A Model Roseobacter, Ruegeria pomeroyi DSS-3, Employs a Diffusible Killing Mechanism To Eliminate Competitors.
ABSTRACT: The Roseobacter clade is a group of alphaproteobacteria that have diverse metabolic and regulatory capabilities. They are abundant in marine environments and have a substantial role in marine ecology and biogeochemistry. However, interactions between roseobacters and other bacterioplankton have not been extensively explored. In this study, we identify a killing mechanism in the model roseobacter Ruegeria pomeroyi DSS-3 by coculturing it with a group of phylogenetically diverse bacteria. The killing mechanism is diffusible and occurs when cells are grown both on surfaces and in suspension and is dependent on cell density. A screen of random transposon mutants revealed that the killing phenotype, as well as resistance to killing, require genes within an ?8-kb putative gamma-butyrolactone synthesis gene cluster, which resembles similar pheromone-sensing systems in actinomycetes that regulate secondary metabolite production, including antimicrobials. Transcriptomics revealed the gene cluster is highly upregulated in wild-type DSS-3 compared to a nonkiller mutant when grown in liquid coculture with a roseobacter target. Our findings show that R. pomeroyi has the capability to eliminate closely and distantly related competitors, providing a mechanism to alter the community structure and function in its native habitats.IMPORTANCE Bacteria carry out critical ecological and biogeochemical processes and form the foundations of ecosystems. Identifying the factors that influence microbial community composition and the functional capabilities encoded within them is key to predicting how microbes impact an ecosystem. Because microorganisms must compete for limited space and nutrients to promote their own propagation, they have evolved diverse mechanisms to outcompete or kill competitors. However, the genes and regulatory strategies that promote such competitive abilities are largely underexplored, particularly in free-living marine bacteria. Here, genetics and omics techniques are used to investigate how a model marine bacterium is capable of quickly eliminating natural competitors in coculture. We determined that a previously uncharacterized horizontally acquired gene cluster is required for this bacterium to kill diverse competitors. This work represents an important step toward understanding the mechanisms bacterial populations can use to become dominant members in marine microbial communities.
Project description:Ruegeria pomeroyi DSS-3 is a model Roseobacter marine bacterium, particularly regarding its catabolism of dimethylsulfoniopropionate (DMSP), an abundant anti-stress molecule made by marine phytoplankton. We found a novel gene, dddW, which encodes a DMSP lyase that cleaves DMSP into acrylate plus the environmentally important volatile dimethyl sulfide (DMS). Mutations in dddW reduced, but did not abolish DMS production. Transcription of dddW was greatly enhanced by pre-growth of cells with DMSP, via a LysR-type regulator. Close DddW homologs occur in only one other Roseobacter species, and there are no close homologs and only a few related sequences in metagenomes of marine bacteria. In addition to DddW, R. pomeroyi DSS-3 had been shown to have two other, different, DMSP lyases, DddP and DddQ, plus an enzyme that demethylates DMSP, emphasizing the importance of this substrate for this model bacterium.
Project description:We report the complete genome sequences of five bacteriophages infecting Ruegeria pomeroyi DSS-3, a member of the marine Roseobacter lineage. The genomic sequences of these five bacteriophages are almost identical and are closely related to members of the Chivirus genus. The genes associated with the lysogenic cycle were also found.
Project description: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.
Project description:Trimethylamine N-oxide (TMAO) is an important nitrogen source for marine bacteria. TMAO can also be metabolized by marine bacteria into volatile methylated amines, the precursors of the greenhouse gas nitrous oxide. However, it was not known how TMAO is recognized and imported by bacteria. Ruegeria pomeroyi DSS-3, a marine Roseobacter, has an ATP-binding cassette transporter, TmoXWV, specific for TMAO. TmoX is the substrate-binding protein of the TmoXWV transporter. In this study, the substrate specificity of TmoX of R. pomeroyi DSS-3 was characterized. We further determined the structure of the TmoX/TMAO complex and studied the TMAO-binding mechanism of TmoX by biochemical, structural, and mutational analyses. A Ca(2+) ion chelated by an extended loop in TmoX was shown to be important for maintaining the stability of TmoX. Molecular dynamics simulations indicate that TmoX can alternate between "open" and "closed" states for binding TMAO. In the substrate-binding pocket, four tryptophan residues interact with the quaternary amine of TMAO by cation-? interactions, and Glu131 forms a hydrogen bond with the polar oxygen atom of TMAO. The ?-? stacking interactions between the side chains of Phe and Trp are also essential for TMAO binding. Sequence analysis suggests that the TMAO-binding mechanism of TmoX may have universal significance in marine bacteria, especially in the marine Roseobacter clade. This study sheds light on how marine microorganisms utilize TMAO.Trimethylamine N-oxide (TMAO) is an important nitrogen source for marine bacteria. The products of TMAO metabolized by bacteria are part of the precursors of the greenhouse gas nitrous oxide. It is unclear how TMAO is recognized and imported by bacteria. TmoX is the substrate-binding protein of a TMAO-specific transporter. Here, the substrate specificity of TmoX of Ruegeria pomeroyi DSS-3 was characterized. The TMAO-binding mechanism of TmoX was studied by biochemical, structural, and mutational analyses. Moreover, our results suggest that the TMAO-binding mechanism may have universal significance in marine bacteria. This study sheds light on how marine microorganisms utilize TMAO and should lead to a better understanding of marine nitrogen cycling.
