Characterization of SFO-1, a plasmid-mediated inducible class A beta-lactamase from Enterobacter cloacae

Abstract

Enterobacter cloacae 8009 produced an inducible class A beta-lactamase which hydrolyzed cefotaxime efficiently. It also hydrolyzed other beta-lactams except cephamycins and carbapenems. The activity was inhibited by clavulanic acid and imipenem. The bla gene was transferable to Escherichia coli by electroporation of plasmid DNA. The molecular mass of the beta-lactamase was 29 kDa and its pI was 7.3. All of these phenotypic characteristics of the enzyme except for inducible production resemble those of some extended-spectrum class A beta-lactamases like FEC-1. The gene encoding this beta-lactamase was cloned and sequenced. The deduced amino acid sequence of the beta-lactamase was homologous to the AmpA sequences of the Serratia fonticola chromosomal enzyme (96%), MEN-1 (78%), Klebsiella oxytoca chromosomal enzymes (77%), TOHO-1 (75%), and FEC-1 (72%). The conserved sequences of class A beta-lactamases, including the S-X(T)-X(S)-K motif, in the active site were all conserved in this enzyme. On the basis of the high degree of homology to the beta-lactamase of S. fonticola, the enzyme was named SFO-1. The ampR gene was located upstream of the ampA gene, and the AmpR sequence of SFO-1 had homology with the AmpR sequences of the chromosomal beta-lactamases from Citrobacter diversus (80%), Proteus vulgaris (68%), and Pseudomonas aeruginosa (60%). SFO-1 was also inducible in E. coli. However, a transformant harboring plasmid without intact ampR produced a small amount of beta-lactamase constitutively, suggesting that AmpR works as an activator of ampA of SFO-1. This is the first report from Japan describing an inducible plasmid-mediated class A beta-lactamase in gram-negative bacteria.

FIG. 1

Agarose gel electrophoresis of plasmid DNAs from E. cloacae 8009. Lane A, HindIII-digested bacteriophage λ DNA; lane B, E. cloacae 8009. L, large plasmid; M, medium-size plasmid; S, small plasmid.

FIG. 2

Construction of recombinant plasmids.

FIG. 3

Alignment of deduced amino acid sequence of SFO-1 (AmpA) with the sequences of homologous enzymes. The amino acid numbering for the class A β-lactamase is used (1). The S-X(T)-X(S)-K motif with the active-site serine is shaded. Asterisks indicate the conserved amino acid residues among these class A sequences.

FIG. 4

Alignment of deduced amino acid sequence of AmpR of SFO-1 with homologous AmpR sequences. Asterisks indicate the conserved amino acid residues.