Silicibacter Pomeroyi Sp. Nov. and Roseovarius Nubinhibens Sp. Nov., Dimethylsulfoniopropionate-demethylating Bacteria from Marine Environments

International Journal of Systematic and Evolutionary Microbiology. Sep, 2003  |  Pubmed ID: 13130004

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 similarity >97 % or DNA-DNA hybridization values >45 % 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.

Dynamics of Bacterial and Fungal Communities on Decaying Salt Marsh Grass

Applied and Environmental Microbiology. Nov, 2003  |  Pubmed ID: 14602628

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 alpha-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 alpha-Proteobacteria and two ascomycete fungi (Phaeosphaeria spartinicola and environmental isolate "4clt").

Diverse Organization of Genes of the Beta-ketoadipate Pathway in Members of the Marine Roseobacter Lineage

Applied and Environmental Microbiology. Mar, 2004  |  Pubmed ID: 15006791

Members of the Roseobacter lineage, an ecologically important marine clade within the class alpha-Proteobacteria, harbor genes for the protocatechuate branch of the beta-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 beta-ketoadipate pathway.

Use of Microautoradiography Combined with Fluorescence in Situ Hybridization to Determine Dimethylsulfoniopropionate Incorporation by Marine Bacterioplankton Taxa

Applied and Environmental Microbiology. Aug, 2004  |  Pubmed ID: 15294798

The fraction of planktonic heterotrophic bacteria capable of incorporating dissolved dimethylsulfoniopropionate (DMSP) and leucine was determined at two coastal sites by microautoradioagraphy (AU). In Gulf of Mexico seawater microcosm experiments, the proportion of prokaryotes that incorporated sulfur from [(35)S]DMSP ranged between 27 and 51% of 4',6-diamidino-2-phenylindole (DAPI)-positive cells, similar to or slightly lower than the proportion incorporating [(3)H]leucine. In the northwest Mediterranean coast, the proportion of cells incorporating sulfur from [(35)S]DMSP increased from 5 to 42% from January to March, coinciding with the development of a phytoplankton bloom. At the same time, the proportion of cells incorporating [(3)H]leucine increased from 21 to 40%. The combination of AU and fluorescence in situ hybridization (FISH) revealed that the Roseobacter clade (alpha-proteobacteria) accounted for 13 to 43% of the microorganisms incorporating [(35)S]DMSP at both sampling sites. Significant uptake of sulfur from DMSP was also found among members of the gamma-proteobacteria and Cytophaga-Flavobacterium groups. Roseobacter and gamma-proteobacteria exhibited the highest percentage of DAPI-positive cells incorporating (35)S from DMSP (around 50%). Altogether, the application of AU with [(35)S]DMSP combined with FISH indicated that utilization of S from DMSP is a widespread feature among active marine bacteria, comparable to leucine utilization. These results point toward DMSP as an important substrate for a broad and diverse fraction of marine bacterioplankton.

Genome Sequence of Silicibacter Pomeroyi Reveals Adaptations to the Marine Environment

Nature. Dec, 2004  |  Pubmed ID: 15602564

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.

Flow-cytometric Cell Sorting and Subsequent Molecular Analyses for Culture-independent Identification of Bacterioplankton Involved in Dimethylsulfoniopropionate Transformations

Applied and Environmental Microbiology. Mar, 2005  |  Pubmed ID: 15746343

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 microM 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, alpha-Proteobacteria); Caulobacter (alpha-Proteobacteria); and Brachymonas and Xenophilus (beta-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, alpha-Proteobacteria) and novel gamma-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.

Analysis of Microbial Gene Transcripts in Environmental Samples

Applied and Environmental Microbiology. Jul, 2005  |  Pubmed ID: 16000831

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 approximately 80% were unambiguously mRNA derived. mRNAs appeared to be from diverse taxonomic groups, including both Bacteria (mainly alpha- and gamma-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.

Overview of the Marine Roseobacter Lineage

Applied and Environmental Microbiology. Oct, 2005  |  Pubmed ID: 16204474

Screening for Bacterial-fungal Associations in a South-eastern US Salt Marsh Using Pre-established Fungal Monocultures

FEMS Microbiology Ecology. Oct, 2005  |  Pubmed ID: 16332317

Both bacteria and fungi play critical roles in decomposition processes in many natural environments, yet only rarely have they been studied as an integrated community. We examined whether physical associations exist between individual bacterial and fungal species that co-occur on decaying smooth cordgrass, Spartina alterniflora, in a south-eastern US salt marsh. Fungal-pervaded decaying Spartina was used as "bait" for potential bacterial associates. The bundles (infiltrated with one of three dominant fungal members of the decomposer assemblage, or an autoclaved control) were placed in a salt marsh and collected biweekly for 6 weeks during the first experiment (late summer 2002), and weekly for 3 weeks during the second experiment (early summer 2003). Terminal-restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to track colonization by bacterial taxa in association with the established fungal species. T-RFLP analysis of 18S-to-28S internal transcribed spacer (ITS) regions was used to monitor changes in fungal communities once bundles had been placed in the field. Results from both years were nearly identical, and showed that invasion by fungi other than the bait species was slow, resulting in a virtual fungal monoculture for several weeks into the experiments. Surprisingly, bacterial communities were unaffected by the identity of the fungal bait. Regardless of the fungal species, and even in the absence of prior fungal colonization, bacterial 16S rRNA profiles were remarkably similar. These results suggest that few species-specific associations, either positive or negative, exist between bacterial and fungal members of the Spartina decomposer community during initial colonization.

Dimethylsulfoniopropionate Turnover is Linked to the Composition and Dynamics of the Bacterioplankton Assemblage During a Microcosm Phytoplankton Bloom

Applied and Environmental Microbiology. Dec, 2005  |  Pubmed ID: 16332737

Processing of the phytoplankton-derived organic sulfur compound dimethylsulfoniopropionate (DMSP) by bacteria was studied in seawater microcosms in the coastal Gulf of Mexico (Alabama). Modest phytoplankton blooms (peak chlorophyll a [Chl a] concentrations of approximately 2.5 microg liter(-1)) were induced in nutrient-enriched microcosms, while phytoplankton biomass remained low in unamended controls (Chl a concentrations of approximately 0.34 microg liter(-1)). Particulate DMSP concentrations reached 96 nM in the enriched microcosms but remained approximately 14 nM in the controls. Bacterial biomass production increased in parallel with the increase in particulate DMSP, and nutrient limitation bioassays in the initial water showed that enrichment with DMSP or glucose caused a similar stimulation of bacterial growth. Concomitantly, increased bacterial consumption rate constants of dissolved DMSP (up to 20 day(-1)) and dimethylsulfide (DMS) (up to 6.5 day(-1)) were observed. Nevertheless, higher DMSP S assimilation efficiencies and higher contribution of DMSP to bacterial S demand were found in the controls compared to the enriched microcosms. This indicated that marine bacterioplankton may rely more on DMSP as a source of S under oligotrophic conditions than under the senescence phase of phytoplankton blooms. Phylogenetic analysis of the bacterial assemblages in all microcosms showed that the DMSP-rich algal bloom favored the occurrence of various Roseobacter members, flavobacteria (Bacteroidetes phylum), and oligotrophic marine Gammaproteobacteria. Our observations suggest that the composition of the bacterial assemblage and the relative contribution of DMSP to the overall dissolved organic sulfur/organic matter pool control how efficiently bacteria assimilate DMSP S and thereby potentially divert it from DMS production.

