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Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpF gene, which codes for a putative class A high-molecular-weight penicillin-binding protein.

ABSTRACT: The partial nucleotide sequence of a gene encoding a Bacillus subtilis homolog to the Escherichia coli ponA gene, encoding penicillin-binding protein 1A, was previously reported. The remaining part of this gene, termed pbpF, was isolated, and its nucleotide sequence was completed. Deletion of this gene did not alter the profile of B. subtilis penicillin-binding proteins observed after gel electrophoresis and resulted in no observable phenotype. A transcriptional pbpF-lacZ fusion was weakly expressed during vegetative growth. Expression diminished during the first hours of sporulation but was slightly induced in the forespore compartment during late sporulation. This sporulation expression was dependent on spoIIIG, which encodes the forespore-specific transcription factor sigma G. A single transcription start site which was apparently directly dependent on E sigma A was detected in vegetative cells.

SUBMITTER: Popham DL

PROVIDER: S-EPMC204940 | biostudies-other | 1993 Aug

REPOSITORIES: biostudies-other

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Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpF gene, which codes for a putative class A high-molecular-weight penicillin-binding protein.

Popham D L DL Setlow P P

Journal of bacteriology 19930801 15

The partial nucleotide sequence of a gene encoding a Bacillus subtilis homolog to the Escherichia coli ponA gene, encoding penicillin-binding protein 1A, was previously reported. The remaining part of this gene, termed pbpF, was isolated, and its nucleotide sequence was completed. Deletion of this gene did not alter the profile of B. subtilis penicillin-binding proteins observed after gel electrophoresis and resulted in no observable phenotype. A transcriptional pbpF-lacZ fusion was weakly expre ...[more]

PMID: 8335642

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Project description:L-Aspartase was purified from Bacillus subtilis, its N-terminal amino acid sequence was determined to construct a probe for the aspartase gene, and the gene (termed ansB) was cloned and sequenced. A second gene (termed ansA) was found upstream of the ansB gene and coded for L-asparaginase. These two genes were in an operon designated the ans operon, which is 80% cotransformed with the previously mapped aspH1 mutation at 215 degrees. Primer extension analysis of in vivo ans mRNA revealed two transcription start sites, depending on the growth medium. In wild-type cells in log-phase growth in 2x YT medium (tryptone-yeast extract rich medium), the ans transcript began at -67 relative to the translation start site, while cells in log-phase growth or sporulating (t1 to t4) in 2x SG medium (glucose nutrient broth-based moderately rich medium) had an ans transcript which began at -73. The level of the -67 transcript was greatly increased in an aspH mutant grown in 2x YT medium; the -67 transcript also predominated when this mutant was grown in 2x SG medium, although the -73 transcript was also present. In vitro transcription of the ans operon by RNA polymerase from log-phase cells grown in 2x YT medium and log-phase or sporulating cells grown in 2x SG medium yielded only the -67 transcript. Depending on the growth medium, the levels of asparaginase and aspartase were from 2- to 40-fold higher in an aspH mutant than in wild-type cells, and evidence was obtained indicating that the gene defined by the aspH1 mutation codes for a trans-acting transcriptional regulatory factor. In wild-type cells grown in 2x SG medium, the levels of both aspartase and asparaginase decreased significantly by t0 of sporulation but then showed a small increase, which was mirrored by changes in the level of beta-galactosidase from an ansB-lacZ fusion. The increase in the activities of ans operon enzymes between t2 and t5 of sporulation was found primarily in the forespore, and the great majority of the increased was found in the mature spore. However, throughout sporulation the only ans transcript detected was the -73 form, and no sporulation-specific RNA polymerase tested yielded a -73 transcript in vitro.

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

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpF gene, which codes for a putative class A high-molecular-weight penicillin-binding protein.

Publications

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpF gene, which codes for a putative class A high-molecular-weight penicillin-binding protein.

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