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Cloning, nucleotide sequence, mutagenesis, and mapping of the Bacillus subtilis pbpD gene, which codes for penicillin-binding protein 4.

ABSTRACT: The gene encoding penicillin-binding protein 4 (PBP 4) of Bacillus subtilis, pbpD, was cloned by two independent methods. PBP 4 was purified, and the amino acid sequence of a cyanogen bromide digestion product was used to design an oligonucleotide probe for identification of the gene. An oligonucleotide probe designed to hybridize to genes encoding class A high-molecular-weight PBPs also identified this gene. DNA sequence analysis of the cloned DNA revealed that (i) the amino acid sequence of PBP 4 was similar to those of other class A high-molecular-weight PBPs and (ii) pbpD appeared to be cotranscribed with a downstream gene (termed orf2) of unknown function. The orf2 gene is followed by an apparent non-protein-coding region which exhibits nucleotide sequence similarity with at least two other regions of the chromosome and which has a high potential for secondary structure formation. Mutations in pbpD resulted in the disappearance of PBP 4 but had no obvious effect on growth, cell division, sporulation, spore heat resistance, or spore germination. Expression of a transcriptional fusion of pbpD to lacZ increased throughout growth, decreased during sporulation, and was induced approximately 45 min into spore germination. A single transcription start site was detected just upstream of pbpD. The pbpD locus was mapped to the 275 to 280 degrees region of the chromosomal genetic map.

SUBMITTER: Popham DL

PROVIDER: S-EPMC197107 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Cloning, nucleotide sequence, mutagenesis, and mapping of the Bacillus subtilis pbpD gene, which codes for penicillin-binding protein 4.

Popham D L DL Setlow P P

Journal of bacteriology 19941201 23

The gene encoding penicillin-binding protein 4 (PBP 4) of Bacillus subtilis, pbpD, was cloned by two independent methods. PBP 4 was purified, and the amino acid sequence of a cyanogen bromide digestion product was used to design an oligonucleotide probe for identification of the gene. An oligonucleotide probe designed to hybridize to genes encoding class A high-molecular-weight PBPs also identified this gene. DNA sequence analysis of the cloned DNA revealed that (i) the amino acid sequence of PB ...[more]

PMID: 7961491

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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, mutagenesis, and mapping of the Bacillus subtilis pbpD gene, which codes for penicillin-binding protein 4.

Publications

Cloning, nucleotide sequence, mutagenesis, and mapping of the Bacillus subtilis pbpD gene, which codes for penicillin-binding protein 4.

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