Bacterial Taxa That Limit Sulfur Flux from the Ocean

Science (New York, N.Y.). Oct, 2006  |  Pubmed ID: 17068264

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.

Comparison of Chitinolytic Enzymes from an Alkaline, Hypersaline Lake and an Estuary

Environmental Microbiology. Jan, 2007  |  Pubmed ID: 17227424

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.

Bacterioplankton Assemblages Transforming Dissolved Organic Compounds in Coastal Seawater

Environmental Microbiology. Aug, 2007  |  Pubmed ID: 17635547

To characterize bacterioplankton functional assemblages that transform specific components of the coastal seawater dissolved organic carbon (DOC) pool, bromodeoxyuridine (BrdU) was used to label the bacterioplankton cells that were active following addition of single-DOC model compounds: two organic osmolytes [dimethylsulfoniopropionate (DMSP) and glycine betaine (GlyB)] and two aromatic monomers [para-hydroxybenzoic acid (pHBA) and vanillic acid (VanA)]. Bacterial populations were analysed based on in situ fluorescent immunodetection of BrdU incorporation followed by fluorescence-activated cell sorting (FACS). Sorted cells were then characterized by 16S rDNA-based analysis. Populations with high BrdU incorporation level (HI) developed within 8 h of introduction of 100 nM model compound. Terminal restriction fragment length polymorphisms (T-RFLP) analysis indicated that the HI populations in all four amendments were composed of bacteria from the same major taxa (phylum and subphylum levels), but the relative abundance of each differed. High-resolution clone libraries (each containing approximately 200 clones) showed that the HI populations in the GlyB and VanA amendments consisted of both metabolic generalists and specialists within the alpha-Proteobacteria (mainly members of the Roseobacter clade), beta-Proteobacteria and gamma-Proteobacteria (mainly members of Altermonadaceae, Chromatiaceae, Oceanospirillaceae and Pseudomonadaceae). The presence of members of OM60/241, OM185, SAR11, SAR86 and SAR116 in the HI populations indicated that members of these groups can assimilate the model DOC compounds, providing some of the first glimpses into heterotrophy by members of these poorly understood environmental clusters.

Resourceful Heterotrophs Make the Most of Light in the Coastal Ocean

Nature Reviews. Microbiology. Oct, 2007  |  Pubmed ID: 17828280

The carbon cycle in the coastal ocean is affected by how heterotrophic marine bacterioplankton obtain their energy. Although it was previously thought that these organisms relied on the organic carbon in seawater for all of their energy needs, several recent discoveries now suggest that pelagic bacteria can depart from a strictly heterotrophic lifestyle by obtaining energy through unconventional mechanisms that are linked to the penetration of sunlight into surface waters. These newly discovered mechanisms involve the harvesting of energy, either directly from light or indirectly from inorganic compounds that are formed when dissolved organic carbon absorbs light. In coastal systems, these mixed metabolic strategies have implications for how efficiently organic carbon is retained in the marine food web and how climatically important gases are exchanged between the ocean and the atmosphere.

Transcriptional Response of Silicibacter Pomeroyi DSS-3 to Dimethylsulfoniopropionate (DMSP)

Environmental Microbiology. Nov, 2007  |  Pubmed ID: 17922758

Dimethylsufoniopropionate (DMSP) is an abundant organic sulfur compound in the ocean and an important substrate for marine bacterioplankton. The Roseobacter clade of marine alphaproteobacteria, including Silicibacter pomeroyi strain DSS-3, are known to be involved in DMSP degradation in situ. The fate of DMSP has important implications for the global sulfur cycle, but the genes involved in this process and their regulation are largely unknown. S. pomeroyi is capable of performing two major pathways of DMSP degradation, making it an important model organism. Based on the full genome sequence of this strain we designed an oligonucleotide-based microarray for the detection of transcripts of nearly all genes. The array was used to study the transcriptional response of S. pomeroyi cultures to additions of DMSP compared to the non-sulfur compound acetate in a time series experiment. We identified a number of upregulated genes that could be assigned to potential roles in the metabolism of DMSP. DMSP also affected the transcription of genes for transport and metabolism of peptides, amino acids and polyamines. DMSP concentration may thus also play a role as a chemical signal, indicating phytoplankton abundance and eliciting a regulatory response aimed at making maximum use of available nutrients.

Variation in Prokaryotic Community Composition As a Function of Resource Availability in Tidal Creek Sediments

Applied and Environmental Microbiology. Mar, 2008  |  Pubmed ID: 18192417

In anaerobic coastal sediments, hydrolytic and/or fermentative bacteria degrade polymeric material and produce labile intermediates, which are used by terminal metabolizers to complete the conversion of organic material to CO(2). We used molecular approaches to evaluate the response of two bacterial terminal metabolizer groups from a coastal tidal creek sediments, sulfate reducers and methanogens, to controlled changes in carbon resource supply. Tidal creek sediment bioreactors were established in April and August 2004. For each date, intact sediment sections were continuously supplied with flowthrough seawater that was either unamended or amended with the high-molecular-weight polysaccharide dextran. Biogeochemical data indicate that the activity of fermenting bacteria and the terminal metabolizers was limited by organic carbon supply during both experiments, with a significant increase in net volatile fatty acid (VFA) production and rates of sulfate reduction and methanogenesis following dextran addition. Community composition (measured by using terminal restriction fragment length polymorphism analysis, and functional gene [dsrA, mcrA] clone libraries) changed from April to August. However, community composition was not different between amended and unamended cores within each month, despite the change in resource level. Moreover, there was no relationship between community richness and evenness with resource level. This lack of variation in community composition with C addition could be attributed to the dynamic environment these sediment communities experience in situ. Fluctuations in VFA concentrations are most likely very high, so that the dominant bacterial species must be able to outcompete other species at both high and low resource levels.

