Penicillinase from Bacillus licheniformis: nucleotide sequence of the gene and implications for the biosynthesis of a secretory protein in a Gram-positive bacterium

Neugebauer, Kristina; Sprengel, Rolf; Schaller, Heinz

doi:10.1093/nar/9.11.2577

Cited by 161 publications

(57 citation statements)

References 32 publications

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“…mRNA has been previously determined by RNA sequencing (30); comparison of this site to the DNA sequence and assuming the use of the putative blaP transcriptional terminator (33) indicates an exact size for in vitro blaP mRNA of 1,180 bases. In contrast, the size of the major in vivo blaP mRNA, as determined on glyoxal gels by comparison with HaeIII-restricted 4X174 DNA, was about 1.3 kb.…”

Section: Methodsmentioning

confidence: 99%

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Transcriptional analysis of beta-lactamase regulation in Bacillus licheniformis

Salerno¹,

Lampen²

1986

J Bacteriol

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The expression of the blaP gene for the ,B-lactamase of Bacillus licheniformis was examined by transcriptional analyses. Radiolabeled probes containing the blaP gene or various regions 3' or 5' to it were used to analyze RNA samples prepared from induced and uninduced cultures of wild-type and mutant B. licheniformis strains.The level of blaP mRNA was low in uninduced wild-type cells. At 37°C, blaP mRNA levels reached a maximum 1 h after induction while rising up to 180-fold and then declined, but remained severalfold above the uninduced level for seyetal hours. The rate of P-lactamase synthesis was roughly proportional to the levels of blaP mRNA in both wild-type and mutant strains, indicating that regulation of (-lactamase formation occurs primarily at the level of transcription. Turnover of blaP mRNA in the presence of rifampin was rapid, giving a blaP mRNA half-life of about 2 min. Yet, high levels of blaP mRNA were maintained for at least 1 h after removal of free inducer. Three blaP mRNAs of 1.2, 2.9, and 3.4 kilobases were produced from the blaP promoter. The most abundant made up about 97% of all blaP transcripts and was also the smnallest, ending at a transcriptional terminator located about 60 bases 3' to the blaP structural gene. Variables such as incubation temperature, cytotoxicity of inducer, and type of strain had essentially no effect on the ratio of large blaP mRNA to total blaP mRNA. The 2.9-and 3.4-kilobase blaP mRNAs identify potential locations of genetically linked regulators of ,l-lactamase synthesis.P-Lactamases can be classified into three groups on the basis of enzyme structure (3,19 (14). To emphasize the evolutionary relationship among class A enzymes and to establish a uniformity in nomenclature, we refer here to the Bacillus licheniformis P-lactamase gene as blaP rather than penP as previously denoted.The blaP gene has been cloned on a 4.2-kilobase (kb) EcoRI DNA fragment (8,15,17) and sequenced (23,33). By complementation analysis of mero-and heteropolyploid strains constructed in B. licheniformis, it was shown that the 4.2-kb EcoRI DNA fragment also contains blaI, the gene encoding the repressor of blaP (17). Dubnau and Pollock (12) had shown by chromosomal DNA transformation that blaI was 90% linked to blaP, and a preliminary linkage map developed by Sherratt and Collins placed blaI 3' to blaP (40). These authors (40) also reported a second regulatory locus (Ri) 50% linked to blaP and a third unlinked locus (R2).Since multiple genetic determinants apparently control the formation of P-lactamase, it is not surprising that induction is complex. Upon induction, production of B. licheniformis ,-lactamase occurs unusually slowly, the rate increasing for * Corresponding author.

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Section: Methodsmentioning

confidence: 99%

“…The blaP gene has been cloned on a 4.2-kilobase (kb) EcoRI DNA fragment (8,15,17) and sequenced (23,33). By complementation analysis of mero-and heteropolyploid strains constructed in B. licheniformis, it was shown that the 4.2-kb EcoRI DNA fragment also contains blaI, the gene encoding the repressor of blaP (17).…”

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confidence: 99%

Transcriptional analysis of beta-lactamase regulation in Bacillus licheniformis

Salerno¹,

Lampen²

1986

J Bacteriol

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“…The penicillinase gene, penP, from Bacillus licheniformis 749/C has been cloned (1, 7) and sequenced (24). We have independently cloned penP and the repressor gene penI from both the wild-type and constitutive strains of B. licheniformis 9945A (14).…”

