linearly related to MeSH concentrations in both coastal sediments (R(2) = 0.7-0.9, P

Summary The contribution of chemotaxis to the competitive colonization of the rhizosphere for the vast majority of the soil community is unknown. We have developed and applied a molecular diagnostic tool, based on a gene encoding the central regulator of bacterial chemotaxis (cheA), to characterize and temporally track specific populations of native microbes with chemotaxis potential that are present in soil exposed to two rhizospheres: wheat and cowpea. The data show that the chemotactic-competent communities present in the rhizospheres of the two plants are distinct and less diverse than the bulk soil, indicating the development of unique microbial communities. Consistent with the supposition that selection and recruitment of specific soil microbes takes place in the rhizosphere, the dynamics of specific cheA phylotypes provides support for the hypothesis that chemotaxis provides a competitive advantage to some soil microbes. This is the first study to examine and profile the genetic diversity of chemotaxis genes in natural populations. As such, it illustrates our limited understanding of microbial chemotaxis for the majority of soil microbes. It also highlights the value of a culture-independent approach for examining chemotaxis populations in order to build empirical lines of evidence for its role in structuring of microbial assemblages.

Genes encoding gene transfer agent (GTA) particles are well conserved in bacteria of the order Rhodobacterales. Members of this order are abundant in diverse marine environments, fre- quently accounting for as much as 25% of the total bacterial community. Conservation of the genes encoding GTAs allows their use as diagnostic markers of Rhodobacterales in biogeographical stud- ies. The first survey of the diversity of Rhodobacterales based on the GTA major capsid gene was con- ducted in a warm temperate estuarine ecosystem, the Chesapeake Bay, but the biogeography of Rhodobacterales has not been explored extensively. This study investigates Rhodobacterales diver- sity in the cold subarctic water near Newfoundland, Canada. Our results suggest that the subarctic region of the North Atlantic contains diverse Rhodobacterales communities in both winter and sum- mer, and that the diversity of the Rhodobacterales community in the summer Newfoundland coastal water is higher than that found in the Chesapeake Bay, in either the summer or winter. Approxi- mately one-third of GTA sequences retrieved from the subarctic waters were most closely related to those from bacteria isolated from sea ice or cold regions. Distinguishable diversity patterns were found between the temperate and subarctic waters, providing further support for niche adaptation of specific Rhodobacterales members to unique environments. We also demonstrate that a number of Rhodobacterales strains, from both the subarctic and temperate locations, express the GTA major capsid protein. This provides robust evidence that the widespread conservation of GTA genes in the Rhodobacterales may result in the production of functionally similar and active GTA systems in these bacteria in different environments.

Within the bacterial class Alphaproteobacteria, the order Rhodobacterales contains the Roseobacter and Rhodobacter clades. Roseobacters are abundant and play important biogeochemical roles in marine environments. Roseobacter and Rhodobacter genomes contain a conserved gene transfer agent (GTA) gene cluster, and GTA-mediated gene transfer has been observed in these groups of bacteria. In this study, we investigated the genetic diversity of these two groups in Chesapeake Bay surface waters using a specific PCR primer set targeting the conserved Rhodobacterales GTA major capsid protein gene (g5). The g5 gene was successfully amplified from 26 Rhodobacterales isolates and the bay microbial communities using this primer set. Four g5 clone libraries were constructed from microbial assemblages representing different regions and seasons of the bay and yielded diverse sequences. In total, 12 distinct g5 clusters could be identified among 158 Chesapeake Bay clones, 11 fall within the Roseobacter clade, and one falls in the Rhodobacter clade. The vast majority of the clusters (10 out of 12) lack cultivated representatives. The composition of g5 sequences varied dramatically along the bay during the wintertime, and a distinct Roseobacter population composition between winter and summer was observed. The congruence between g5 and 16S rRNA gene phylogenies indicates that g5 may serve as a useful genetic marker to investigate diversity and abundance of Roseobacter and Rhodobacter in natural environments. The presence of the g5 gene in the natural populations of Roseobacter and Rhodobacter implies that genetic exchange through GTA transduction could be an important mechanism for maintaining the metabolic flexibility of these groups of bacteria.