Bacterial Carbon Processing by Generalist Species in the Coastal Ocean

Nature. Feb, 2008  |  Pubmed ID: 18223640

The assimilation and mineralization of dissolved organic carbon (DOC) by marine bacterioplankton is a major process in the ocean carbon cycle. However, little information exists on the specific metabolic functions of participating bacteria and on whether individual taxa specialize on particular components of the marine DOC pool. Here we use experimental metagenomics to show that coastal communities are populated by taxa capable of metabolizing a wide variety of organic carbon compounds. Genomic DNA captured from bacterial community subsets metabolizing a single model component of the DOC pool (either dimethylsulphoniopropionate or vanillate) showed substantial overlap in gene composition as well as a diversity of carbon-processing capabilities beyond the selected phenotypes. Our direct measure of niche breadth for bacterial functional assemblages indicates that, in accordance with ecological theory, heterogeneity in the composition and supply of organic carbon to coastal oceans may favour generalist bacteria. In the important interplay between microbial community structure and biogeochemical cycling, coastal heterotrophic communities may be controlled less by transient changes in the carbon reservoir that they process and more by factors such as trophic interactions and physical conditions.

Functional Metagenomic Profiling of Nine Biomes

Nature. Apr, 2008  |  Pubmed ID: 18337718

Microbial activities shape the biogeochemistry of the planet and macroorganism health. Determining the metabolic processes performed by microbes is important both for understanding and for manipulating ecosystems (for example, disruption of key processes that lead to disease, conservation of environmental services, and so on). Describing microbial function is hampered by the inability to culture most microbes and by high levels of genomic plasticity. Metagenomic approaches analyse microbial communities to determine the metabolic processes that are important for growth and survival in any given environment. Here we conduct a metagenomic comparison of almost 15 million sequences from 45 distinct microbiomes and, for the first time, 42 distinct viromes and show that there are strongly discriminatory metabolic profiles across environments. Most of the functional diversity was maintained in all of the communities, but the relative occurrence of metabolisms varied, and the differences between metagenomes predicted the biogeochemical conditions of each environment. The magnitude of the microbial metabolic capabilities encoded by the viromes was extensive, suggesting that they serve as a repository for storing and sharing genes among their microbial hosts and influence global evolutionary and metabolic processes.

Occurrence and Expression of Gene Transfer Agent Genes in Marine Bacterioplankton

Applied and Environmental Microbiology. May, 2008  |  Pubmed ID: 18359833

Genes with homology to the transduction-like gene transfer agent (GTA) were observed in genome sequences of three cultured members of the marine Roseobacter clade. A broader search for homologs for this host-controlled virus-like gene transfer system identified likely GTA systems in cultured Alphaproteobacteria, and particularly in marine bacterioplankton representatives. Expression of GTA genes and extracellular release of GTA particles ( approximately 50 to 70 nm) was demonstrated experimentally for the Roseobacter clade member Silicibacter pomeroyi DSS-3, and intraspecific gene transfer was documented. GTA homologs are surprisingly infrequent in marine metagenomic sequence data, however, and the role of this lateral gene transfer mechanism in ocean bacterioplankton communities remains unclear.

Abundant and Diverse Bacteria Involved in DMSP Degradation in Marine Surface Waters

Environmental Microbiology. Sep, 2008  |  Pubmed ID: 18510552

An expanded analysis of oceanic metagenomic data indicates that the majority of prokaryotic cells in marine surface waters have the genetic capability to demethylate dimethylsulfoniopropionate (DMSP). The 1701 homologues of the DMSP demethylase gene, dmdA, identified in the (2007) Global Ocean Sampling (GOS) metagenome, are sufficient for 58% (+/-9%) of sampled cells to participate in this critical step in the marine sulfur cycle. This remarkable frequency of DMSP-demethylating cells is in accordance with biogeochemical data indicating that marine phytoplankton direct up to 10% of fixed carbon to DMSP synthesis, and that most of this DMSP is subsequently degraded by bacteria via demethylation. The GOS metagenomic data also revealed a new cluster of dmdA sequences (designated Clade E) that implicates marine gammaproteobacteria in DMSP demethylation, along with previously recognized alphaproteobacterial groups Roseobacter and SAR11. Analyses of G+C content and gene order indicate that lateral gene transfer is likely responsible for the wide distribution of dmdA among diverse taxa, contributing to the homogenization of biogeochemical roles among heterotrophic marine bacterioplankton. Candidate genes for the competing bacterial degradation process that converts DMSP to the climate-active gas dimethylsulfide (DMS) (dddD and dddL) occur infrequently in the (2007) GOS metagenome, suggesting either that the key DMS-producing bacterial genes are yet to be identified or that DMS formation by free-living bacterioplankton is insignificant relative to their demethylation activity.

Toward a Standards-compliant Genomic and Metagenomic Publication Record

Omics : a Journal of Integrative Biology. Jun, 2008  |  Pubmed ID: 18564916

Increasingly, we are aware as a community of the growing need to manage the avalanche of genomic and metagenomic data, in addition to related data types like ribosomal RNA and barcode sequences, in a way that tightly integrates contextual data with traditional literature in a machine-readable way. It is for this reason that the Genomic Standards Consortium (GSC) formed in 2005. Here we suggest that we move beyond the development of standards and tackle standards compliance and improved data capture at the level of the scientific publication. We are supported in this goal by the fact that the scientific community is in the midst of a publishing revolution. This revolution is marked by a growing shift away from a traditional dichotomy between "journal articles" and "database entries" and an increasing adoption of hybrid models of collecting and disseminating scientific information. With respect to genomes and metagenomes and related data types, we feel the scientific community would be best served by the immediate launch of a central repository of short, highly structured "Genome Notes" that must be standards compliant. This could be done in the context of an existing journal, but we also suggest the more radical solution of launching a new journal. Such a journal could be designed to cater to a wide range of standards-related content types that are not currently centralized in the published literature. It could also support the demand for centralizing aspects of the "gray literature" (documents developed by institutions or communities) such as the call by the GSC for a central repository of Standard Operating Procedures describing the genomic annotation pipelines of the major sequencing centers. We argue that such an "eJournal," published under the Open Access paradigm by the GSC, could be an attractive publishing forum for a broader range of standardization initiatives within, and beyond, the GSC and thereby fill an unoccupied yet increasingly important niche within the current research landscape.