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confidence: 99%

Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor

Himeno¹,

Imanaka²,

Aiba³

1986

J Bacteriol

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Bacillus licheniformis penicillinase genes, penP and penl, are coded on a 4.2-kilobase EcoRI fragment of pTTE21 (T. Imanaka, T. Tanaka, H. Tsunekawa, and S. Aiba, J. Bacteriol. 147:776-186, 1981). The EcoRI fragment was subcloned in a low-copy-number plasmid pTB522 in Bacillus subtilis. B. subtilis carrying the recombinant plasmid pPTB60 (Tcr penP+ penI+) was chemically mutagenized. Of about 150,000 colonies, two penl(Ts) mutant plasmids, pPTB60D13 and pPTB60E24, were screened by the plate assay at 30 and 48°C for penicillinase. By constructing recombinant plasmids between wild-type and mutant plasmids, the mutation points were shown to be located in a 1.7-kilobase EcoRI-PstI fragment. The EcoRI-PstI fragments of the wild-type plasmid and two mutant plasmids were sequenced. A large open reading frame, composed of 384 bases and 128 amino acid residues (molecular weight, 14,983), was found. Since the mutation points were located at different positions in the protein coding region (Ala to Val for pPTB60D13 and Pro to Leu for pPTB6OE24), the coding region was concluded to be the pen! gene. A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site (ATG). A probable promoter sequence which is very similar to the consensus sequence was also found upstream of the penP promoter, but in the opposite direction. A consensus twofold symmetric sequence (AAAGTATTA CATATGTAAGNTTT) which might have been used as a repressor binding region was found downstream and in the midst of the penP promoter and also downstream of the penl promoter. The regulation of penP and pen! by the repressor is discussed.

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“…The sequence of the NH2-terminal portion of the penicillinase, as deduced from the DNA sequence of the cloned gene, is shown in Fig. 1B (6). The sequence of prepenicillinase includes the tetrapeptide Leu-Ala-Gly-Cys.…”

mentioning

confidence: 99%

Bacillus licheniformis penicillinase synthesized in Escherichia coli contains covalently linked fatty acid and glyceride.

Lai¹,

Sarvas²,

Brammar³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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DNA sequence analysis of the structural gene for Bacillus licheniformis penicillinase has revealed a tetrapeptide sequence of Leu-Ala-Gly-Cys within the NH2-terminal part of the precursor form of penicillinase (pencillin amido-i3-lactamhydrolase, EC 3.5.2.6). The same tetrapeptide occurs in the signal sequence of the prolipoprotein of Escherichia coli, and the cysteine residue in the tetrapeptide of prolipoprotein is modified to form glyceride-cysteine which becomes the NH2 terminus of Braun's lipoprotein. On Penicillinase (penicillin amido-g-lactamhydrolase, EC 3.5.2.6) from Bacillus licheniformis exists in two forms, the exoenzyme which is secreted into the culture medium and the membranebound form which is associated with the membrane (1, 2) presumably via the hydrophobic segment at the NH2 terminus (3). The membrane-bound penicillinase is not a phospholipoprotein because it contains less than 0.02 mol of phosphate per mol of enzyme (3). The structural gene for the penicillinase from B. licheniformis has been cloned on a A-transducing phage (4). The availability of this clone allows study of the expression of this gene both in vitro and in Escherichia coli. In vitro transcription and translation of the penicillinase gene reveals the synthesis of a penicillinase precursor that is larger than the membranebound form (5). The sequence of the NH2-terminal portion of the penicillinase, as deduced from the DNA sequence of the cloned gene, is shown in Fig. 1B (6). The sequence of prepenicillinase includes the tetrapeptide Leu-Ala-Gly-Cys. This is the same tetrapeptide surrounding the site ofposttranslational modification and processing ofprolipoprotein in E. coli (Fig. LA) (1 ,uCi/ ml, 23.5 Ci/mmol), [2-3H]glycerol (50 ,uCi/ml, 6.35 Ci/ 3506The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Penicillinase from Bacillus licheniformis: nucleotide sequence of the gene and implications for the biosynthesis of a secretory protein in a Gram-positive bacterium

Cited by 161 publications

References 32 publications

Transcriptional analysis of beta-lactamase regulation in Bacillus licheniformis

Transcriptional analysis of beta-lactamase regulation in Bacillus licheniformis

Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor

Bacillus licheniformis penicillinase synthesized in Escherichia coli contains covalently linked fatty acid and glyceride.

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