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Interactions among Redox Regulators and the CtrA Phosphorelay in Dinoroseobacter shibae and Rhodobacter capsulatus.

ABSTRACT: Bacteria employ regulatory networks to detect environmental signals and respond appropriately, often by adjusting gene expression. Some regulatory networks influence many genes, and many genes are affected by multiple regulatory networks. Here, we investigate the extent to which regulatory systems controlling aerobic-anaerobic energetics overlap with the CtrA phosphorelay, an important system that controls a variety of behavioral processes, in two metabolically versatile alphaproteobacteria, Dinoroseobacter shibae and Rhodobacter capsulatus. We analyzed ten available transcriptomic datasets from relevant regulator deletion strains and environmental changes. We found that in D. shibae, the CtrA phosphorelay represses three of the four aerobic-anaerobic Crp/Fnr superfamily regulator-encoding genes (fnrL, dnrD, and especially dnrF). At the same time, all four Crp/Fnr regulators repress all three phosphorelay genes. Loss of dnrD or dnrF resulted in activation of the entire examined CtrA regulon, regardless of oxygen tension. In R. capsulatus FnrL, in silico and ChIP-seq data also suggested regulation of the CtrA regulon, but it was only with loss of the redox regulator RegA where an actual transcriptional effect on the CtrA regulon was observed. For the first time, we show that there are complex interactions between redox regulators and the CtrA phosphorelays in these bacteria and we present several models for how these interactions might occur.

SUBMITTER: Koppenhofer S

PROVIDER: S-EPMC7232146 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Interactions among Redox Regulators and the CtrA Phosphorelay in Dinoroseobacter shibae and Rhodobacter capsulatus.

Koppenhöfer Sonja S Lang Andrew S AS

Microorganisms 20200414 4

Bacteria employ regulatory networks to detect environmental signals and respond appropriately, often by adjusting gene expression. Some regulatory networks influence many genes, and many genes are affected by multiple regulatory networks. Here, we investigate the extent to which regulatory systems controlling aerobic-anaerobic energetics overlap with the CtrA phosphorelay, an important system that controls a variety of behavioral processes, in two metabolically versatile alphaproteobacteria, ...[more]

PMID: 32295208

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Project description:BACKGROUND: Dinoroseobacter shibae, a member of the Roseobacter clade abundant in marine environments, maintains morphological heterogeneity throughout growth, with small cells dividing by binary fission and large cells dividing by budding from one or both cell poles. This morphological heterogeneity is lost if the quorum sensing (QS) system is silenced, concurrent with a decreased expression of the CtrA phosphorelay, a regulatory system conserved in Alphaproteobacteria and the master regulator of the Caulobacter crescentus cell cycle. It consists of the sensor histidine kinase CckA, the phosphotransferase ChpT and the transcriptional regulator CtrA. Here we tested if the QS induced differentiation of D. shibae is mediated by the CtrA phosphorelay. RESULTS: Mutants for ctrA, chpT and cckA showed almost homogeneous cell morphology and divided by binary fission. For ctrA and chpT, expression in trans on a plasmid caused the fraction of cells containing more than two chromosome equivalents to increase above wild-type level, indicating that gene copy number directly controls chromosome number. Transcriptome analysis revealed that CtrA is a master regulator for flagellar biosynthesis and has a great influence on the transition to stationary phase. Interestingly, the expression of the autoinducer synthase genes luxI2 and luxI3 was strongly reduced in all three mutants, resulting in loss of biosynthesis of acylated homoserine-lactones with C14 side-chain, but could be restored by expressing these genes in trans. Several phylogenetic clusters of Alphaproteobacteria revealed a CtrA binding site in the promoters of QS genes, including Roseobacters and Rhizobia. CONCLUSIONS: The CtrA phosphorelay induces differentiation of a marine Roseobacter strain that is strikingly different from that of C. crescentus. Instead of a tightly regulated cell cycle and a switch between two morphotypes, the morphology and cell division of Dinoroseobacter shibae are highly heterogeneous. We discovered for the first time that the CtrA phosphorelay controls the biosynthesis of signaling molecules. Thus cell-cell communication and differentiation are interlinked in this organism. This may be a common strategy, since we found a similar genetic set-up in other species in the ecologically relevant group of Alphaproteobacteria. D. shibae will be a valuable model organism to study bacterial differentiation into pleomorphic cells.

Project description:Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphotransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus, these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads.IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphotransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

Project description:The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is controlling genes for a putative Na+/H+ antiporter, indicating a potential role of a Na+ gradient under anaerobic conditions. The formation of the electron donating primary dehydrogenases is coordinated by FnrL and DnrE. Many plasmid encoded genes were DnrE regulated. DnrF is controlling directly two regulator genes including the Fe-S cluster biosynthesis regulator iscR, genes of the electron transport chain and the glutathione metabolism. The genes for nitrate reductase and CO dehydrogenase are repressed by DnrD and DnrF. Both regulators in concert with FnrL are inducing the photosynthesis genes. One of the major denitrification operon control regions, the intergenic region between nirS and nosR2, contains one Fnr/Dnr binding site. Using regulator gene mutant strains, lacZ-reporter gene fusions in combination with promoter mutagenesis, the function of the single Fnr/Dnr binding site for FnrL-, DnrD-, and partly DnrF-dependent nirS and nosR2 transcriptional activation was shown. Overall, the unique regulatory network of the marine bacterium D. shibae for the transition from aerobic to anaerobic growth composed of four Crp/Fnr family regulators was elucidated.

Project description:Several members of the Rhodobacterales (Alphaproteobacteria) produce a conserved horizontal gene transfer vector, called the gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study GTA biology is the Rhodobacter capsulatus GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the Alphaproteobacteria and is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production in R. capsulatus We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels in R. capsulatus and identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the RhodobacteralesIMPORTANCE Members of the Rhodobacterales are abundant in ocean and freshwater environments. The conserved GTA produced by many Rhodobacterales may have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA in R. capsulatus extends the model of CtrA regulation from Caulobacter crescentus to a member of the Rhodobacterales We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles.

Dataset Information

Interactions among Redox Regulators and the CtrA Phosphorelay in Dinoroseobacter shibae and Rhodobacter capsulatus.

Publications

Interactions among Redox Regulators and the CtrA Phosphorelay in <i>Dinoroseobacter shibae</i> and <i>Rhodobacter capsulatus</i>.

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