Marine viruses are critical drivers of ocean biogeochemistry, and their abundances vary spatiotemporally in the global oceans, with upper estimates exceeding 108 per ml. Over many years, a consensus has emerged that virus abundances are typically tenfold higher than microbial cell abundances. However, the true explanatory power of a linear relationship and its robustness across diverse ocean environments is unclear. Here, we compile 5,508 microbial cell and virus abundance estimates from 22 distinct marine surveys and find substantial variation in the virus-to-microbial cell ratio, in which a 10:1 model has either limited or no explanatory power. Instead, virus abundances are better described as nonlinear, power-law functions of microbial cell abundances. The fitted scaling exponents are typically less than 1, implying that the virus-to-microbial cell ratio decreases with microbial cell density, rather than remaining fixed. The observed scaling also implies that viral effect sizes derived from ‘representative’ abundances require substantial refinement to be extrapolated to regional or global scales. Analysis of microbial cell and virus abundance estimates from 25 distinct marine surveys reveals that virus-to-microbial cell ratio decreases with microbial cell density, questioning the idea that viral abundance is always 10-fold higher.

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.

Marine viruses are critical drivers of ocean biogeochemistry and their abundances vary spatiotem- porally in the global oceans, with upper estimates exceeding 10 8 per ml. Over many years, a con- sensus has emerged that virus abundances are typically 10-fold higher than prokaryote abundances. The use of a fixed-ratio suggests that the relationship between virus and prokaryote abundances is both predictable and linear. However, the true explanatory power of a linear relationship and its robustness across diverse ocean environments is unclear. Here, we compile 5671 prokaryote and virus abundance estimates from 25 distinct marine surveys to characterize the relationship between virus and prokaryote abundances. We find that the median virus-to-prokaryote ratio (VPR) is 10:1 and 16:1 in the near- and sub-surface oceans, respectively. Nonetheless, we observe substantial variation in the VPR and find either no or limited explanatory power using fixed-ratio models. Instead, virus abundances are better described as nonlinear, power-law functions of prokaryote abundances - par- ticularly when considering relationships within distinct marine surveys. Estimated power-laws have scaling exponents that are typically less than 1, signifying that the VPR decreases with prokaryote density, rather than remaining fixed. The emergence of power-law scaling presents a challenge for mechanistic models seeking to understand the ecological causes and consequences of marine virus- microbe interactions. Such power-law scaling also implies that efforts to average viral effects on microbial mortality and biogeochemical cycles using “representative” abundances or abundance- ratios need to be refined if they are to be utilized to make quantitative predictions at regional or global ocean scales.

Coliphage N4 is a lytic bacteriophage discovered nearly half a century ago, and it was considered to be a “genetic orphan” until very recently, when several additional N4-like phages were discovered to infect nonenteric bacterial hosts. Interest in this genus of phages is stimulated by their unique genetic features and propagation strategies. To better understand the ecology of N4-like phages, we investigated the diversity and geographic patterns of N4-like phages by examining 56 Chesapeake Bay viral communities, using a PCR-clone library approach targeting a diagnostic N4-like DNA polymerase gene. Many new lineages of N4-like phages were found in the bay, and their genotypes shift from the lower to the upper bay. Interestingly, signature sequences of N4-like phages were recovered only from winter month samples, when water temperatures were below 4°C. An analysis of existing metagenomic libraries from various aquatic environments supports the hypothesis that N4-like phages are most prolific in colder waters. In particular, a high number of N4-like phages were detected in Organic Lake, Antarctica, a cold and hypersaline system. The prevalence of N4-like phages in the cold biosphere suggests these viruses possess yet-to-be-determined mechanisms that facilitate lytic infections under cold conditions.

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”).

