Alison Buchan

Overview of the Marine Roseobacter Lineage

Despite the overwhelming bacterial diversity present in the worlds oceans, the majority of recognized marine bacteria fall into as few as nine major clades (36), many of which have yet to be cultivated in the laboratory. Molecular-based approaches targeting 16S rRNA genes demonstrate that the Roseobacter clade is one of these major marine groups, typically comprising upwards of 20% of coastal and 15% of mixed-layer ocean bacterioplankton communities (see, e.g., references 36, 37, 42, 98, and 109). Roseobacters are well represented across diverse marine habitats, from coastal to open oceans and from sea ice to sea floor (see, e.g., references 16, 28, 37, 42, 52, and 98). Members have been found to be free living, particle associated, or in commensal relationships with marine phytoplankton, invertebrates, and vertebrates (see, e.g., references 4, 6, 7, 44, 49, 115, and 119). Furthermore, representatives of the clade stand out as representing one of the most readily cultivated of the major marine lineages (36). These isolated representatives are serving as the foundation for an improved understanding of marine bacterial ecology and physiology.

Ecological Genomics of Marine Roseobacters

ABSTRACT 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.

Master recyclers: features and functions of bacteria associated with phytoplankton blooms.

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

Analysis of microbial gene transcripts in environmental samples.

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

Key aromatic-ring-cleaving enzyme, protocatechuate 3,4-dioxygenase, in the ecologically important marine Roseobacter lineage.

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

Dynamics of Bacterial and Fungal Communities on Decaying Salt Marsh Grass

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

Chitinase Gene Sequences Retrieved from Diverse Aquatic Habitats Reveal Environment-Specific Distributions

ABSTRACT 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.

Surface Colonization by Marine Roseobacters: Integrating Genotype and Phenotype

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

The elemental composition of virus particles: implications for marine biogeochemical cycles.

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

A multitrophic model to quantify the effects of marine viruses on microbial food webs and ecosystem processes

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