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Microbial biofilms associated with marine particulate organic matter carry out transformations that influence local and regional biogeochemical cycles. Early microbial colonizers are often hypothesized to “set the stage” for biofilm structure, dynamics, and function via N-acyl homoserine lactone (AHL)-mediated quorum sensing (QS). Production of AHLs, as well as antimicrobials, contributes to the colonization success of members of the Roseobacter clade. One member of this group of abundant marine bacteria, Rhodobacterales sp. Y4I, possesses two QS systems, phaRI (QS1) and pgaRI (QS2). Here, we characterize mutants in both QS systems to provide genetic evidence that the two systems work in hierarchical fashion to coordinate production of the antimicrobial indigoidine as well as biofilm formation. A mutation in pgaR (QS2) results in decreased expression of genes encoding both QS systems as well as those governing the biosynthesis of indigoidine. In contrast, mutations in QS1 did not significantly influence gene expression of QS2. Addition of exogenous AHLs to QS1 and QS2 mutants led to partial restoration of indigoidine production (45–60% of WT) for QS1 but not QS2. Mutational disruptions of QS1 had a more pronounced effect on biofilm development than those in QS2. Finally, we demonstrate that c-di-GMP levels are altered in QS and indigoidine biosynthesis Y4I mutants. Together, these results indicate that pgaRI (QS2) is at the top of a regulatory hierarchy governing indigoidine biosynthesis and that the global regulatory metabolite, c-di-GMP, is likely integrated into the QS circuitry of this strain. These findings provide mechanistic understanding of physiological processes that are important in elucidating factors driving competitiveness of Roseobacters in nature.

Geochemical models typically represent organic matter (OM) as consisting of multiple, independent pools of compounds, each accessed by microorganisms at different rates. However, recent findings indicate that organic compounds can interact within microbial metabolisms. The relevance of interactive effects within marine systems is debated and a mechanistic understanding of its complexities, including microbe-substrate relationships, is lacking. As a first step toward uncovering mediating processes, the interactive effects of distinct pools of OM on the growth and respiration of marine bacteria, individual strains and a simple, constructed community of Roseobacter lineage members were tested. Isolates were provided with natural organic matter (NOM) and different concentrations (1, 4, 40, 400 μM-C) and forms of labile organic matter (acetate, casamino acids, tryptone, coumarate). The microbial response to the mixed substrate regimes was assessed using viable counts and respiration in two separate experiments. Two marine bacteria and a six-member constructed community were assayed with these experiments. Both synergistic and antagonistic growth responses were evident for all strains, but all were transient. The specific substrate conditions promoting a response, and the direction of that response, varied amongst species. These findings indicate that the substrate conditions that result in OM interactive effects are both transient and species-specific and thus influenced by both the composition and metabolic potential of a microbial community.

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.

Geochemical models typically represent organic matter (OM) as consisting of multiple, independent pools of compounds, each accessed by microorganisms at different rates. However, recent findings indicate that organic compounds can interact within microbial metabolisms. The relevance of interactive effects within marine systems is debated and a mechanistic understanding of its complexities, including microbe-substrate relationships, is lacking. As a first step toward uncovering mediating processes, the interactive effects of distinct pools of OM on the growth and respiration of marine bacteria, individual strains and a simple, constructed community of Roseobacter lineage members were tested. Isolates were provided with natural organic matter (NOM) and different concentrations (1, 4, 40, 400 μM-C) and forms of labile organic matter (acetate, casamino acids, tryptone, coumarate). The microbial response to the mixed substrate regimes was assessed using viable counts and respiration in two separate experiments. Two marine bacteria and a six-member constructed community were assayed with these experiments. Both synergistic and antagonistic growth responses were evident for all strains, but all were transient. The specific substrate conditions promoting a response, and the direction of that response, varied amongst species. These findings indicate that the substrate conditions that result in OM interactive effects are both transient and species-specific and thus influenced by both the composition and metabolic potential of a microbial community.

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.

Contact Dr. Alison Buchan (abuchan@utk.edu) with any questions.

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.

The "priming effect", in which addition of labile substances changes the remineralization rate of recalcitrant organic matter, has been intensively studied in soils, but is less well-documented in aquatic systems. We investigated the extent to which additions of nutrients or labile organic carbon could influence remineralization rates of 14C-labeled, microbially-degraded, phytoplankton-derived organic matter (OM) in microcosms inoculated with microbial communities drawn from Groves Creek Estuary in coastal Georgia, USA. We found that amendment with labile protein plus phosphorus increased remineralization rates of degraded, phytoplankton-derived OM by up to 100%, whereas acetate slightly decreased remineralization rates relative to an unamended control. Addition of ammonium and phosphate induced a smaller effect, whereas addition of ammonium alone had no effect. Counterintuitively, alkaline phosphatase activities increased in response to the addition of protein under P-replete conditions, indicating that production of enzymes unrelated to the labile priming compound may be a mechanism for the priming effect. The observed priming effect was transient: after 36 days of incubation roughly the same quantity of organic carbon had been mineralized in all treatments including no-addition controls. This timescale is on the order of the typical hydrologic residence times of well-flushed estuaries suggesting that priming in estuaries has the potential to influence whether OC is remineralized in situ or exported to the coastal ocean.