Viral lysis of microbial hosts releases organic matter that can then be assimilated by nontargeted microorganisms. Quantitative estimates of virus-mediated recycling of carbon in marine waters, first established in the late 1990s, were originally extrapolated from marine host and virus densities, host carbon content and inferred viral lysis rates. Yet, these estimates did not explicitly incorporate the cascade of complex feedbacks associated with virus-mediated lysis. To evaluate the role of viruses in shaping community structure and ecosystem functioning, we extend dynamic multitrophic ecosystem models to include a virus component, specifically parameterized for processes taking place in the ocean euphotic zone. Crucially, we are able to solve this model analytically, facilitating evaluation of model behavior under many alternative parameterizations. Analyses reveal that the addition of a virus component promotes the emergence of complex communities. In addition, biomass partitioning of the emergent multitrophic community is consistent with well-established empirical norms in the surface oceans. At steady state, ecosystem fluxes can be probed to characterize the effects that viruses have when compared with putative marine surface ecosystems without viruses. The model suggests that ecosystems with viruses will have (1) increased organic matter recycling, (2) reduced transfer to higher trophic levels and (3) increased net primary productivity. These model findings support hypotheses that viruses can have significant stimulatory effects across whole-ecosystem scales. We suggest that existing efforts to predict carbon and nutrient cycling without considering virus effects are likely to miss essential features of marine food webs that regulate global biogeochemical cycles.

In marine environments, virus-mediated lysis of host cells leads to the release of cellular carbon and nutrients and is hypothesized to be a major driver of carbon recycling on a global scale. However, efforts to characterize the effects of viruses on nutrient cycles have overlooked the geochemical potential of the virus particles themselves, particularly with respect to their phosphorus content. In this Analysis article, we use a biophysical scaling model of intact virus particles that has been validated using sequence and structural information to quantify differences in the elemental stoichiometry of marine viruses compared with their microbial hosts. By extrapolating particle-scale estimates to the ecosystem scale, we propose that, under certain circumstances, marine virus populations could make an important contribution to the reservoir and cycling of oceanic phosphorus.

Specific SYBR green-based quantitative-PCR assays targeting conserved regions in the 16S-23S rRNA internal transcribed spacer regions were developed for five subgroups of the environmentally abundant and biogeochemically active Roseobacter clade of marine bacteria. The assays were applied to field samples demonstrating their utility in investigations of abundant Roseobacter group phylotypes in the environment.

The Roseobacter clade is a broadly distributed, abundant, and biogeochemically relevant group of marine bacteria. Representatives are often associated with organic surfaces in disparate marine environments, suggesting that a sessile lifestyle is central to the ecology of lineage members. The importance of surface association and colonization has been demonstrated recently for select strains, and it has been hypothesized that production of antimicrobial agents, cell density-dependent regulatory mechanisms, and morphological features contribute to the colonization success of roseobacters. Drawing on these studies, insight into a broad representation of strains is facilitated by the availability of a substantial collection of genome sequences that provides a holistic view of these features among clade members. These genome data often corroborate phenotypic data but also reveal significant variation in terms of gene content and synteny among group members, even among closely related strains (congeners and conspecifics). Thus, while detailed studies of representative strains are serving as models for how roseobacters transition between planktonic and sessile lifestyles, it is becoming clear that additional studies are needed if we are to have a more comprehensive view of how these transitions occur in different lineage members. This is important if we are to understand how associations with surfaces influence metabolic activities contributing to the cycling of carbon and nutrients in the world's oceans.

Random PCR mutagenesis is a powerful tool for structure-function analysis of targeted proteins, especially when coupled with DNA integration through natural transformation followed by selection for loss of function. The technique has been applied successfully to structure-function analysis of transcriptional regulators, enzymes, and transporters in Acinetobacter sp. strain ADP1. However, the mismatch repair system prevents the full spectrum of nucleotide substitutions that may be selected at the level of protein function from being recovered. This barrier may be overcome by introducing PCR-mutagenized genes into strains in which the corresponding genes have been deleted.

