Novel class A beta-lactamase Sed-1 from Citrobacter sedlakii: genetic diversity of beta-lactamases within the Citrobacter genus

Abstract

Citrobacter sedlakii 2596, a clinical strain resistant to aminopenicillins, carboxypenicillins, and early cephalosporins such as cephalothin, but remaining susceptible to acylureidopenicillins, carbapenems, and later cephalosporins such as cefotaxime, was isolated from the bile of a patient treated with beta-lactam and quinolone antibiotics. The isolate produced an inducible class A beta-lactamase of pI 8.6, named Sed-1, which was purified. Characterized by a molecular mass of 30 kDa, Sed-1 preferentially hydrolyzed benzylpenicillin, cephalothin, and cloxacillin. The corresponding gene, bla(Sed-1), was cloned and sequenced. Its deduced amino acid sequence shared more than 60% identity with the chromosome-encoded beta-lactamases from Citrobacter koseri (formerly C. diversus) (84%), Klebsiella oxytoca (74%), Serratia fonticola (67%), and Proteus vulgaris (63%) and 71% identity with the plasmid-mediated enzyme MEN-1. A gene coding for a LysR transcriptional regulator was found upstream from bla(Sed-1). This regulator, named SedR, displayed 90% identity with the AmpR sequence of the chromosomal beta-lactamase from C. koseri and 63 and 50% identity with the AmpR sequences of P. vulgaris and Enterobacter cloacae, respectively. By using DNA-DNA hybridization, a bla(Sed-1)-like gene was identified in two reference strains, C. sedlakii (CIP-105037) and Citrobacter rodentium (CIP-104675), but not in the 18 strains of C. koseri studied. Two DNA fragments were amplified and sequenced from the reference strains of C. sedlakii CIP-105037 and C. rodentium CIP-104675 using two primers specific for bla(Sed-1). They shared 98 and 80% identity with bla(Sed-1), respectively, confirming the diversity of the chromosomally encoded class A beta-lactamases found in Citrobacter.

FIG. 1

Nucleotide sequences of bla_Sed-I and bla_SedR from E. coli(pBC2596). The deduced amino acid sequence is indicated in single-letter code above the nucleotide sequence. RBS indicates a potential ribosome-binding site. The Sed-1 signal peptide extends from amino acid 1 to 28, and the postulated cleavage site is indicated by a vertical arrow. The residues conserved in the active site of serine β-lactamases are boxed. Two numbering schemes are indicated: the one in boldface is for the Sed-1 amino acid sequence, and the other one is for the Sed-1 and SedR nucleotide sequences.

FIG. 2

Multiple alignment of amino acid sequences for nine class A β-lactamases. Dashes indicate gaps inserted in the alignment; asterisks indicate identical residues. Numbering is according to Ambler (R. P. Ambler et al., Letter, Biochem. J. 276:269–270, 1991). Boldface residues are the highly conserved residues involved in the catalytic site of the protein. The β-lactamases included in the alignment are Sed-1 from C. sedlakii (present study), CdiA from C. diversus (32), OXY-1 and OXY-2 from K. oxytoca (25), MEN-1 from E. coli (8), TOHO-1 from E. coli (30), CUV from S. fonticola (44), CUM from P. vulgaris (22), and TEM from E. coli (54).

FIG. 3

Alignment of LysR-type proteins. The AmpR sequences involved in the class A β-lactamase regulation systems were from C. diversus (CdiR), E. cloacae (NMCR), P. vulgaris (CumR), and Serratia marcescens (SmeR). LysR, the type regulator protein, was from E. coli. The predicted helix-turn-helix domain is boxed. Residues identical in SedR and the AmpR family sequences are indicated in boldface, whereas those identical in SedR and the LysR family sequences are indicated with asterisks. The residues highly conserved in LTTRs are shaded in gray (the amino acid numbering is indicated below the multiple alignment).