Dataset Information

Cloning, nucleotide sequence, and mutagenesis of the Bacillus subtilis ponA operon, which codes for penicillin-binding protein (PBP) 1 and a PBP-related factor.

ABSTRACT: An oligonucleotide probe designed to hybridize to genes encoding class A high-molecular-weight penicillin-binding proteins (PBPs) was used to identify the ponA gene encoding PBP1a and -1b (PBP1) of Bacillus subtilis. The identity of the ponA product was established by (i) the presence of a sequence coding for a peptide generated from PBP1 and (ii) the disappearance of PBP1 in a ponA mutant. DNA sequence analysis revealed that the amino acid sequence of PBP1 was similar to those of other class A high-molecular-weight PBPs and that ponA appeared to be cotranscribed with an upstream gene (termed prfA) of unknown function. Null mutations in ponA resulted in a slight decrease in growth rate and a change in colony morphology but had no significant effect on cell morphology, cell division, sporulation, spore heat resistance, or spore germination. Mutations in prfA which did not effect ponA expression produced a more significant decrease in growth rate but had no other significant phenotypic effects. Deletion of both prfA and ponA resulted in extremely slow growth and a reduction in sporulation efficiency. Studies of expression of transcriptional fusions of ponA and prfA to lacZ demonstrated that these two genes constitute an operon. Expression of these genes was relatively constant during growth, decreased during sporulation, and was induced approximately 15 min into spore germination. The ponA locus was mapped to the 200 degrees region of the chromosomal physical map.

SUBMITTER: Popham DL

PROVIDER: S-EPMC176595 | biostudies-other | 1995 Jan

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Cloning, nucleotide sequence, and mutagenesis of the Bacillus subtilis ponA operon, which codes for penicillin-binding protein (PBP) 1 and a PBP-related factor.

Popham D L DL Setlow P P

Journal of bacteriology 19950101 2

An oligonucleotide probe designed to hybridize to genes encoding class A high-molecular-weight penicillin-binding proteins (PBPs) was used to identify the ponA gene encoding PBP1a and -1b (PBP1) of Bacillus subtilis. The identity of the ponA product was established by (i) the presence of a sequence coding for a peptide generated from PBP1 and (ii) the disappearance of PBP1 in a ponA mutant. DNA sequence analysis revealed that the amino acid sequence of PBP1 was similar to those of other class A ...[more]

PMID: 7814321

Similar Datasets

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 mutagenesis of the Bacillus subtilis ponA operon, which codes for penicillin-binding protein (PBP) 1 and a PBP-related factor.

Publications

Cloning, nucleotide sequence, and mutagenesis of the Bacillus subtilis ponA operon, which codes for penicillin-binding protein (PBP) 1 and a PBP-related factor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets