Metabolic flexibility revealed in the genome of the cyst-forming α-1 proteobacterium Rhodospirillum centenum

<p>Abstract</p> <p>Background</p> <p><it>Rhodospirillum centenum </it>is a photosynthetic non-sulfur purple bacterium that favors growth in an anoxygenic, photosynthetic N₂-fixing environment. It is emerging as a genetically amenable model organism for molecular genetic analysis of cyst formation, photosynthesis, phototaxis, and cellular development. Here, we present an analysis of the genome of this bacterium.</p> <p>Results</p> <p><it>R. centenum </it>contains a singular circular chromosome of 4,355,548 base pairs in size harboring 4,105 genes. It has an intact Calvin cycle with two forms of Rubisco, as well as a gene encoding phosphoenolpyruvate carboxylase (PEPC) for mixotrophic CO₂fixation. This dual carbon-fixation system may be required for regulating internal carbon flux to facilitate bacterial nitrogen assimilation. Enzymatic reactions associated with arsenate and mercuric detoxification are rare or unique compared to other purple bacteria. Among numerous newly identified signal transduction proteins, of particular interest is a putative bacteriophytochrome that is phylogenetically distinct from a previously characterized <it>R. centenum </it>phytochrome, Ppr. Genes encoding proteins involved in chemotaxis as well as a sophisticated dual flagellar system have also been mapped.</p> <p>Conclusions</p> <p>Remarkable metabolic versatility and a superior capability for photoautotrophic carbon assimilation is evident in <it>R. centenum</it>.</p

Phosphate Flow between Hybrid Histidine Kinases CheA₃ and CheS₃ Controls <i>Rhodospirillum centenum</i> Cyst Formation

<div><p>Genomic and genetic analyses have demonstrated that many species contain multiple chemotaxis-like signal transduction cascades that likely control processes other than chemotaxis. The Che₃ signal transduction cascade from <i>Rhodospirillum centenum</i> is one such example that regulates development of dormant cysts. This Che-like cascade contains two hybrid response regulator-histidine kinases, CheA₃ and CheS₃, and a single-domain response regulator CheY₃. We demonstrate that <i>cheS₃</i> is epistatic to <i>cheA₃</i> and that only CheS₃∼P can phosphorylate CheY₃. We further show that CheA₃ derepresses cyst formation by phosphorylating a CheS₃ receiver domain. These results demonstrate that the flow of phosphate as defined by the paradigm <i>E. coli</i> chemotaxis cascade does not necessarily hold true for non-chemotactic Che-like signal transduction cascades.</p></div

Identification and characterization of putative Aeromonas spp. T3SS effectors.

The genetic determinants of bacterial pathogenicity are highly variable between species and strains. However, a factor that is commonly associated with virulent Gram-negative bacteria, including many Aeromonas spp., is the type 3 secretion system (T3SS), which is used to inject effector proteins into target eukaryotic cells. In this study, we developed a bioinformatics pipeline to identify T3SS effector proteins, applied this approach to the genomes of 105 Aeromonas strains isolated from environmental, mutualistic, or pathogenic contexts and evaluated the cytotoxicity of the identified effectors through their heterologous expression in yeast. The developed pipeline uses a two-step approach, where candidate Aeromonas gene families are initially selected using Hidden Markov Model (HMM) profile searches against the Virulence Factors DataBase (VFDB), followed by strict comparisons against positive and negative control datasets, greatly reducing the number of false positives. This approach identified 21 Aeromonas T3SS likely effector families, of which 8 represent known or characterized effectors, while the remaining 13 have not previously been described in Aeromonas. We experimentally validated our in silico findings by assessing the cytotoxicity of representative effectors in Saccharomyces cerevisiae BY4741, with 15 out of 21 assayed proteins eliciting a cytotoxic effect in yeast. The results of this study demonstrate the utility of our approach, combining a novel in silico search method with in vivo experimental validation, and will be useful in future research aimed at identifying and authenticating bacterial effector proteins from other genera

Model for regulation of Che₃ signal transduction pathway.

