1887

Abstract

Summary: Clinical isolates as well as transformants producing the ß-lactamases SHV-2 or SHV-2a demonstrate MIC values for cefotaxime of 4 mg l or 64 to >128 mg l, respectively. The ß-lactamases differ by one possibly insignificant amino acid exchange at position 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 transformants harbouring these genes cloned into the same vector plasmid gave a positive signal down to 1·2 μg (SHV-2) and 0·32 to 0·16 μg (SHV-2a), 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 ß-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 pBGS19 using a HI restriction site introduced by three point mutations into the cloned sequences was employed to transform DH5a. The MIC value for cefotaxime of transformants harbouring the SHV-2 promoter and SHV-2 structural gene was 8 mg l, but was 64 mg 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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1991-07-01
2021-08-05
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