Different promoters of SHV-2 and SHV-2a beta-lactamase lead to diverse levels of cefotaxime resistance in their bacterial producers.
Clinical Klebsiella pneumoniae isolates as well as Escherichia coli transformants producing the beta-lactamases SHV-2 or SHV-2a demonstrate MIC values for cefotaxime of 4 mg l-1 or 64 to greater than 128 mg l-1, respectively. The beta-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 E. coli transformants harbouring these genes cloned into the same vector plasmid gave a positive signal down to 1.2 micrograms (SHV-2) and 0.32 to 0.16 micrograms (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 to 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 beta-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 BamHI restriction site introduced by three point mutations into the cloned sequences was employed to transforms E. coli DH5 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
Podbielski
A
A
Institute of Medical Microbiology, Medical Faculty, Technical University, Aachen, FRG.
Schönling
J
J
Melzer
B
B
Haase
G
G
D001483:Base Sequence; D015152:Blotting, Northern; D002439:Cefotaxime / Q000494:pharmacology*; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016415:Sequence Alignment; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D015719:Single-Strand Specific DNA and RNA Endonucleases; D001618:beta-Lactamases; D002439:Cefotaxime