Project description:We extracted and sequenced the RNA during different stages of an 8 hour cyst germination in Rhodospirillum centenum. It has aided in defining the chronology of molecular events during dormant cell germination in this model Gram-negative, cyst-forming organism.
Project description:Adenylyl cyclases are widely distributed across all kingdoms whereas guanylyl cyclases are generally thought to be restricted to eukaryotes. Here we report that the α-proteobacterium Rhodospirillum centenum secretes cGMP when developing cysts and that a guanylyl cyclase deletion strain fails to synthesize cGMP and is defective in cyst formation. The R. centenum cyclase was purified and shown to effectively synthesize cGMP from GTP in vitro, demonstrating that it is a functional guanylyl cyclase. A homologue of the Escherichia coli cAMP receptor protein (CRP) is linked to the guanylyl cyclase and when deleted is deficient in cyst development. Isothermal calorimetry (ITC) and differential scanning fluorimetry (DSF) analyses demonstrate that the recombinant CRP homologue preferentially binds to, and is stabilized by cGMP, but not cAMP. This study thus provides evidence that cGMP has a crucial role in regulating prokaryotic development. The involvement of cGMP in regulating bacterial development has broader implications as several plant-interacting bacteria contain a similar cyclase coupled by the observation that Azospirillum brasilense also synthesizes cGMP when inducing cysts.
Project description:Rhodospirillum centenum is a purple photosynthetic bacterium that forms resting cyst cells when starved for nutrients. In this study, we demonstrate that chalcone synthase gene (chsA) expression is developmentally regulated, with expression of chsA increasing up to 86-fold upon induction of the cyst developmental cycle. Screening for mini-Tn5-induced mutants that exhibit elevated chsA::lacZ expression has led to the isolation of a set of R. centenum mutants that display increased chsA gene expression concomitant with constitutive induction of the cyst developmental cycle. These "hypercyst" mutants have lost the ability to regulate cyst cell formation in response to nutrient availability. Sequence analysis indicates that the mini-Tn5-disrupted genes code for a variety of factors, including metabolic enzymes and a large set of potential regulatory factors, including four gene products with homology to histidine sensor kinases and three with homology to response regulators. Several of the disrupted genes also have sequence similarity to che-like signal transduction components.
Project description:A chemotaxis gene cluster from the photosynthetic bacterium Rhodospirillum centenum has been cloned, sequenced, and analyzed for the control of transcription during swimmer-to-swarm cell differentiation. The first gene of the operon (cheAY) codes for a large 108-kDa polypeptide with an amino-terminal domain that is homologous to CheA and a carboxyl terminus that is homologous to CheY. cheAY is followed by cheW, an additional homolog of cheY, cheB, and cheR. Sequence analysis indicated that all of the che genes are tightly compacted with the same transcriptional polarity, suggesting that they are organized in an operon. Cotranscription of the che genes was confirmed by demonstrating through Western blot analysis that insertion of a polar spectinomycin resistance gene in cheAY results in loss of cheR expression. The promoter for the che operon was mapped by primer extension analysis as well as by the construction of promoter reporter plasmids that include several deletion intervals. This analysis indicated that the R. centenum che operon utilizes two promoters; one exhibits a sigma 70-like sequence motif, and the other exhibits a sigma 54-like motif. Expression of the che operon is shown to be relatively constant for swimmer cells which contain a single flagellum and for swarm cells that contain multiple lateral flagella.
Project description:Genomic and genetic analyses have demonstrated that many species contain multiple chemotaxis-like signal transduction cascades that likely control processes other than chemotaxis. The Che? signal transduction cascade from Rhodospirillum centenum is one such example that regulates development of dormant cysts. This Che-like cascade contains two hybrid response regulator-histidine kinases, CheA? and CheS?, and a single-domain response regulator CheY?. We demonstrate that cheS? is epistatic to cheA? and that only CheS??P can phosphorylate CheY?. We further show that CheA? derepresses cyst formation by phosphorylating a CheS? receiver domain. These results demonstrate that the flow of phosphate as defined by the paradigm E. coli chemotaxis cascade does not necessarily hold true for non-chemotactic Che-like signal transduction cascades.
Project description:Genes coding for putative CrtJ and AerR homologs were identified and characterized in the purple photosynthetic bacterium Rhodospirillum centenum (also known as Rhodocista centenaria), an organism that synthesizes photopigments even under highly aerated conditions. Mutational analysis indicated that in Rsp. centenum, gene crtJ codes for a repressor for photosynthesis gene expression as in Rhodobacter capsulatus, which exhibits a high level of oxygen repression of photosystem synthesis. In contrast to Rba. capsulatus, AerR in Rsp. centenum appears to be an aerobic activator; an aerR mutation resulted in significantly reduced levels of photopigment synthesis. Both aerR and crtJ mutants retained essentially normal levels of photosystem synthesis under anaerobic conditions, indicating that their activities are specific for aerobic photosystem synthesis. The readthrough transcript from crtE promoter, which is regulated by AerR and CrtJ, seems to be significant in maintaining the expression levels of the light harvesting I (puf) genes in Rsp. centenum. We suggest that AerR and CrtJ regulate aerobic photosystem synthesis primarily through controlling activity of the transcriptional readthrough.