Silicibacter Sp Research Articles

Development of a real-time quantitative PCR assay for detection and quantification of the marine bacterium Alteromonas macleodii from coastal environments

Alteromonas macleodii is a ubiquitous marine bacterial species found in a variety of habitats that displays both planktonic and particle-associated lifestyles. Transcriptomic studies demonstrate that, even when present at low abundance, it can make significant contributions to biogeochemical cycles, and its specific association with key marine phytoplankton species indicates other ecological roles as well. It has also been shown to be one of the early colonizers of copper-treated marine vessels. There currently exist no rapid, reliable molecular assays for the detection and quantification of A. macleodii from its different environments. We developed a real-time PCR assay, specific to A. macleodii. This assay targets the DNA gyrase B subunit (gyrB) gene, which occurs as a single copy in the genome. The assay possesses an amplification efficiency of 94.3%, with a limit of detection of 2.5 gyrB copies per μL. Assay specificity was validated by melt curve analysis, followed by sequencing of the amplified product. The assay was specific to thirteen A. macleodii strains and did not amplify other marine bacteria, including Roseobacter denitrificans, Silicibacter sp. TM1040, Vibrio coralliilyticus, Vibrio harveyi, and Vibrio alginolyticus. It also did not amplify Alteromonas mediterranea, a close relative that can occur in the same environment as A. macleodii. This assay was used to determine the presence and abundance of A. macleodii from a range of coastal habitats. The assay was also used to monitor the A. macleodii growth in biofilm and planktonic cultures over time in the presence of elevated copper. This assay provides a rapid and reliable means to assess the presence and abundance of a ubiquitous marine bacterium that, even at low abundance, has been shown to make significant contributions to key marine processes.

A novel pathway for bacterial nitric oxide signaling (614.3)

Nitric oxide (NO) serves dual biological roles as both a signaling molecule and cytotoxic agent. In higher animals including mammals, NO is synthesized by nitric oxide synthase (NOS), and NO signaling involves the heme‐nitric oxide/oxygen‐binding (H‐NOX) domain of soluble guanylate cyclase as a selective sensor. Bacterial NOS and H‐NOX proteins have been reported, but their functions are less well understood. We have recently identified two organisms that contain a predicted NOS, H‐NOX, and downstream histidine kinase, which are constituents in a two‐component signaling system, suggesting that these organisms are capable of both NO synthesis and sensing. In Silicibacter sp. TrichCH4B, we have shown that NO regulates cellular c‐di‐GMP levels and biofilm formation through two‐component signaling. Furthermore, this NOS is activated by a protein secreted by the algae Trichodesmium, which occupies the same habitat as Silicibacter. Therefore, NO could serve as a quorum sensing molecule that mediates Silicibacter biofilm behavior in a symbiotic relationship with Trichodesmium.Grant Funding Source: Supported by the NSF Graduate Research Fellowship

Chemotactic response of marine bacteria to the extracellular products of Synechococcus and Prochlorococcus

The cyanobacterial genera Prochlorococcus and Synechococcus are key phototrophic organisms in the open ocean, and ecological interactions between these groups and heterotrophic bacteria have fundamental importance for marine carbon and nutrient cycling. We applied a microfluidic chemotaxis assay to study the chemotactic response of 3 marine bacterial isolates (Pseudoalteromonas haloplanktis, Vibrio alginolyticus and Silicibacter sp. TM1040) to the chemical products of Synechococcus elongatus and Prochlorococcus marinus MED 4Ax. In the ocean, chemi- cal products from these cyanobacteria may be released into the water column via extracellular exu- dation or cell lysis. Strong and rapid chemotactic responses by all 3 bacterial strains occurred in reac- tion to the Synechococcus products. P. haloplanktis cells exhibited the strongest attraction, accumulating within the chemoattractant band in concentrations of up to 9-fold above background levels. Positive chemotaxis to Prochlorococcus chemical products also occurred, with P. haloplanktis and Silicibacter sp. TM1040 exhibiting the strongest responses. These observations indicate that marine bacteria can exhibit behavioural responses to the chemical products of Synechococcus and Prochlorococcus, which may support ecological associations between these important populations of marine prokaryotes in the environment.