Project description:The marine roseobacter-clade affiliated cluster (RCA) represents one of the most abundant groups of bacterioplankton in the global oceans, particularly in temperate and sub-polar regions. They play a key role in the biogeochemical cycling of various elements and are important players in oceanic climate-active trace gas metabolism. In contrast to copiotrophic roseobacter counterparts such as <i>Ruegeria pomeroyi</i> DSS-3 and <i>Phaeobacter</i> sp. MED193, RCA bacteria are truly pelagic and have smaller genomes. We have previously shown that RCA bacteria do not appear to encode the PlcP-mediated lipid remodeling pathway, whereby marine heterotrophic bacteria remodel their membrane lipid composition in response to phosphorus (P) stress by substituting membrane glycerophospholipids with alternative glycolipids or betaine lipids. In this study, we report lipidomic analysis of six RCA isolates. In addition to the commonly found glycerophospholipids such as phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), RCA bacteria synthesize a relatively uncommon phospholipid, acylphosphatidylglycerol, which is not found in copiotrophic roseobacters. Instead, like the abundant SAR11 clade, RCA bacteria upregulate ornithine lipid biosynthesis in response to P stress, suggesting a key role of this aminolipid in the adaptation of marine heterotrophs to oceanic nutrient limitation.
Project description:Bacteria in the Roseobacter lineage have been studied extensively due to their significant biogeochemical roles in the marine ecosystem. However, our knowledge on bacteriophage which infects the Roseobacter clade is still very limited. Here, we report a new bacteriophage, phage DSS3?8, which infects marine roseobacter Ruegeria pomeroyi DSS-3. DSS3?8 is a lytic siphovirus. Genomic analysis showed that DSS3?8 is most closely related to a group of siphoviruses, CbK-like phages, which infect freshwater bacterium Caulobacter crescentus. DSS3?8 contains a smaller capsid and has a reduced genome size (146?kb) compared to the CbK-like phages (205-279?kb). DSS3?8 contains the DNA polymerase gene which is closely related to T7-like podoviruses. DSS3?8 also contains the integrase and repressor genes, indicating its potential to involve in lysogenic cycle. In addition, four GTA (gene transfer agent) genes were identified in the DSS3?8 genome. Genomic analysis suggests that DSS3?8 is a highly mosaic phage that inherits the genetic features from siphoviruses, podoviruses, prophages and GTAs. This is the first report of CbK-like phages infecting marine bacteria. We believe phage isolation is still a powerful tool that can lead to discovery of new phages and help interpret the overwhelming unknown sequences in the viral metagenomics.
Project description:Most of the energy that is introduced into the oceans by photosynthetic primary producers is in the form of organic matter that then sustains the rest of the food web, from micro to macro-organisms. However, it is the interactions between phototrophs and heterotrophs that are vital to maintaining the nutrient balance of marine microbiomes that ultimately feed these higher trophic levels. The primary produced organic matter is mostly remineralized by heterotrophic microorganisms but, because most of the oceanic dissolved organic matter is in the form of biopolymers, and microbial membrane transport systems operate with molecules <0.6 kDa, it must be hydrolyzed outside the cell before a microorganism can acquire it. As a simili of the marine microbiome, we analyzed, using state-of-the-art proteomics, the exoproteomes obtained from synthetic communities combining specific Roseobacter (Ruegeria pomeroyi DSS-3, Roseobacter denitrificans OCh114, and Dinoroseobacter shibae DFL-12) and Synechococcus strains (WH7803 and WH8102). This approach identified the repertoire of hydrolytic enzymes secreted by Roseobacter, opening up the black box of heterotrophic transformation/remineralization of biopolymers generated by marine phytoplankton. As well as highlighting interesting exoenzymes this strategy also allowed us to infer clues on the molecular basis of niche partitioning.
Project description:Dimethylsufoniopropionate (DMSP) is an important and abundant organic sulfur compound and an important substrate for marine bacterioplankton. The Roseobacter clade of marine alpha-proteobacteria, including Silicibacter pomeroyi strain DSS3, are known to be a key phylogenetic group involved in DMSP degradaton. 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 interesting 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 or Acetate in a time series experiment. We identified a number of DMSP-upregulated genes that could be assigned to potential roles in the metabolization of DMSP. DMSP also affected the transcription of other groups of genes, including genes for transport and metabolization of peptides, amino-acids and polyamines. High DMSP concentrations may be a chemical signal indicating phytoplankton abundance and elicit a regulatory response aimed at making maximum use of the available nutrients under these conditions. Keywords: Microarray, marine bacterium, messenger RNA, transcription, sulfur metabolism The array design is based on the complete genome sequence of S. pomeroyi strain DSS 3 and available from Genbank (Accession numbers CP000031 and CP000032). Probes for all identified potential genes were designed by Combimatrix using proprietary software. A total of 4161 genes out of the 4348 identified potential genes on the S. pomeroyi genome are represented on the array. When possible, two probes per gene were designed.
Project description:Bacteria of the marine Roseobacter clade are characterised by their ability to utilise a wide range of organic and inorganic compounds to support growth. Trimethylamine (TMA) and trimethylamine N-oxide (TMAO) are methylated amines (MA) and form part of the dissolved organic nitrogen pool, the second largest source of nitrogen after N2 gas, in the oceans. We investigated if the marine heterotrophic bacterium, Ruegeria pomeroyi DSS-3, could utilise TMA and TMAO as a supplementary energy source and whether this trait had any beneficial effect on growth. In R. pomeroyi, catabolism of TMA and TMAO resulted in the production of intracellular ATP which in turn helped to enhance growth rate and growth yield as well as enhancing cell survival during prolonged energy starvation. Furthermore, the simultaneous use of two different exogenous energy sources led to a greater enhancement of chemoorganoheterotrophic growth. The use of TMA and TMAO primarily as an energy source resulted in the remineralisation of nitrogen in the form of ammonium, which could cross feed into another bacterium. This study provides greater insight into the microbial metabolism of MAs in the marine environment and how it may affect both nutrient flow within marine surface waters and the flux of these climatically important compounds into the atmosphere.
Project description:Members of the marine Roseobacter clade can degrade dimethylsulfoniopropionate (DMSP) via competing pathways releasing either methanethiol (MeSH) or dimethyl sulfide (DMS). Deuterium-labeled [(2)H6]DMSP and the synthetic DMSP analogue dimethyltelluriopropionate (DMTeP) were used in feeding experiments with the Roseobacter clade members Phaeobacter gallaeciensis DSM 17395 and Ruegeria pomeroyi DSS-3, and their volatile metabolites were analyzed by closed-loop stripping and solid-phase microextraction coupled to GC-MS. Feeding experiments with [(2)H6]DMSP resulted in the incorporation of a deuterium label into MeSH and DMS. Knockout of relevant genes from the known DMSP demethylation pathway to MeSH showed in both species a residual production of [(2)H3]MeSH, suggesting that a second demethylation pathway is active. The role of DMSP degradation pathways for MeSH and DMS formation was further investigated by using the synthetic analogue DMTeP as a probe in feeding experiments with the wild-type strain and knockout mutants. Feeding of DMTeP to the R. pomeroyi knockout mutant resulted in a diminished, but not abolished production of demethylation pathway products. These results further corroborated the proposed second demethylation activity in R. pomeroyi. Isotopically labeled [(2)H3]methionine and (34)SO4 (2-), synthesized from elemental (34)S8, were tested to identify alternative sulfur sources besides DMSP for the MeSH production in P. gallaeciensis. Methionine proved to be a viable sulfur source for the MeSH volatiles, whereas incorporation of labeling from sulfate was not observed. Moreover, the utilization of selenite and selenate salts by marine alphaproteobacteria for the production of methylated selenium volatiles was explored and resulted in the production of numerous methaneselenol-derived volatiles via reduction and methylation. The pathway of selenate/selenite reduction, however, proved to be strictly separated from sulfate reduction.