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.

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

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.

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.

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.

Oxidized low density lipoprotein (LDL) and acetyl LDL are recognized by the scavenger receptor class A type I/II (SR-AI/II) on macrophages and liver endothelial cells. Several investigators have suggested that there are additional receptors specific for oxidized LDL, but characterization of these alternate receptors for oxidized LDL and evaluation of their quantitative importance in uptake of oxidized LDL has been difficult because of overlapping ligand specificity with SR-AI/II. The purpose of this study was to determine the importance of SR-AI/II in the removal of modified LDL from the bloodstream in vivo. The clearance rate of oxidized LDL from plasma in normal mice was very rapid, and > 90% of injected dose was removed from the blood within 5 min. Clearance rates of oxidized LDL were equally high in SR-AI/II knockout mice, indicating that this receptor is not required for removal of oxidized LDL from plasma. Surprisingly, there was no difference in the clearance rate of acetyl LDL in wild-type and SR-AI/II knockout animals. The plasma clearance of radioiodinated acetyl LDL was almost fully blocked by a 50-fold excess of unlabeled acetyl LDL, but the latter only inhibited oxidized LDL clearance by approximately 5%. Both modified LDLs were cleared mostly by the liver, and there was no difference in the tissue distribution of modified LDL in control and knockout mice. Studies in isolated nonparenchymal liver cells showed that Kupffer cells accounted for most of the uptake of oxidized LDL. Extensively oxidized LDL and LDL modified by exposure to fatty acid peroxidation products were efficient competitors for the uptake of labeled oxidized LDL by SR-AI/II-deficient Kupffer cells, while acetyl LDL and malondialdehyde-modified LDL were relatively poor competitors.

Bacteria have been widely reported to use quorum sensing (QS) systems, which employ small diffusible metabolites to coordinate gene expression in a population density dependent manner. In Proteobacteria, the most commonly described QS signaling molecules are N-acyl-homoserine lactones (AHLs). Recent studies suggest that members of the abundant marine Roseobacter lineage possess AHL-based QS systems and are environmentally relevant models for relating QS to ecological success. As reviewed here, these studies suggest that the roles of QS in roseobacters are varied and complex. An analysis of the 43 publically available Roseobacter genomes shows conservation of QS protein sequences and overall gene topologies, providing support for the hypothesis that quorum sensing is a conserved and widespread trait in the clade.

We examined the genetic and physiological characteristics of chitin degrading enzymes expressed by fosmids cloned from two strains of chitinolytic gammaproteobacteria isolated from alkaline, hypersaline Mono Lake, California; and from a metagenomic library derived from an estuarine bacterial community (Dean Creek, Sapelo Island, GA, USA). The Mono Lake chitinolytic enzymes presented unique adaptations in terms of halo- and alkalitolerance. The sequence from one of the Mono Lake isolates (strain 12A) was a conventional family 18 glycosyl hydrolase; however, the expressed protein had a novel secondary activity peak at pH 10. We obtained a novel family 20 glycosyl hydrolase sequence from Mono Lake strain AI21. The activity of the expressed protein had a pH optimum of 10, several pH units higher than any other enzyme currently assigned to this family, and the enzyme retained 80% of its activity at pH 11. The enzyme was also halotolerant, retaining activity in salt solutions of up to 225 g l(-1). Sequence analysis indicated a molecular weight of approximately 90 kDa for the protein, and that it contained two active sites. Culture supernatant contained two chitinolytic proteins, 45 and 31 kDa, suggesting possible post-expression modification of the gene product. In contrast, the sequence found in the estuarine metagenomic library and the functional characteristics of the protein expressed from it were those of a conventional family 18 glycosyl hydrolase.

Flux of dimethylsulfide (DMS) from ocean surface waters is the predominant natural source of sulfur to the atmosphere and influences climate by aerosol formation. Marine bacterioplankton regulate sulfur flux by converting the precursor dimethylsulfoniopropionate (DMSP) either to DMS or to sulfur compounds that are not climatically active. Through the discovery of a glycine cleavage T-family protein with DMSP methyltransferase activity, marine bacterioplankton in the Roseobacter and SAR11 taxa were identified as primary mediators of DMSP demethylation to methylmercaptopropionate. One-third of surface ocean bacteria harbor a DMSP demethylase homolog and thereby route a substantial fraction of global marine primary production away from DMS formation and into the marine microbial food web.

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.