Dimethylsulfoniopropionate-dependent Demethylase (DmdA) from Pelagibacter Ubique and Silicibacter Pomeroyi

Journal of Bacteriology. Dec, 2008  |  Pubmed ID: 18849431

The ubiquitous algal metabolite dimethylsulfoniopropionate (DMSP) is a major source of carbon and reduced sulfur for marine bacteria. Recently, the enzyme responsible for the demethylation of DMSP, designated DmdA, was identified, and homologs were found to be common in marine bacterioplankton cells. The recombinant DmdA proteins from the cultured marine bacteria Pelagibacter ubique HTCC1062 and Silicibacter pomeroyi DSS-3 were purified with a three-step procedure using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatographies. The P. ubique enzyme possessed an M(r) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 38,500. Under nondenaturing conditions, the M(r) was 68,000, suggesting that the enzyme was likely to be a dimer. The purified enzyme exhibited strict substrate specificity for DMSP, as DmdA from both S. pomeroyi and P. ubique possessed no detectable demethylase activity with glycine betaine, dimethyl glycine, methylmercaptopropionate, methionine, or dimethylsulfonioacetate. Less than 1% activity was found with dimethylsulfoniobutanoate and dimethylsulfoniopentanoate. The apparent K(m)s for DMSP were 13.2 +/- 2.0 and 5.4 +/- 2.3 mM for the P. ubique and S. pomeroyi enzymes, respectively. In cell extracts of S. pomeroyi DSS-3, the apparent K(m) for DMSP was 8.6 +/- 1.2 mM, similar to that of purified recombinant DmdA. The intracellular concentration of DMSP in chemostat-grown S. pomeroyi DSS-3 was 70 mM. These results suggest that marine bacterioplankton may actively accumulate DMSP to osmotically significant concentrations that favor near-maximal rates of DMSP demethylation activity.

Comparative Day/night Metatranscriptomic Analysis of Microbial Communities in the North Pacific Subtropical Gyre

Environmental Microbiology. Jun, 2009  |  Pubmed ID: 19207571

Metatranscriptomic analyses of microbial assemblages (< 5 microm) from surface water at the Hawaiian Ocean Time-Series (HOT) revealed community-wide metabolic activities and day/night patterns of differential gene expression. Pyrosequencing produced 75 558 putative mRNA reads from a day transcriptome and 75 946 from a night transcriptome. Taxonomic binning of annotated mRNAs indicated that Cyanobacteria contributed a greater percentage of the transcripts (54% of annotated sequences) than expected based on abundance (35% of cell counts and 21% 16S rRNA of libraries), and may represent the most actively transcribing cells in this surface ocean community in both the day and night. Major heterotrophic taxa contributing to the community transcriptome included alpha-Proteobacteria (19% of annotated sequences, most of which were SAR11-related) and gamma-Proteobacteria (4%). The composition of transcript pools was consistent with models of prokaryotic gene expression, including operon-based transcription patterns and an abundance of genes predicted to be highly expressed. Metabolic activities that are shared by many microbial taxa (e.g. glycolysis, citric acid cycle, amino acid biosynthesis and transcription and translation machinery) were well represented among the community transcripts. There was an overabundance of transcripts for photosynthesis, C1 metabolism and oxidative phosphorylation in the day compared with night, and evidence that energy acquisition is coordinated with solar radiation levels for both autotrophic and heterotrophic microbes. In contrast, housekeeping activities such as amino acid biosynthesis, membrane synthesis and repair, and vitamin biosynthesis were overrepresented in the night transcriptome. Direct sequencing of these environmental transcripts has provided detailed information on metabolic and biogeochemical responses of a microbial community to solar forcing.

The Diverse Bacterial Community in Intertidal, Anaerobic Sediments at Sapelo Island, Georgia

Microbial Ecology. Aug, 2009  |  Pubmed ID: 19212699

The phylogenetic diversity and composition of the bacterial community in anaerobic sediments from Sapelo Island, GA, USA were examined using 16S rRNA gene libraries. The diversity of this community was comparable to that of soil, and 1,186 clones formed 817 OTUs at 99% sequence similarity. Chao1 estimators for the total richness were also high, at 3,290 OTUs at 99% sequence similarity. The program RDPquery was developed to assign clones to taxonomic groups based upon comparisons to the RDP database. While most clones could be assigned to describe phyla, fewer than 30% of the clones could be assigned to a described order. Similarly, nearly 25% of the clones were only distantly related (<90% sequence similarity) to other environmental clones, illustrating the unique composition of this community. One quarter of the clones were related to one or more undescribed orders within the gamma-Proteobacteria. Other abundant groups included the delta-Proteobacteria, Bacteroidetes, and Cyanobacteria. While these phyla were abundant in other estuarine sediments, the specific members at Sapelo Island appeared to be different from those previously described in other locations, suggesting that great diversity exists between as well as within estuarine intertidal sediments. In spite of the large differences in pore water chemistry with season and depth, differences in the bacterial community were modest over the temporal and spatial scales examined and generally restricted to only certain taxa.

In Situ Transcriptomic Analysis of the Globally Important Keystone N2-fixing Taxon Crocosphaera Watsonii

The ISME Journal. May, 2009  |  Pubmed ID: 19225552

The diazotrophic cyanobacterium Crocosphaera watsonii supplies fixed nitrogen (N) to N-depleted surface waters of the tropical oceans, but the factors that determine its distribution and contribution to global N(2) fixation are not well constrained for natural populations. Despite the heterogeneity of the marine environment, the genome of C. watsonii is highly conserved in nucleotide sequence in contrast to sympatric planktonic cyanobacteria. We applied a whole assemblage shotgun transcript sequencing approach to samples collected from a bloom of C. watsonii observed in the South Pacific to understand the genomic mechanisms that may lead to high population densities. We obtained 999 C. watsonii transcript reads from two metatranscriptomes prepared from mixed assemblage RNA collected in the day and at night. The C. watsonii population had unexpectedly high transcription of hypothetical protein genes (31% of protein-encoding genes) and transposases (12%). Furthermore, genes were expressed that are necessary for living in the oligotrophic ocean, including the nitrogenase cluster and the iron-stress-induced protein A (isiA) that functions to protect photosystem I from high-light-induced damage. C. watsonii transcripts retrieved from metatranscriptomes at other locations in the southwest Pacific Ocean, station ALOHA and the equatorial Atlantic Ocean were similar in composition to those recovered in the enriched population. Quantitative PCR and quantitative reverse transcriptase PCR were used to confirm the high expression of these genes within the bloom, but transcription patterns varied at shallower and deeper horizons. These data represent the first transcript study of a rare individual microorganism in situ and provide insight into the mechanisms of genome diversification and the ecophysiology of natural populations of keystone organisms that are important in global nitrogen cycling.

