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Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpE operon, which codes for penicillin-binding protein 4* and an apparent amino acid racemase.

ABSTRACT: Penicillin-binding protein 4* (PBP 4*) was purified from Bacillus subtilis, its N-terminal sequence was determined, and the coding gene, termed pbpE, was cloned and sequenced. The predicted amino acid sequence of PBP 4* exhibited similarity to those of other penicillin-recognizing enzymes. Downstream of pbpE there was a second gene, termed orf2, which exhibited sequence similarity with aspartate racemase. The two genes were found to constitute an operon adjacent to and divergently transcribed from the sacB gene at 296 degrees on the chromosomal map. A weak beta-lactamase activity was associated with PBP 4*, but no enzymatic activity was found for the product of orf2. Mutation of pbpE, orf2, or both genes resulted in no observable effect on growth, sporulation, spore heat resistance, or spore germination. A translational pbpE-lacZ fusion was weakly expressed during vegetative growth and was significantly induced at the onset of sporulation. This induction depended on the activity of the spo0A product in relieving repression by the abrB repressor. A single transcription start site which was apparently dependent on E sigma A was detected upstream of pbpE.

SUBMITTER: Popham DL

PROVIDER: S-EPMC204609 | biostudies-other | 1993 May

REPOSITORIES: biostudies-other

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Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpE operon, which codes for penicillin-binding protein 4* and an apparent amino acid racemase.

Popham D L DL Setlow P P

Journal of bacteriology 19930501 10

Penicillin-binding protein 4* (PBP 4*) was purified from Bacillus subtilis, its N-terminal sequence was determined, and the coding gene, termed pbpE, was cloned and sequenced. The predicted amino acid sequence of PBP 4* exhibited similarity to those of other penicillin-recognizing enzymes. Downstream of pbpE there was a second gene, termed orf2, which exhibited sequence similarity with aspartate racemase. The two genes were found to constitute an operon adjacent to and divergently transcribed fr ...[more]

PMID: 8491712

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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.

Dataset Information

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpE operon, which codes for penicillin-binding protein 4* and an apparent amino acid racemase.

Publications

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpE operon, which codes for penicillin-binding protein 4* and an apparent amino acid racemase.

Similar Datasets

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