Genetic Determinants ofSilicibactersp. TM1040 Motility

Silicibacter sp. TM1040 is a member of the marine Roseobacter clade of Alphaproteobacteria that forms symbioses with unicellular eukaryotic phytoplankton, such as dinoflagellates. The symbiosis is complex and involves a series of steps that physiologically change highly motile bacteria into cells that readily form biofilms on the surface of the host. The initial phases of symbiosis require bacterial motility and chemotaxis that drive the swimming bacteria toward their planktonic host. Cells lacking wild-type motility fail to establish biofilms on host cells and do not produce effective symbioses, emphasizing the importance of understanding the molecular mechanisms controlling flagellar biosynthesis and the biphasic "swim-or-stick" switch. In the present study, we used a combination of bioinformatic and genetic approaches to identify the genes critical for swimming of Silicibacter sp. TM1040. More than 40 open reading frames with homology to known flagellar structural and regulatory genes were identified, most of which are organized into approximately eight operons comprising a 35.4-kb locus, with surprising similarity to the fla2 locus of Rhodobacter sphaeroides. The genome has homologs of CckA, CtrA, FlbT, and FlaF, proteins that in Caulobacter crescentus regulate flagellum biosynthesis. In addition, we uncovered three novel genes, flaB, flaC, and flaD, which encode flagellar regulatory proteins whose functions are likely to involve regulation of motor function (FlaD) and modulation of the swim-or-stick switch (FlaC). The data support the conclusion that Silicibacter sp. TM1040 uses components found in other Alphaproteobacteria, as well as novel molecular mechanisms, to regulate the expression of the genes required for motility and biofilm formation. These unique molecular mechanisms may enhance the symbiosis and survival of Roseobacter clade bacteria in the marine environment.

Phylogenomics and signature proteins for the alpha Proteobacteria and its main groups

BackgroundAlpha proteobacteria are one of the largest and most extensively studied groups within bacteria. However, for these bacteria as a whole and for all of its major subgroups (viz. Rhizobiales, Rhodobacterales, Rhodospirillales, Rickettsiales, Sphingomonadales and Caulobacterales), very few or no distinctive molecular or biochemical characteristics are known.ResultsWe have carried out comprehensive phylogenomic analyses by means of Blastp and PSI-Blast searches on the open reading frames in the genomes of several α-proteobacteria (viz. Bradyrhizobium japonicum, Brucella suis, Caulobacter crescentus, Gluconobacter oxydans, Mesorhizobium loti, Nitrobacter winogradskyi, Novosphingobium aromaticivorans, Rhodobacter sphaeroides 2.4.1, Silicibacter sp. TM1040, Rhodospirillum rubrum and Wolbachia (Drosophila) endosymbiont). These studies have identified several proteins that are distinctive characteristics of all α-proteobacteria, as well as numerous proteins that are unique repertoires of all of its main orders (viz. Rhizobiales, Rhodobacterales, Rhodospirillales, Rickettsiales, Sphingomonadales and Caulobacterales) and many families (viz. Rickettsiaceae, Anaplasmataceae, Rhodospirillaceae, Acetobacteraceae, Bradyrhiozobiaceae, Brucellaceae and Bartonellaceae). Many other proteins that are present at different phylogenetic depths in α-proteobacteria provide important information regarding their evolution. The evolutionary relationships among α-proteobacteria as deduced from these studies are in excellent agreement with their branching pattern in the phylogenetic trees and character compatibility cliques based on concatenated sequences for many conserved proteins. These studies provide evidence that the major groups within α-proteobacteria have diverged in the following order: (Rickettsiales(Rhodospirillales (Sphingomonadales (Rhodobacterales (Caulobacterales-Parvularculales (Rhizobiales)))))). We also describe two conserved inserts in DNA Gyrase B and RNA polymerase beta subunit that are distinctive characteristics of the Sphingomonadales and Rhodosprilllales species, respectively. The results presented here also provide support for the grouping of Hyphomonadaceae and Parvularcula species with the Caulobacterales and the placement of Stappia aggregata with the Rhizobiaceae group.ConclusionThe α-proteobacteria-specific proteins and indels described here provide novel and powerful means for the taxonomic, biochemical and molecular biological studies on these bacteria. Their functional studies should prove helpful in identifying novel biochemical and physiological characteristics that are unique to these bacteria.

Motility is involved in Silicibacter sp. TM1040 interaction with dinoflagellates

Silicibacter sp. TM1040, originally isolated from a culture of the dinoflagellate Pfiesteria piscicida, senses and responds to the dinoflagellate secondary metabolite dimethylsulfoniopropionate (DMSP) by flagella-mediated chemotaxis behaviour. In this report we show that swimming motility is important for initiating the interaction between the bacterium and dinoflagellate. Following transposon mutagenesis, three mutants defective in wild-type swimming motility (Mot-) were identified. The defects in motility were found to be in homologues of cckA and ctrA, encoding a two-component regulatory circuit, and in a novel gene, flaA, likely to function in flagellar export or biogenesis. Mutation of flaA or cckA results in the loss of flagella and non-motile cells (Fla-), while CtrA- cells possess flagella, but have reduced motility due to increased cell length. All three Mot- mutants were defective in attaching to the dinoflagellate, particularly to regions that colocalized with intracellular organelles. The growth rate of the dinoflagellates was reduced in the presence of the Fla- mutants compared with Fla+ cells. These results indicate that bacterial motility is important for the Silicibacter sp. TM1040-P. piscicida interaction.