Microbial Community Response to Seawater Amendment in Low-salinity Tidal Sediments

Microbial Ecology. Oct, 2009  |  Pubmed ID: 19629578

Rising sea levels and excessive water withdrawals upstream are making previously freshwater coastal ecosystems saline. Plant and animal responses to variation in the freshwater-saline interface have been well studied in the coastal zone; however, microbial community structure and functional response to seawater intrusion remains relatively unexplored. Here, we used molecular approaches to evaluate the response of the prokaryotic community to controlled changes in porewater salinity levels in freshwater sediments from the Altamaha River, Georgia, USA. This work is a companion to a previously published study describing results from an experiment using laboratory flow-through sediment core bioreactors to document biogeochemical changes as porewater salinity was increased from 0 to 10 over 35 days. As reported in Weston et al. (Biogeochemistry, 77:375-408, 62), porewater chemistry was monitored, and cores were sacrificed at 0, 9, 15, and 35 days, at which time we completed terminal restriction fragment length polymorphism and 16S rRNA clone library analyses of sediment microbial communities. The biogeochemical study documented changes in mineralization pathways in response to artificial seawater additions, with a decline in methanogenesis, a transient increase in iron reduction, and finally a dominance of sulfate reduction. Here, we report that, despite these dramatic and significant changes in microbial activity at the biogeochemical level, no significant differences were found between microbial community composition of control vs. seawater-amended treatments for either Bacterial or Archaeal members. Further, taxa in the seawater-amended treatment community did not become more "marine-like" through time. Our experiment suggests that, as seawater intrudes into freshwater sediments, observed changes in metabolic activity and carbon mineralization on the time scale of weeks are driven more by shifts in gene expression and regulation than by changes in the composition of the microbial community.

Transporter Genes Expressed by Coastal Bacterioplankton in Response to Dissolved Organic Carbon

Environmental Microbiology. Mar, 2010  |  Pubmed ID: 19930445

Coastal ocean bacterioplankton control the flow of dissolved organic carbon (DOC) from terrestrial and oceanic sources into the marine food web, and regulate the release of inorganic carbon to atmospheric and offshore reservoirs. While the fate of the chemically complex coastal DOC reservoir has long been recognized as a critical feature of the global carbon budget, it has been problematic to identify both the compounds that serve as major conduits for carbon flux and the roles of individual bacterioplankton taxa in mediating that flux. Here we analyse random libraries of expressed genes from a coastal bacterial community to identify sequences representing DOC-transporting proteins. Predicted substrates of expressed transporter genes indicated that carboxylic acids, compatible solutes, polyamines and lipids may be key components of the biologically labile DOC pool in coastal waters, in addition to canonical bacterial substrates such as amino acids, oligopeptides and carbohydrates. Half of the expressed DOC transporter sequences in this coastal ocean appeared to originate from just eight taxa: Roseobacter, SAR11, Flavobacteriales and five orders of gamma-Proteobacteria. While all major taxa expressed transporter genes for some DOC components (e.g. amino acids), there were indications of specialization within the bacterioplankton community for others (e.g. carbohydrates, carboxylic acids and polyamines). Experimental manipulations of the natural DOC pool that increased the concentration of phytoplankton- or vascular plant-derived compounds invoked a readily measured response in bacterial transporter gene expression. This highly resolved view of the potential for carbon flux into heterotrophic bacterioplankton cells identifies possible bioreactive components of the coastal DOC pool and highlights differing ecological roles in carbon turnover for the resident bacterial taxa.

Deep Sequencing of a Dimethylsulfoniopropionate-degrading Gene (dmdA) by Using PCR Primer Pairs Designed on the Basis of Marine Metagenomic Data

Applied and Environmental Microbiology. Jan, 2010  |  Pubmed ID: 19948858

In silico design and testing of environmental primer pairs with metagenomic data are beneficial for capturing a greater proportion of the natural sequence heterogeneity in microbial functional genes, as well as for understanding limitations of existing primer sets that were designed from more restricted sequence data. PCR primer pairs targeting 10 environmental clades and subclades of the dimethylsulfoniopropionate (DMSP) demethylase protein, DmdA, were designed using an iterative bioinformatic approach that took advantage of thousands of dmdA sequences captured in marine metagenomic data sets. Using the bioinformatically optimized primers, dmdA genes were amplified from composite free-living coastal bacterioplankton DNA (from 38 samples over 5 years and two locations) and sequenced using 454 technology. An average of 6,400 amplicons per primer pair represented more than 700 clusters of environmental dmdA sequences across all primers, with clusters defined conservatively at >90% nucleotide sequence identity (approximately 95% amino acid identity). Degenerate and inosine-based primers did not perform better than specific primer pairs in determining dmdA richness and sometimes captured a lower degree of richness of sequences from the same DNA sample. A comparison of dmdA sequences in free-living versus particle-associated bacteria in southeastern U.S. coastal waters showed that sequence richness in some dmdA subgroups differed significantly between size fractions, though most gene clusters were shared (52 to 91%) and most sequences were affiliated with the shared clusters (approximately 90%). The availability of metagenomic sequence data has significantly enhanced the design of quantitative PCR primer pairs for this key functional gene, providing robust access to the capabilities and activities of DMSP demethylating bacteria in situ.

Genome Characteristics of a Generalist Marine Bacterial Lineage

The ISME Journal. Jun, 2010  |  Pubmed ID: 20072162

Members of the marine Roseobacter lineage have been characterized as ecological generalists, suggesting that there will be challenges in assigning well-delineated ecological roles and biogeochemical functions to the taxon. To address this issue, genome sequences of 32 Roseobacter isolates were analyzed for patterns in genome characteristics, gene inventory, and individual gene/pathway distribution using three predictive frameworks: phylogenetic relatedness, lifestyle strategy and environmental origin of the isolate. For the first framework, a phylogeny containing five deeply branching clades was obtained from a concatenation of 70 conserved single-copy genes. Somewhat surprisingly, phylogenetic tree topology was not the best model for organizing genome characteristics or distribution patterns of individual genes/pathways, although it provided some predictive power. The lifestyle framework, established by grouping isolates according to evidence for heterotrophy, photoheterotrophy or autotrophy, explained more of the gene repertoire in this lineage. The environment framework had a weak predictive power for the overall genome content of each strain, but explained the distribution of several individual genes/pathways, including those related to phosphorus acquisition, chemotaxis and aromatic compound degradation. Unassembled sequences in the Global Ocean Sampling metagenomic data independently verified this global-scale geographical signal in some Roseobacter genes. The primary findings emerging from this comparative genome analysis are that members of the lineage cannot be easily collapsed into just a few ecologically differentiated clusters (that is, there are almost as many clusters as isolates); the strongest framework for predicting genome content is trophic strategy, but no single framework gives robust predictions; and previously unknown homologs to genes for H(2) oxidation, proteorhodopsin-based phototrophy, xanthorhodpsin-based phototrophy, and CO(2) fixation by Form IC ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) expand the possible mechanisms for energy and carbon acquisition in this remarkably versatile bacterial lineage.

Transcriptomic Analysis of a Marine Bacterial Community Enriched with Dimethylsulfoniopropionate

The ISME Journal. Nov, 2010  |  Pubmed ID: 20463763

Dimethylsulfoniopropionate (DMSP) is an important source of reduced sulfur and carbon for marine microbial communities, as well as the precursor of the climate-active gas dimethylsulfide (DMS). In this study, we used metatranscriptomic sequencing to analyze gene expression profiles of a bacterial assemblage from surface waters at the Bermuda Atlantic Time-series Study (BATS) station with and without a short-term enrichment of DMSP (25 nM for 30 min). An average of 303 143 reads were obtained per treatment using 454 pyrosequencing technology, of which 51% were potential protein-encoding sequences. Transcripts from Gammaproteobacteria and Bacteroidetes increased in relative abundance on DMSP addition, yet there was little change in the contribution of two bacterioplankton groups whose cultured members harbor known DMSP degradation genes, Roseobacter and SAR11. The DMSP addition led to an enrichment of transcripts supporting heterotrophic activity, and a depletion of those encoding light-related energy generation. Genes for the degradation of C3 compounds were significantly overrepresented after DMSP addition, likely reflecting the metabolism of the C3 component of DMSP. Mapping these transcripts to known biochemical pathways indicated that both acetyl-CoA and succinyl-CoA may be common entry points of this moiety into the tricarboxylic acid cycle. In a short time frame (30 min) in the extremely oligotrophic Sargasso Sea, different gene expression patterns suggest the use of DMSP by a diversity of marine bacterioplankton as both carbon and sulfur sources.

Quantitative Analysis of a Deeply Sequenced Marine Microbial Metatranscriptome

The ISME Journal. Mar, 2011  |  Pubmed ID: 20844569

The potential of metatranscriptomic sequencing to provide insights into the environmental factors that regulate microbial activities depends on how fully the sequence libraries capture community expression (that is, sample-sequencing depth and coverage depth), and the sensitivity with which expression differences between communities can be detected (that is, statistical power for hypothesis testing). In this study, we use an internal standard approach to make absolute (per liter) estimates of transcript numbers, a significant advantage over proportional estimates that can be biased by expression changes in unrelated genes. Coastal waters of the southeastern United States contain 1 × 10(12) bacterioplankton mRNA molecules per liter of seawater (~200 mRNA molecules per bacterial cell). Even for the large bacterioplankton libraries obtained in this study (~500,000 possible protein-encoding sequences in each of two libraries after discarding rRNAs and small RNAs from >1 million 454 FLX pyrosequencing reads), sample-sequencing depth was only 0.00001%. Expression levels of 82 genes diagnostic for transformations in the marine nitrogen, phosphorus and sulfur cycles ranged from below detection (<1 × 10(6) transcripts per liter) for 36 genes (for example, phosphonate metabolism gene phnH, dissimilatory nitrate reductase subunit napA) to >2.7 × 10(9) transcripts per liter (ammonia transporter amt and ammonia monooxygenase subunit amoC). Half of the categories for which expression was detected, however, had too few copy numbers for robust statistical resolution, as would be required for comparative (experimental or time-series) expression studies. By representing whole community gene abundance and expression in absolute units (per volume or mass of environment), 'omics' data can be better leveraged to improve understanding of microbially mediated processes in the ocean.

Analysis of Sulfur-related Transcription by Roseobacter Communities Using a Taxon-specific Functional Gene Microarray

Environmental Microbiology. Feb, 2011  |  Pubmed ID: 20880331

The fraction of dissolved dimethylsulfoniopropionate (DMSPd) converted by marine bacterioplankton into the climate-active gas dimethylsulfide (DMS) varies widely in the ocean, with the factors that determine this value still largely unknown. One current hypothesis is that the ratio of DMS formation: DMSP demethylation is determined by DMSP availability, with 'availability' in both an absolute sense (i.e. concentration in seawater) and in a relative sense (i.e. proportionally to other labile organic S compounds) proposed as the critical factor. We investigated these models during an experimentally induced phytoplankton bloom using a taxon-specific microarray targeting DMSP-related gene transcription in members of the Roseobacter clade, a group hypothesized to play an important role in the surface ocean sulfur cycle and well represented by genome sequences. The array consisted of 1578 probes to 431 genes and was designed to target diverse Roseobacter communities in natural seawater by using hierarchical probe design based on 13 genome sequences. The prevailing pattern of Roseobacter gene transcription showed relative depletion of DMSP-related transcripts during the peak of the bloom, despite increasing absolute concentrations and flux of DMSP-related compounds. DMSPd thus appeared to be assimilated by Roseobacter populations in proportion to its relative abundance in the organic matter pool (the 'relative sense' hypothesis), rather than assimilated in preference to other labile organic sulfur or carbon compounds produced during the bloom. The relative investment of the Roseobacter community in DMSP demethylation was not useful for predicting the formation of DMS, however, suggesting a complex regulatory process that may involve multiple taxa and alternative fates of DMSPd.

Metatranscriptomic Analysis of Ammonia-oxidizing Organisms in an Estuarine Bacterioplankton Assemblage

The ISME Journal. May, 2011  |  Pubmed ID: 21085199

Quantitative PCR (qPCR) analysis revealed elevated relative abundance (1.8% of prokaryotes) of marine group 1 Crenarchaeota (MG1C) in two samples of southeastern US coastal bacterioplankton, collected in August 2008, compared with samples collected from the same site at different times (mean 0.026%). We analyzed the MG1C sequences in metatranscriptomes from these samples to gain an insight into the metabolism of MG1C population growing in the environment, and for comparison with ammonia-oxidizing bacteria (AOB) in the same samples. Assemblies revealed low diversity within sequences assigned to most individual MG1C open reading frames (ORFs) and high homology with 'Candidatus Nitrosopumilus maritimus' strain SCM1 genome sequences. Reads assigned to ORFs for ammonia uptake and oxidation accounted for 37% of all MG1C transcripts. We did not recover any reads for Nmar_1354-Nmar_1357, proposed to encode components of an alternative, nitroxyl-based ammonia oxidation pathway; however, reads from Nmar_1259 and Nmar_1667, annotated as encoding a multicopper oxidase with homology to nirK, were abundant. Reads assigned to two homologous ORFs (Nmar_1201 and Nmar_1547), annotated as hypothetical proteins were also abundant, suggesting that their unknown function is important to MG1C. Superoxide dismutase and peroxiredoxin-like transcripts were more abundant in the MG1C transcript pool than in the complete metatranscriptome, suggesting an enhanced response to oxidative stress by the MG1C population. qPCR indicated low AOB abundance (0.0010% of prokaryotes), and we found no transcripts related to ammonia oxidation and only one RuBisCO transcript among the transcripts assigned to AOB, suggesting they were not responding to the same environmental cues as the MG1C population.