Despite the overwhelming bacterial diversity present in the world's oceans, the majority of recognized marine bacteria fall into as few as nine major clades ([36][1]), many of which have yet to be cultivated in the laboratory. Molecular-based approaches targeting 16S rRNA genes demonstrate that the

We analyzed gene expression in marine and freshwater bacterioplankton communities by the direct retrieval and analysis of microbial transcripts. Environmental mRNA, obtained from total RNA by subtractive hybridization of rRNA, was reverse transcribed, amplified with random primers, and cloned. Approximately 400 clones were analyzed, of which ∼80% were unambiguously mRNA derived. mRNAs appeared to be from diverse taxonomic groups, including both Bacteria (mainly α- and γ- Proteobacteria ) and Archaea (mainly Euryarchaeota ). Many transcripts could be linked to environmentally important processes such as sulfur oxidation ( soxA ), assimilation of C1 compounds ( fdh1B ), and acquisition of nitrogen via polyamine degradation ( aphA ). Environmental transcriptomics is a means of exploring functional gene expression within natural microbial communities without bias toward known sequences, and provides a new approach for obtaining community-specific variants of key functional genes.

Chitin is an abundant biopolymer whose degradation is mediated primarily by bacterial chitinases. We developed a degenerate PCR primer set to amplify a ∼900-bp fragment of family 18, group I chitinase genes and used it to retrieve these gene fragments from environmental samples. Clone libraries of presumptive chitinase genes were created for nine water and six sediment samples from 10 aquatic environments including freshwater and saline lakes, estuarine water and sediments, and the central Arctic Ocean. Putative chitinase sequences were also retrieved from the Sargasso Sea metagenome sequence database. We were unable to obtain PCR product with these primers from an alkaline, hypersaline lake (Mono Lake, California). In total, 108 partial chitinase gene sequences were analyzed, with a minimum of 5 and a maximum of 13 chitinase sequences obtained from each library. All chitinase sequences were novel compared to previously identified sequences. Intralibrary sequence diversity was low, while we found significant differences between libraries from different water column samples and between water column and sediment samples. However, identical sequences were retrieved from samples collected at widely distributed locations that did not necessarily represent similar environments, suggesting homogeneity of chitinoclastic communities between some environments.

The diversity of ascomycete laccase sequences was surveyed in a southeastern US salt marsh using a degenerate primer set designed around copper binding sites conserved in fungal laccases. This gene was targeted for diversity analysis because of its potential function in lignin degradation in the salt marsh ecosystem and because few studies have assessed functional gene diversity in natural fungal communities. Laccase sequences were amplified from genomic DNA extracted from 24 isolates (representing 10 ascomycete species) cultured from decaying blades of Spartina alterniflora, and from DNA extracted directly from the decaying blades. Among the ascomycete isolates, 21 yielded a PCR product of expected size (˜900 bp) that was tentatively identified as laccase based on sequence similarities to previously published laccase sequences from related organisms. Overall, 13 distinct sequence types, containing 39 distinct sequences, were identified among the isolates, with several species yielding multiple distinct laccase types. PCR amplifications from early and late decay blades of S. alterniflora yielded seven laccase types. Of these, five were composed of sequences >96% similar at the amino acid level to sequences from three cultured ascomycetes previously found to be dominant members of the fungal communities on decaying S. alterniflora blades. Two of the laccase types from the natural-decay clone library were novel and did not match any of the sequences obtained from the cultured ascomycetes. The 39 distinct sequences and 15 distinct laccase sequence types retrieved from the S. alterniflora decay system demonstrate high sequence diversity of this functional gene in a natural fungal community.

Degradation of lignin-related aromatic compounds is an important ecological process in the highly productive salt marshes of the southeastern United States, yet little is known about the mediating organisms or their catabolic pathways. Here we report the diversity of a gene encoding a key ring-cleaving enzyme of the β-ketoadipate pathway, pcaH , amplified from bacterial communities associated with decaying Spartina alterniflora , the salt marsh grass that dominates these coastal systems, as well as from enrichment cultures with aromatic substrates ( p -hydroxybenzoate, anthranilate, vanillate, and dehydroabietate). Sequence analysis of 149 pcaH clones revealed 85 unique sequences. Thirteen of the 53 amino acid residues compared were invariant in the PcaH proteins, suggesting that these residues have a required catalytic or structural function. Fifty-eight percent of the clones matched sequences amplified from a collection of 36 bacterial isolates obtained from seawater, marine sediments, or senescent Spartina . Fifty-two percent of the pcaH clones could be assigned to the roseobacter group, a marine lineage of the class α- Proteobacteria abundant in coastal ecosystems. Another 6% of the clones matched genes retrieved from isolates belonging to the genera Acinetobacter, Bacillus , and Stappia , and 42% of the clones could not be assigned to a cultured bacterium based on sequence identity. These results suggest that the diversity of the genes encoding a single step in aromatic compound degradation in the coastal marsh examined is high.

