Beta-lactamase expression in Streptomyces cacaoi

Urabe, Hiroaki; Lenzini, Mauro V.; Mukaide, Motokazu; Dusart, Jean; Nakano, Michiko; Ghuysen, Jean‐Marie; Ogawara, Hiroshi

doi:10.1128/jb.172.11.6427-6434.1990

Cited by 10 publications

(11 citation statements)

References 43 publications

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“…14 and 15 for the blaU upstream region. The sequences of the oligonucleotides 1 to 15 are given in C. The A10 and A35 promoter regions of blaL (Urabe et al 1990), blaA (Urabe and Ogawara 1992) and blaU (Forsman 1991) genes are shown, as well as the transcription starts and the ribosomebinding sites of blaL and blaU (rbs). For BlaA, the transcription start coincides with the translation start (Urabe and Ogawara 1992) (lanes 7, 8).…”

Section: A T-n 11 -A Motif Is Also Present Upstream Of Blaumentioning

confidence: 99%

The two β-lactamase genes of Streptomyces cacaoi, blaL and blaU, are under the control of the same regulatory system

et al. 1997

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The production of beta-lactamase in Streptomyces cacaoi, which contains two beta-lactamase-encoding genes, blaL and blaU, is inducible by beta-lactam compounds. The two genes have been cloned independently in S. lividans TK24, a beta-lactamase-negative species. The blaU clone did not respond to the presence of beta-lactams, whereas the blaL clone appeared to be inducible in S. lividans. The latter clone contains two open reading frames, blaA and blaB, located just upstream of but transcribed divergently from blaL, which were shown to be required for the production as well as the induction of BlaL. The deduced BlaA protein belongs to the LysR family of transcription regulators. In order to examine the role of BlaA in regulation, we here report on over-expression of a GST-BlaA fusion protein in Escherichia coli and its use for antibody preparation. The GST-BlaA fusion protein was partially purified and bandshift assays showed that it bound the 197-bp blaL-blaA intergenic region. The BlaA DNA binding-site was further restricted to a 30-bp sequence containing a T-N11-A motif, a characteristic of LysR-type promoters. Another T-N11-A motif upstream of the blaU gene was also shown to bind BlaA. The affinities of these two T-N11-A motifs in BlaA binding were comparable. A plasmid bearing the blaU structural gene and the blaA-blaB regulatory region was constructed and shown to confer on an S. lividans host the capacity to produce inducible beta-lactamase. It can thus be concluded that the S. cacaoi blaL and blaU genes are controlled by the same regulatory system.

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Section: A T-n 11 -A Motif Is Also Present Upstream Of Blaumentioning

confidence: 99%

The two β-lactamase genes of Streptomyces cacaoi, blaL and blaU, are under the control of the same regulatory system

et al. 1997

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“…In previous studies, the gene encoding the P-lactamase of Streptomyces cacaoi (38) was cloned and sequenced (26,27). During these studies, we found that the upstream sequence of the structural gene had an enhancing effect on 3-lactamase production by a StreptomWces host, Streptomyces lividans (51). In addition, the same nucleotide sequence is necessary for the inducible expression of the S. cacaoi * Corresponding author.…”

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Nucleotide sequence and transcriptional analysis of activator-regulator proteins for beta-lactamase in Streptomyces cacaoi