<p>(A) In the absence of unknown signals, CheA₃ is deactivated; CheS₃ autophosphorylates and transfers phosphates to its cognate response regulator CheY₃; activated CheY₃ then interacts with downstream components to repress cyst formation. (B) In the presence of an unknown signal (denoted by a red star), CheA₃ autophosphorylation is activated; His-phosphorylated CheA₃ constantly transfers the phosphates to its C-terminal REC domain, which serves as a phosphate sink. CheA₃∼P also phosphorylates the REC1 domain of CheS₃, inhibiting CheS₃ kinase activity and CheY₃ remains unphosphorylated. Cyst formation is therefore derepressed without activated CheY₃. The thickness of the arrows represents the level of phosphate flow.</p

Gene arrangement of the <i>R. centenum che₃</i> cluster and domain organizations of CheA₃, CheS₃, and CheY₃.

<p>Arrow length is proportional to gene length. Abbreviations: REC, receiver domain; PAS, <u>P</u>er, <u>A</u>rnt, <u>S</u>im domains; HWE_HK, HWE superfamily of histidine kinases; Hpt, histidine phosphotransfer domain; CA, catalytic and ATP-binding domain. Conserved histidine and aspartate residues as putative phosphorylation sites are denoted for each protein. The start and end amino acid positions of the receiver domains as well as those of the full proteins are also labeled according to the prediction by SMART <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004002#pgen.1004002-Schultz1" target="_blank">[48]</a>.</p

Identification of intramolecular phosphoryl transfer within CheA₃ and CheS₃.

<p>(A) CheA₃∼P is acid- and alkaline-labile, whereas the REC mutant CheA₃:D663A∼P is acid-labile and base-resistant. (B) Both CheS₃∼P and its REC mutant CheS₃:D54A are acid-labile and alkaline-stable. (C) CheA₃:D663A∼P phosphorylates CheA₃-REC truncation protein in Buffer 15 containing K⁺ and 18 mM Mg²⁺. (D) Phosphoryl transfer from CheS₃:D54A∼P to CheS₃-REC1 truncation protein was not observed in Buffer 15.</p

RMeseeartcah Abrtoiclelic Flexibility Revealed in the Genome of the Cyst-forming α-1 Proteobacterium Rhodospirillum Centenum

Background: Rhodospirillum centenum is a photosynthetic non-sulfur purple bacterium that favors growth in an anoxygenic, photosynthetic N2-fixing environment. It is emerging as a genetically amenable model organism for molecular genetic analysis of cyst formation, photosynthesis, phototaxis, and cellular development. Here, we present an analysis of the genome of this bacterium. Results: R. centenum contains a singular circular chromosome of 4,355,548 base pairs in size harboring 4,105 genes. It has an intact Calvin cycle with two forms of Rubisco, as well as a gene encoding phosphoenolpyruvate carboxylase (PEPC) for mixotrophic CO2 fixation. This dual carbon-fixation system may be required for regulating internal carbon flux to facilitate bacterial nitrogen assimilation. Enzymatic reactions associated with arsenate and mercuric detoxification are rare or unique compared to other purple bacteria. Among numerous newly identified signal transduction proteins, of particular interest is a putative bacteriophytochrome that is phylogenetically distinct from a previously characterized R. centenum phytochrome, Ppr. Genes encoding proteins involved in chemotaxis as well as a sophisticated dual flagellar system have also been mapped. Conclusions: Remarkable metabolic versatility and a superior capability for photoautotrophic carbon assimilation is evident in R. centenum.This work was supported by the U.S. National Science Foundation Phototrophic Prokaryotes Sequencing Project, grant number 0412824, by a Grantin- Aid for Creative Scientific Research (No. 17GS0314) from the Japanese Society for Promotion of Science, and a Indiana University MetaCyt grant. W.D.S. is funded by the Japanese Society for Promotion of Science Postdoctoral Fellowship for Foreign Researchers (No. P07141)