The ascomycete community colonizing decaying Spartina alterniflora blades in a southeastern U.S. salt marsh was characterized by analysis of internal transcribed spacer (ITS) regions of fungal rRNA genes. ITS sequences were amplified with ascomycete-specific primers from DNA extracted from S. alterniflora blades at two stages of decay (early and late) and were identified based on sequence analysis of a companion ascomycete culture collection. The S. alterniflora ITS libraries were dominated by clones from three species of ascomycetes: Mycosphaerella sp. 2, Phaeosphaeria spartinicola, and Phaeosphaeria halima. ITS sequences from five other less abundant ascomycete species were also found in the clone libraries, only two of which could be identified based on the culture collection, Hydropisphaera erubescens and a new species nick-named '4clt'. Ascospore expulsion assays indicated dominance by the same three species as the ITS analysis, although this non-molecular approach differed from the molecular method in relative ranking of the dominant species and in characterization of minor species. Analysis of ITS amplicons from three replicate plots by terminal restriction fragment length polymorphism (T-RFLP) analysis showed significant spatial homogeneity in ascomycete community composition for both early- and late-stage decay. ITS sequence analysis identified morphologically cryptic subgroups for two of the three dominant salt marsh ascomycetes.

Denaturing gradient gel electrophoresis (DGGE) was applied to the 16S-23S rRNA intergenic spacer region (ISR) as a means to evaluate strain level differences in Escherichia coli. The ISRs of 81 environmental E. coli isolates obtained from bovine, poultry, and human sources yielded a total of 41 unique DGGE banding patterns, with identical patterns and common bands within each source and no overlapping patterns among sources. An additional 51 isolates from two nearby streams yielded 45 unique banding patterns with no overlap between sites. However, two of the isolates from the streams had identical banding patterns to those from two of the source isolates, resulting in a total of 84 unique DGGE banding patterns out of 132 isolates identified in this study. These results revealed high diversity among environmental E. coli isolates, which made it difficult to unambiguously ascribe strains found in water samples to specific host organisms.

Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the α-proteobacteria was investigated. Initial screens suggested that isolates in the Roseobacter lineage can degrade aromatic compounds via the β-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six Roseobacter isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the β-ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate, p -hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the Roseobacter group isolates had inducible protocatechuate 3,4-dioxygenase activity in cell extracts when grown on p -hydroxybenzoate. The pcaGH genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates, Sagittula stellata E-37 and isolate Y3F, and in both cases the genes could be expressed in Escherichia coli to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the β-ketoadipate pathway ( pcaC , pcaQ , and pobA ) were found to cluster with pcaGH in these two isolates. Pairwise sequence analysis of the pca genes revealed greater similarity between the two Roseobacter group isolates than between genes from either Roseobacter strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of pcaH from two additional Roseobacter group isolates, and Southern hybridization indicated the presence of pcaH in the remaining two isolates. This evidence of protocatechuate 3,4-dioxygenase and the β-ketoadipate pathway was found in all six Roseobacter isolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.

Background The Roseobacter clade is a widespread, metabolically versatile, and easily cultured lineage of marine microbes. Furthermore, cultured isolates are generally considered to be representative of the naturally occurring diversity in the environment. Roseobacter is thus considered an ideal clade for exploring and understanding microbial function in a representative model system. Nevertheless, recent studies question whether cultured strains are truly representative of natural populations. Support for one of these studies was based on the potentially biased samples of a Global Ocean Sampling (GOS) expedition [1]. The marine metagenomic studies of the GOS expeditions initially focused on microbes collected from a single filter range (0.1um – 0.8um) and it has been previously suggested that this excludes many Roseobacters, thus biasing the dataset towards smaller cells.