Dataset Information

Architecture of divergent flagellar promoters controlled by CtrA in Rhodobacter sphaeroides.

ABSTRACT:

Background

Rhodobacter sphaeroides has two sets of flagellar genes, fla1 and fla2, that are responsible for the synthesis of two different flagellar structures. The expression of the fla2 genes is under control of CtrA. In several ?-proteobacteria CtrA is also required for the expression of the flagellar genes, but the architecture of CtrA-dependent promoters has only been studied in detail in Caulobacter crescentus. In many cases the expression of fla genes originates from divergent promoters located a few base pairs apart, suggesting a particular arrangement of the cis-acting sites.

Results

Here we characterized several control regions of the R. sphaeroides fla2 genes and analyzed in detail two regions containing the divergent promoters flgB2p-fliI2p, and fliL2p-fliF2p. Binding sites for CtrA of these promoters were identified in silico and tested by site directed mutagenesis. We conclude that each one of these promoter regions has a particular arrangement, either a single CtrA binding site for activation of fliL2p and fliF2p, or two independent sites for activation of flgB2p and fliI2p. ChIP experiments confirmed that CtrA binds to the control region containing the flgB2 and fliI2 promoters, supporting the notion that CtrA directly controls the expression of the fla2 genes. The flgB and fliI genes are syntenic and show a short intercistronic region in closely related bacterial species. We analyzed these regions and found that the arrangement of the CtrA binding sites varies considerably.

Conclusions

The results in this work reveal the arrangement of the fla2 divergent promoters showing that CtrA promotes transcriptional activation using more than a single architecture.

SUBMITTER: Rivera-Osorio A

PROVIDER: S-EPMC6180460 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Publications

Architecture of divergent flagellar promoters controlled by CtrA in Rhodobacter sphaeroides.

Rivera-Osorio Anet A Osorio Aurora A Poggio Sebastian S Dreyfus Georges G Camarena Laura L

BMC microbiology 20181010 1

<h4>Background</h4>Rhodobacter sphaeroides has two sets of flagellar genes, fla1 and fla2, that are responsible for the synthesis of two different flagellar structures. The expression of the fla2 genes is under control of CtrA. In several α-proteobacteria CtrA is also required for the expression of the flagellar genes, but the architecture of CtrA-dependent promoters has only been studied in detail in Caulobacter crescentus. In many cases the expression of fla genes originates from divergent pro ...[more]

PMID: 30305031

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Project description:Activation of the two-component system formed by CckA, ChpT, and CtrA (kinase, phosphotransferase, and response regulator, respectively) in Rhodobacter sphaeroides does not occur under the growth conditions commonly used in the laboratory. However, it is possible to isolate a gain-of-function mutant in CckA that turns the system on. Using massive parallel transcriptome sequencing (RNA-seq), we identified 321 genes that are differentially regulated by CtrA. From these genes, 239 were positively controlled and 82 were negatively regulated. Genes encoding the Fla2 polar flagella and gas vesicle proteins are strongly activated by CtrA. Genes involved in stress responses as well as several transcriptional factors are also positively controlled, whereas the photosynthetic and CO2 fixation genes are repressed. Potential CtrA-binding sites were bioinformatically identified, leading to the proposal that at least 81 genes comprise the direct regulon. Based on our results, we ponder that the transcriptional response orchestrated by CtrA enables a lifestyle in which R. sphaeroides will effectively populate the surface layer of a water body enabled by gas vesicles and will remain responsive to chemotactic stimuli using the chemosensoring system that controls the Fla2 flagellum. Simultaneously, fine-tuning of photosynthesis and stress responses will reduce the damage caused by heat and high light intensity in this water stratum. In summary, in this bacterium CtrA has evolved to control physiological responses that allow its adaptation to a particular lifestyle instead of controlling the cell cycle as occurs in other species.IMPORTANCE Cell motility in Alphaproteobacteria is frequently controlled by the CckA, ChpT, and CtrA two-component system. Under the growth conditions commonly used in the laboratory, ctrA is transcriptionally inactive in Rhodobacter sphaeroides, and motility depends on the Fla1 flagellar system that was acquired by a horizontal transfer event. Likely, the incorporation of this flagellar system released CtrA from the strong selective pressure of being the main motility regulator, allowing this two-component system to specialize and respond to some specific conditions. Identifying the genes that are directly regulated by CtrA could help us understand the conditions in which the products of this regulon are required. Massive parallel transcriptome sequencing (RNA-seq) revealed that CtrA orchestrates an adaptive response that contributes to the colonization of a particular environmental niche.

