Cloning and characterization of chromosomal class C -Lactamase and its regulatory gene in Laribacter hongkongensis

by Li, Wai-shan

(Uncorrected OCR) Abstract Abstract of thesis entitled ?loning and Characterization of Chromosomal Class C ?Lactamase and Its Regulatory Gene in Laribacter hongkongensis?Submitted by Li Wai Shan for the Degree of Master of Philosophy at The University of Hong Kong in March 2005 Laribacter hongkongensis is a newly discovered bacterium recently shown to be associated with community-acquired gastroenteritis. The bacterium displays general resistance to most ?lactams, including penicillins and cephalosporins, except the carbapenems. However, the mechanism of its ?lactam resistance remains unknown. The cloning and characterization of a novel chromosomal class C ?lactamase and its regulatory gene in L. hongkongensis are described in this study. Two genes, ampC and ampR, were cloned by inserting restriction fragments of genomic DNA from L. hongkongensis strain HLHK5 into pBK-CMV to yield the recombinant plasmid pBK-LHK-5. The ampR precedes the ampC gene; both genes are reversely oriented and divergently transcribed from overlapping promoters located in the intercistronic region. An intercistronic Lys-R motif, typical of inducible ampC-ampR regulatory systems, was also identified. The deduced amino acid sequence of the cloned ampC ?lactamase (pI, 8.1) contained consensus motifs characteristic of class C ?lactamases, but had no greater than 46% identities to known class C ?lactamases. The kinetic properties of this ampC were also in parallel with class C ?lactamases. PCR of 20 clinical isolates of L. hongkongensis, including HLHK5, were performed, with results indicating the existence of both ampC and ampR genes in all isolates. Southern hybridization also provided evidence that the ampC gene of HLHK5 was chromosomally encoded. Subcloning experiments demonstrated that the expression of the ampC gene of HLHK5 was regulated by its ampR which acts as a repressor. The ?lactamase characterized from strain HLHK5 was named LHK-5 (gene, blaLHK-5) and represented the first example of ampC ?lactamase in the ?subdivision of proteobacteria.