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Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

ABSTRACT: The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes.

SUBMITTER: Zuberi AR

PROVIDER: S-EPMC207063 | biostudies-other | 1991 Jan

REPOSITORIES: biostudies-other

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Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

Zuberi A R AR Bischoff D S DS Ordal G W GW

Journal of bacteriology 19910101 2

The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch les ...[more]

PMID: 1898932

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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.

Project description:The regulatory gene (degT) from Bacillus stearothermophilus NCA1503 which enhanced production of extracellular alkaline protease (Apr) was cloned in Bacillus subtilis with pTB53 as a vector. When B. subtilis MT-2 (Npr- [deficiency of neutral protease] Apr+) was transformed with the recombinant plasmid, pDT145, the plasmid carrier produced about three times more alkaline protease than did the wild-type strain. In contrast, when B. subtilis DB104 (Npr- Apr-) was used as a host, the transformant with pDT145 could not exhibit any protease activity. After construction of the deletion plasmids, DNA sequencing was done. A large open reading frame was found, and nucleotide sequence analysis showed that the degT gene was composed of 1,116 bases (372 amino acid residues, molecular weight of 41,244). A Shine-Dalgarno sequence was found nine bases upstream from the open reading frame. A B. subtilis strain carrying degT showed the following pleiotropic phenomena: (i) enhancement of production of extracellular enzymes such as alkaline protease and levansucrase, (ii) repression of autolysin activity, (iii) decrease of transformation efficiency for B. subtilis (competent cell procedure), (iv) altered control of sporulation, (v) loss of flagella, and (vi) abnormal cell division. When B. stearothermophilus SIC1 was transformed with the recombinant plasmid carrying degT, the transformants exhibited abnormal cell division. These phenomena are similar to those of the phenotypes of degSU(Hy) (hyperproduction), degQ(Hy), and degR mutants of B. subtilis. However, the amino acid sequence of the degT product (DegT) is different from those of the reported gene products. Furthermore, DegT includes a hydrophobic core region in the N-terminal portion (amino acid numbers 50 to 160), a consensus sequence for a DNA binding region (amino acid numbers 160 to 179), and a region homologous to transcription activator proteins (amino acid numbers 351 to 366). We discuss the possibility that the membrane protein DegT functions as a sensor protein and transfers the signal of environmental stimuli to the regulatory region of target genes to activate or repress transcription of the genes.

Dataset Information

Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

Publications

Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

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