Project description:The flagellar lipoprotein FlgP has been identified in several species of bacteria, and its absence provokes different phenotypes. In this study, we show that in the alphaproteobacterium Rhodobacter sphaeroides, a ΔflgP mutant is unable to assemble the hook and the filament. In contrast, the membrane/supramembrane (MS) ring and the flagellar rod appear to be assembled. In the absence of FlgP a severe defect in the transition from rod to hook polymerization occurs. In agreement with this idea, we noticed a reduction in the amount of intracellular flagellin and the chemotactic protein CheY4, both encoded by genes dependent on σ28 This suggests that in the absence of flgP the switch to export the anti-sigma factor, FlgM, does not occur. The presence of FlgP was detected by Western blot in samples of isolated wild-type filament basal bodies, indicating that FlgP is an integral part of the flagellar structure. In this regard, we show that FlgP interacts with FlgH and FlgT, indicating that FlgP should be localized closely to the L and H rings. We propose that FlgP could affect the architecture of the L ring, which has been recently identified to be responsible for the rod-hook transition.IMPORTANCE Flagellar based motility confers a selective advantage on bacteria by allowing migration to favorable environments or in pathogenic species to reach the optimal niche for colonization. The flagellar structure has been well established in Salmonella However, other accessory components have been identified in other species. Many of these have been implied in adapting the flagellar function to enable faster rotation, or higher torque. FlgP has been proposed to be the main component of the basal disk located underlying the outer membrane in Campylobacter jejuni and Vibrio fischeri Its role is still unclear, and its absence impacts motility differently in different species. The study of these new components will bring a better understanding of the evolution of this complex organelle.

Project description:In this work, we have characterized the soluble lytic transglycosylase (SltF) from Rhodobacter sphaeroides that interacts with the scaffolding protein FlgJ in the periplasm to open space at the cell wall peptidoglycan heteropolymer for the emerging rod. The characterization of the genetic context of flgJ and sltF in alphaproteobacteria shows that these two separate genes coexist frequently in a flagellar gene cluster. Two domains of unknown function in SltF were studied, and the results show that the deletion of a 17-amino-acid segment near the N terminus does not show a recognizable phenotype, whereas the deletion of 47 and 95 amino acids of the C terminus of SltF disrupts the interaction with FlgJ without affecting the transglycosylase catalytic activity of SltF. These mutant proteins are unable to support swimming, indicating that the physical interaction between SltF and FlgJ is central for flagellar formation. In a maximum likelihood tree of representative lytic transglycosylases, all of the flagellar SltF proteins cluster in subfamily 1F. From this analysis, it was also revealed that the lytic transglycosylases related to the type III secretion systems present in pathogens cluster with the closely related flagellar transglycosylases.IMPORTANCE Flagellar biogenesis is a highly orchestrated event where the flagellar structure spans the bacterial cell envelope. The rod diameter of approximately 4 nm is larger than the estimated pore size of the peptidoglycan layer; hence, its insertion requires the localized and controlled lysis of the cell wall. We found that a 47-residue domain of the C terminus of the lytic transglycosylase (LT) SltF of R. sphaeroides is involved in the recognition of the rod chaperone FlgJ. We also found that in many alphaproteobacteria, the flagellar cluster includes a homolog of SltF and FlgJ, indicating that association of an LT with the flagellar machinery is ancestral. A maximum likelihood tree shows that family 1 of LTs segregates into seven subfamilies.

Dataset Information

Architecture of divergent flagellar promoters controlled by CtrA in Rhodobacter sphaeroides.

Background

Results

Conclusions

Publications

Architecture of divergent flagellar promoters controlled by CtrA in Rhodobacter sphaeroides.

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