Cloning and Characterization of a Chromosomal Class C β-Lactamase and Its Regulatory Gene in Laribacter hongkongensis

Abstract

Laribacter hongkongensis, a newly discovered bacterium recently shown to be associated with community-acquired gastroenteritis, is generally resistant to most beta-lactams except the carbapenems. We describe the cloning and characterization of a novel chromosomal class C beta-lactamase and its regulatory gene in L. hongkongensis. Two genes, ampC and ampR, were cloned by inserting restriction fragments of genomic DNA from L. hongkongensis strain HLHK5 into pBK-CMV to give the recombinant plasmid pBK-LHK-5. The ampR and ampC genes and their promoters were divergently oriented, with the ampR gene immediately upstream of the ampC gene and an intercistronic Lys-R motif, typical of inducible ampC-ampR regulatory systems. The deduced amino acid sequence of the cloned AmpC beta-lactamase (pI 8.1) contained consensus motifs characteristic of class C beta-lactamases but had identities no greater than 46% to known class C beta-lactamases. The kinetic properties of this AmpC were also compatible with those of a class C beta-lactamase. PCR of 20 clinical isolates of L. hongkongensis, including HLHK5, showed the presence of both ampC and ampR genes in all isolates. Southern hybridization suggested that the ampC gene of HLHK5 was chromosomally encoded. Subcloning experiments showed that the expression of the ampC gene of HLHK5 was regulated by its ampR gene, which acts as a repressor. The beta-lactamase characterized from strain HLHK5 was named LHK-5 (gene, bla(LHK-5)) and represents the first example of AmpC beta-lactamase in the beta subdivision of proteobacteria.