Changes in Dimethylsulfoniopropionate Demethylase Gene Assemblages in Response to an Induced Phytoplankton Bloom

Applied and Environmental Microbiology. Jan, 2011  |  Pubmed ID: 21097583

Over half of the bacterioplankton cells in ocean surface waters are capable of carrying out a demethylation of the phytoplankton metabolite dimethylsulfoniopropionate (DMSP) that routes the sulfur moiety away from the climatically active gas dimethylsulfide (DMS). In this study, we tracked changes in dmdA, the gene responsible for DMSP demethylation, over the course of an induced phytoplankton bloom in Gulf of Mexico seawater microcosms. Analysis of >91,000 amplicon sequences indicated 578 different dmdA sequence clusters at a conservative clustering criterion of ≥90% nucleotide sequence identity over the 6-day study. The representation of the major clades of dmdA, several of which are linked to specific taxa through genomes of cultured marine bacterioplankton, remained fairly constant. However, the representation of clusters within these major clades shifted significantly in response to the bloom, including two Roseobacter-like clusters and a SAR11-like cluster, and the best correlate with shifts of the dominant dmdA clades was chlorophyll a concentration. Concurrent 16S rRNA amplification and sequencing indicated the presence of Roseobacter, SAR11, OM60, and marine Rhodospirillales populations, all of which are known to harbor dmdA genes, although the largest taxonomic change was an increase in Flavobacteriaceae, a group not yet demonstrated to have DMSP-demethylating capabilities. Sequence heterogeneity in dmdA and other functional gene populations is becoming increasingly evident with the advent of high-throughput sequencing technologies, and understanding the ecological implications of this heterogeneity is a major challenge for marine microbial ecology.

Novel Pathway for Assimilation of Dimethylsulphoniopropionate Widespread in Marine Bacteria

Nature. May, 2011  |  Pubmed ID: 21562561

Dimethylsulphoniopropionate (DMSP) accounts for up to 10% of carbon fixed by marine phytoplankton in ocean surface waters, producing an estimated 11.7-103 Tmol S per year, most of which is processed by marine bacteria through the demethylation/demethiolation pathway. This pathway releases methanethiol (MeSH) instead of the climatically active gas dimethylsulphide (DMS) and enables marine microorganisms to assimilate the reduced sulphur. Despite recognition of this critical microbial transformation for over two decades, the biochemical pathway and enzymes responsible have remained unidentified. Here we show that three new enzymes related to fatty acid β-oxidation constitute the pathway that assimilates methylmercaptopropionate (MMPA), the first product of DMSP demethylation/demethiolation, and that two previously unknown coenzyme A (CoA) derivatives, 3-methylmercaptopropionyl-CoA (MMPA-CoA) and methylthioacryloyl-CoA (MTA-CoA), are formed as novel intermediates. A member of the marine roseobacters, Ruegeria pomeroyi DSS-3, requires the MMPA-CoA pathway for MMPA assimilation and MeSH production. This pathway and the ability to produce MeSH from MMPA are present in diverse bacteria, and the ubiquitous SAR11 clade bacterium Pelagibacter ubique possesses enzymes for at least the first two steps. Analysis of marine metagenomic data indicates that the pathway is widespread among bacterioplankton in the ocean surface waters, making it one of the most important known routes for acquisition of reduced carbon and sulphur by surface ocean heterotrophs.

Bacterial Catabolism of Dimethylsulfoniopropionate (DMSP)

Frontiers in Microbiology. 2011  |  Pubmed ID: 21886640

Dimethylsulfoniopropionate (DMSP) is a metabolite produced primarily by marine phytoplankton and is the main precursor to the climatically important gas dimethylsulfide (DMS). DMS is released upon bacterial catabolism of DMSP, but it is not the only possible fate of DMSP sulfur. An alternative demethylation/demethiolation pathway results in the eventual release of methanethiol, a highly reactive volatile sulfur compound that contributes little to the atmospheric sulfur flux. The activity of these pathways control the natural flux of sulfur released to the atmosphere. Although these biochemical pathways and the factors that regulate them are of great interest, they are poorly understood. Only recently have some of the genes and pathways responsible for DMSP catabolism been elucidated. Thus far, six different enzymes have been identified that catalyze the cleavage of DMSP, resulting in the release of DMS. In addition, five of these enzymes appear to produce acrylate, while one produces 3-hydroxypropionate. In contrast, only one enzyme, designated DmdA, has been identified that catalyzes the demethylation reaction producing methylmercaptopropionate (MMPA). The metabolism of MMPA is performed by a series of three coenzyme-A mediated reactions catalyzed by DmdB, DmdC, and DmdD. Interestingly, CandidatusPelagibacter ubique, a member of the SAR11 clade of Alphaproteobacteria that is highly abundant in marine surface waters, possessed functional DmdA, DmdB, and DmdC enzymes. Microbially mediated transformations of both DMS and methanethiol are also possible, although many of the biochemical and molecular genetic details are still unknown. This review will focus on the recent discoveries in the biochemical pathways that mineralize and assimilate DMSP carbon and sulfur, as well as the areas for which a comprehensive understanding is still lacking.

Bacterial Community Transcription Patterns During a Marine Phytoplankton Bloom

Environmental Microbiology. Jan, 2012  |  Pubmed ID: 21985473

Bacterioplankton consume a large proportion of photosynthetically fixed carbon in the ocean and control its biogeochemical fate. We used an experimental metatranscriptomics approach to compare bacterial activities that route energy and nutrients during a phytoplankton bloom compared with non-bloom conditions. mRNAs were sequenced from duplicate bloom and control microcosms 1 day after a phytoplankton biomass peak, and transcript copies per litre of seawater were calculated using an internal mRNA standard. Transcriptome analysis revealed a potential novel mechanism for enhanced efficiency during carbon-limited growth, mediated through membrane-bound pyrophosphatases [V-type H(+)-translocating; hppA]; bloom bacterioplankton participated less in this metabolic energy scavenging than non-bloom bacterioplankton, with possible implications for differences in growth yields on organic substrates. Bloom bacterioplankton transcribed more copies of genes predicted to increase cell surface adhesiveness, mediated by changes in bacterial signalling molecules related to biofilm formation and motility; these may be important in microbial aggregate formation. Bloom bacterioplankton also transcribed more copies of genes for organic acid utilization, suggesting an increased importance of this compound class in the bioreactive organic matter released during phytoplankton blooms. Transcription patterns were surprisingly faithful within a taxon regardless of treatment, suggesting that phylogeny broadly predicts the ecological roles of bacterial groups across 'boom' and 'bust' environmental backgrounds.

