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SlrA/SinR/SlrR inhibits motility gene expression upstream of a hypersensitive and hysteretic switch at the level of ?(D) in Bacillus subtilis.

ABSTRACT: Exponentially growing Bacillus subtilis cultures are epigenetically differentiated into two subpopulations in which cells are either ON or OFF for ?(d) -dependent gene expression: a pattern suggestive of bistability. The gene encoding ?(D) , sigD, is part of the 31-gene fla/che operon where its location at the 3' end, 25 kb away from the strong P(fla/che) promoter, determines its expression level relative to a threshold. Here we show that addition of a single extra copy of the slrA gene in the chromosome inhibited ?(d) -dependent gene expression. SlrA together with SinR and SlrR reduced sigD transcript by potentiating a distance-dependent decrease in fla/che operon transcript abundance that was not mediated by changes in expression from the P(fla/che) promoter. Consistent with acting upstream of ?(D) , SlrA/SinR/SlrR was bypassed by artificial ectopic expression of sigD and hysteretically maintained for 20 generations by engaging the sigD gene at the native locus. SlrA/SinR/SlrR was also bypassed by increasing fla/che transcription and resulted in a hypersensitive output in flagellin expression. Thus, flagellin gene expression demonstrated hypersensitivity and hysteresis and we conclude that ?(d) -dependent gene expression is bistable.

SUBMITTER: Cozy LM

PROVIDER: S-EPMC3303961 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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SlrA/SinR/SlrR inhibits motility gene expression upstream of a hypersensitive and hysteretic switch at the level of σ(D) in Bacillus subtilis.

Cozy Loralyn M LM Phillips Andrew M AM Calvo Rebecca A RA Bate Ashley R AR Hsueh Yi-Huang YH Bonneau Richard R Eichenberger Patrick P Kearns Daniel B DB

Molecular microbiology 20120222 6

Exponentially growing Bacillus subtilis cultures are epigenetically differentiated into two subpopulations in which cells are either ON or OFF for σ(d) -dependent gene expression: a pattern suggestive of bistability. The gene encoding σ(D) , sigD, is part of the 31-gene fla/che operon where its location at the 3' end, 25 kb away from the strong P(fla/che) promoter, determines its expression level relative to a threshold. Here we show that addition of a single extra copy of the slrA gene in the c ...[more]

PMID: 22329926

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Project description:BackgroundMenaquinone (MK-7) is a highly valuable vitamin K2 produced by Bacillus subtilis. Common static metabolic engineering approaches for promoting the production of MK-7 have been studied previously. However, these approaches caused an accumulation of toxic substances and reduced product yield. Hence, dynamic regulation by the quorum sensing (QS) system is a promising method for achieving a balance between product synthesis and cell growth.ResultsIn this study, the QS transcriptional regulator SinR, which plays a significant role in biofilm formation and MK production simultaneously, was selected, and its site-directed mutants were constructed. Among these mutants, sinR knock out strain (KO-SinR) increased the biofilm biomass by 2.8-fold compared to the wild-type. SinRquad maximized the yield of MK-7 (102.56 ± 2.84 mg/L). To decipher the mechanism of how this mutant regulates MK-7 synthesis and to find additional potential regulators that enhance MK-7 synthesis, RNA-seq was used to analyze expression changes in the QS system, biofilm formation, and MK-7 synthesis pathway. The results showed that the expressions of tapA, tasA and epsE were up-regulated 9.79-, 0.95-, and 4.42-fold, respectively. Therefore, SinRquad formed more wrinkly and smoother biofilms than BS168. The upregulated expressions of glpF, glpk, and glpD in this biofilm morphology facilitated the flow of glycerol through the biofilm. In addition, NADH dehydrogenases especially sdhA, sdhB, sdhC and glpD, increased 1.01-, 3.93-, 1.87-, and 1.11-fold, respectively. The increased expression levels of NADH dehydrogenases indicated that more electrons were produced for the electron transport system. Electrical hyperpolarization stimulated the synthesis of the electron transport chain components, such as cytochrome c and MK, to ensure the efficiency of electron transfer. Wrinkly and smooth biofilms formed a network of interconnected channels with a low resistance to liquid flow, which was beneficial for the uptake of glycerol, and facilitated the metabolic flux of four modules of the MK-7 synthesis pathway.ConclusionsIn this study, we report for the first time that SinRquad has significant effects on MK-7 synthesis by forming wrinkly and smooth biofilms, upregulating the expression level of most NADH dehydrogenases, and providing higher membrane potential to stimulate the accumulation of the components in the electron transport system.

Project description:While the protein complex responsible for controlling the direction (clockwise [CW] or counterclockwise [CCW]) of flagellar rotation has been fairly well studied in Escherichia coli and Salmonella, less is known about the switch complex in Bacillus subtilis or other Gram-positive species. Two component proteins (FliG and FliM) are shared between E. coli and B. subtilis, but in place of the protein FliN found in E. coli, the B. subtilis complex contains the larger protein FliY. Notably, in B. subtilis the signaling protein CheY-phosphate induces a switch from CW to CCW rotation, opposite to its action in E. coli Here, we have examined the architecture and function of the switch complex in B. subtilis using targeted cross-linking, bacterial two-hybrid protein interaction experiments, and characterization of mutant phenotypes. In major respects, the B. subtilis switch complex appears to be organized similarly to that in E. coli The complex is organized around a ring built from the large middle domain of FliM; this ring supports an array of FliG subunits organized in a similar way to that of E. coli, with the FliG C-terminal domain functioning in the generation of torque via conserved charged residues. Key differences from E. coli involve the middle domain of FliY, which forms an additional, more outboard array, and the C-terminal domains of FliM and FliY, which are organized into both FliY homodimers and FliM heterodimers. Together, the results suggest that the CW and CCW conformational states are similar in the Gram-negative and Gram-positive switches but that CheY-phosphate drives oppositely directed movements in the two cases.IMPORTANCE Flagellar motility plays key roles in the survival of many bacteria and in the harmful action of many pathogens. Bacterial flagella rotate; the direction of flagellar rotation is controlled by a multisubunit protein complex termed the switch complex. This complex has been extensively studied in Gram-negative model species, but little is known about the complex in Bacillus subtilis or other Gram-positive species. Notably, the switch complex in Gram-positive species responds to its effector CheY-phosphate (CheY-P) by switching to CCW rotation, whereas in E. coli or Salmonella CheY-P acts in the opposite way, promoting CW rotation. In the work here, the architecture of the B. subtilis switch complex has been probed using cross-linking, protein interaction measurements, and mutational approaches. The results cast light on the organization of the complex and provide a framework for understanding the mechanism of flagellar direction control in B. subtilis and other Gram-positive species.

Dataset Information

SlrA/SinR/SlrR inhibits motility gene expression upstream of a hypersensitive and hysteretic switch at the level of ?(D) in Bacillus subtilis.

Publications

SlrA/SinR/SlrR inhibits motility gene expression upstream of a hypersensitive and hysteretic switch at the level of σ(D) in Bacillus subtilis.

Similar Datasets

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