NUCLEOTIDE-SEQUENCE AND PHYLOGENY OF SHV-2 BETA-LACTAMASE

We determine the nucleotide sequence of the bla(SHV-2(pBP60-1)) gene from Klebsiella ozaenae which confers resistance to broad-spectrum cephalosporins. The structural gene encodes a polypeptide produce of 286 amino acids, and the estimated molecular weight of the mature protein is 28,900. Amino acid sequence comparison of the SHV-2(pBP60-1) enzyme with all known class A β-lactamases and homology studies showed that the residues were highly conserved. Furthermore, SHV-2(pBP60-1) was clearly related to SHV-1, LEN-1, and OHIO-1. The SHV-2(pBP60-1) enzyme differed from SHV-1 isolated from Klebsiella pneumoniae by seven amino acid substitutions. One of the substitutions, the Gly → Ser substitution at position 234, is probably a key region for the novel activity of cefotaxime hydrolysis. A phylogenetic tree was constructed by using all class A β-lactamases of known sequences by a progressive alignment method. The data suggested that the β-lcatamases of gram-positive Streptomyces, Staphylococcus, and Bacillus species appeared early in evolution, followed by the PSE and CARB enzymes of Pseudomonas species and, more recently, by the SHV-type and TEM-type enzymes found in enteric bacteria. Larger evolutionary distances separated clusters of the gram-positive β-lactamases than separated clusters of the gram-negative enzymes. Results of this phylogenetic study suggested that extended-spectrum enzymes are recent derivatives that are selected by the use of new cephalosporins.