Structures of Dimethylsulfoniopropionate-dependent Demethylase from the Marine Organism Pelagabacter Ubique

Protein Science : a Publication of the Protein Society. Feb, 2012  |  Pubmed ID: 22162093

Dimethylsulfoniopropionate (DMSP) is a ubiquitous algal metabolite and common carbon and sulfur source for marine bacteria. DMSP is a precursor for the climatically active gas dimethylsulfide that is readily oxidized to sulfate, sulfur dioxide, methanesulfonic acid, and other products that act as cloud condensation nuclei. Although the environmental importance of DMSP metabolism has been known for some time, the enzyme responsible for DMSP demethylation by marine bacterioplankton, dimethylsufoniopropionate-dependent demethylase A (DmdA, EC 2.1.1.B5), has only recently been identified and biochemically characterized. In this work, we report the structure for the apoenzyme DmdA from Pelagibacter ubique (2.1 Å), as well as for DmdA co-crystals soaked with substrate DMSP (1.6 Å) or the cofactor tetrahydrofolate (THF) (1.6 Å). Surprisingly, the overall fold of the DmdA is not similar to other enzymes that typically utilize the reduced form of THF and in fact is a triple domain structure similar to what has been observed for the glycine cleavage T protein or sarcosine oxidase. Specifically, while the THF binding fold appears conserved, previous biochemical studies have shown that all enzymes with a similar fold produce 5,10-methylene-THF, while DmdA catalyzes a redox-neutral methyl transfer reaction to produce 5-methyl-THF. On the basis of the findings presented herein and the available biochemical data, we outline a mechanism for a redox-neutral methyl transfer reaction that is novel to this conserved THF binding domain.

Environmental, Biochemical and Genetic Drivers of DMSP Degradation and DMS Production in the Sargasso Sea

Environmental Microbiology. Feb, 2012  |  Pubmed ID: 22324779

Dimethylsulfide (DMS) is a climatically relevant trace gas produced and cycled by the surface ocean food web. Mechanisms driving intraannual variability in DMS production and dimethylsulfoniopropionate (DMSP) degradation in open-ocean, oligotrophic regions were investigated during a 10-month time-series at the Bermuda Atlantic Time-series Study site in the Sargasso Sea. Abundance and transcription of bacterial DMSP degradation genes, DMSP lyase enzyme activity, and DMS and DMSP concentrations, consumption rates and production rates were quantified over time and depth. This interdisciplinary data set was used to test current hypotheses of the role of light and carbon supply in regulating upper-ocean sulfur cycling. Findings supported UV-A-dependent phytoplankton DMS production. Bacterial DMSP degraders may also contribute significantly to DMS production when temperatures are elevated and UV-A dose is moderate, but may favour DMSP demethylation under low UV-A doses. Three groups of bacterial DMSP degraders with distinct intraannual variability were identified and niche differentiation was indicated. The combination of genetic and biochemical data suggest a modified 'bacterial switch' hypothesis where the prevalence of different bacterial DMSP degradation pathways is regulated by a complex set of factors including carbon supply, temperature and UV-A dose.

Bacterial Dimethylsulfoniopropionate-degrading Genes in the Oligotropic North Pacific Subtropical Gyre

Applied and Environmental Microbiology. Feb, 2012  |  Pubmed ID: 22327587

Dimethylsulfoniopropionate (DMSP) is an organic sulfur compound that is rapidly metabolized by marine bacteria either by cleavage to dimethylsulfide (DMS) or demethylation to 3-methiolpropionate. The abundance and diversity of genes encoding bacterial DMS production (dddP) and demethylation (dmdA) were measured in the North Pacific Subtropical Gyre (NPSG) between May 2008 and February 2009 at Station ALOHA (22° 45' N, 158° 00' W) at two depths: 25 m and the Deep Chlorophyll Maximum (DCM; ∼100 m). The highest abundance of dmdA genes was in May 2008 at 25 m with ∼16.5% of cells harboring a gene in one of the eight subclades surveyed, while the highest abundance of dddP genes was in July 2008 at 25 m with ∼2% of cells harboring a gene. The dmdA gene pool was consistently dominated by homologs from SAR11 subclades, which was supported by findings in metagenomic datasets derived from Station ALOHA. Expression of the SAR11 dmdA genes was low, with typical transcript:gene ratios between 1:350 and 1:1,400. Abundance of DMSP genes was statistically different between 25 m and the DCM and correlated with a number of environmental variables including primary production, photosynthetically active radiation, particulate DMSP, and DMS concentrations. At 25 m, dddP abundance was positively correlated with pigments that are diagnostic of diatoms; at the DCM, dmdA abundance was positively correlated with temperature. Based on gene abundance, we hypothesize that SAR11 bacterioplankton dominate DMSP cycling in the oligotrophic NPSG, with lesser but consistent involvement of other members of the bacterioplankton community.

Omics for Understanding Microbial Functional Dynamics

Environmental Microbiology. Jan, 2012  |  Pubmed ID: 21651688

Genome Content of Uncultivated Marine Roseobacters in the Surface Ocean

Environmental Microbiology. Jan, 2012  |  Pubmed ID: 21854517

Understanding of the ecological roles and evolutionary histories of marine bacterial taxa can be complicated by mismatches in genome content between wild populations and their better-studied cultured relatives. We used computed patterns of non-synonymous (amino acid-altering) nucleotide diversity in marine metagenomic data to provide high-confidence identification of DNA fragments from uncultivated members of the Roseobacter clade, an abundant taxon of heterotrophic marine bacterioplankton in the world's oceans. Differences in gene stoichiometry in the Global Ocean Survey metagenomic data set compared with 39 sequenced isolates indicated that natural Roseobacter populations differ systematically in several genomic attributes from their cultured representatives, including fewer genes for signal transduction and cell surface modifications but more genes for Sec-like protein secretion systems, anaplerotic CO(2) incorporation, and phosphorus and sulfate uptake. Several of these trends match well with characteristics previously identified as distinguishing r- versus K-selected ecological strategies in bacteria, suggesting that the r-strategist model assigned to cultured roseobacters may be less applicable to their free-living oceanic counterparts. The metagenomic Roseobacter DNA fragments revealed several traits with evolutionary histories suggestive of horizontal gene transfer from other marine bacterioplankton taxa or viruses, including pyrophosphatases and glycosylation proteins.