Urabe

Ogawara

1992

J Bacteriol

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The nucleotide sequence of the 2.7-kb DNA fragment upstream of the structural gene of beta-lactamase in Streptomyces cacaoi was determined. Computer-aided "FRAME" analysis revealed four possible open reading frames (ORFs), three in one direction and one in the opposite direction. One of them (ORF1, BlaA) encoded an activator-regulator protein whose deduced amino acid sequence was similar to that of other activator-regulator proteins in bacteria. Insertion of an 8-bp BamHI linker into the BlaA region decreased the beta-lactamase activity sharply, from 50 U to 1 U/ml. This protein (BlaA) was found to bind to the nucleotide sequence between the bla (beta-lactamase structural gene) and blaA genes. Another ORF (ORF2, BlaB) in the same orientation had a couple of amino acid sequences similar to that of pBR322 beta-lactamase. However, insertion of the 8-bp BamHI linker indicated that this ORF was functional as an activator-regulator but not as a beta-lactamase. Therefore, there were two activator-regulator proteins in the upstream region of the structural gene of the beta-lactamase. Nuclease S1 mapping predicted that transcription for the activator proteins commenced at the translational initiation codon or within a few nucleotides from the translational start site. Transcription was in the opposite direction to that of the beta-lactamase structural gene.

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“…On the other hand we cannot exclude the influence of upstream regulatory sequences like those described by Urabe et al (1990) because the amplified promotercontaining DNA fragments are long enough to comprise such regions and the sequences 500 to 300 bp upstream of the start codon are diverse, so they may exert different influences on the downstream promoters. This influence would have to be caused by cis-acting elements, because we did not amplify the open reading frame situated upstream of the SHV-2a P-lactamase gene (1 135 to 899 nucleotides in front of the start codon; there is none in front of the SHV-2 gene).…”

Section: Discussionmentioning

confidence: 99%

Different promoters of SHV-2 and SHV-2a -lactamase lead to diverse levels of cefotaxime resistance in their bacterial producers

Podbielski¹,

Schönling²,

Melzer³

et al. 1991

Journal of General Microbiology

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Clinical Klebsiellapneumoniae isolates as well as Escherichia coli transformants producing the p-lactamases SHV-2 or SHV-2a demonstrate MIC values for cefotaxime of 4 mg 1-l or 64 to > 128 mg P I , respectively. The plactamases differ by one possibly insignificant amino acid exchange at p i t i o n number 10 of the mature protein; their kinetic parameters are rather similar. The 5' untranslated regions of both corresponding genes show no homology starting 74 nucleotides upstream to the start codon. Hybridization of intragenically annealing oligonucleotides to dot-blotted serial dilutions of total cellular RNA from E. coli transformants harbouring these genes cloned into the same vector plasmid gave a positive signal down to 1.2 pg (SHV-2) and 0.32 to 0.16 pg (SHV2a), indicating a four to eight times higher amount of specific transcript in the case of SHV-2a. By primer extension analysis and S1 nuclease digestion the starting point of transcription was located 100 nucleotides (SHV-2) and 50 nucleotides (SHV-2a) in front of the start codon. No other transcripts of different length could be detected after prolonged exposure. Northern blot analysis demonstrated the length of the p-lactamase mRNA to be about 1.6 kb in both cases, thus comprising a potential open reading frame downstream of the two enzymes' genes. Selective PCR amplification of both promoter regions and of the structural gene of SHV-2 and subsequent combined cloning of each of the promoters and the SHV-2 gene into pBGSl9 using a B m H I restriction site introduced by three point mutations into the cloned sequences was employed to transform E. coli DH5a. The MIC value for cefotaxime of transformants harbouring the SHV-2 promoter and SHV-2 structural gene was 8 mg 1-l , but was 64 mg 1-l in the case of the SHV-2a promoter and SHV-2 structural gene combination, indicating that the quantitative change of resistance to cefotaxime of SHV-2a-producing bacteria is caused solely by a significant promoter mutation.

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Beta-lactamase expression in Streptomyces cacaoi

Cited by 10 publications

References 43 publications

The two β-lactamase genes of Streptomyces cacaoi, blaL and blaU, are under the control of the same regulatory system

The two β-lactamase genes of Streptomyces cacaoi, blaL and blaU, are under the control of the same regulatory system

Nucleotide sequence and transcriptional analysis of activator-regulator proteins for beta-lactamase in Streptomyces cacaoi

Different promoters of SHV-2 and SHV-2a -lactamase lead to diverse levels of cefotaxime resistance in their bacterial producers

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