Biochemical-genetic characterization and regulation of expression of an ACC-1-like chromosome-borne cephalosporinase from Hafnia alvei

A naturally occurring AmpC beta-lactamase (cephalosporinase) gene was clone d from the Hafnia alvei 1 clinical isolate and expressed in Escherichia coi l, The deduced AmpC beta-lactamase (ACC-2) had a pI of 8 and a relative mol ecular mass of 37 kDa and showed 50 and 47% amino acid identity with the ch romosome-encoded AmpCs from Serratia marcescens and Providentia stuartii, r espectively. It had 94% amino acid identity with the recently described pla smid-borne cephalosporinase ACC-1 from Klebsiella pneumoniae, suggesting th e chromosomal origin of ACC-1, The hydrolysis constants (k(cat) and K-m) sh owed that ACC-2 was a peculiar cephalosporinase. since it significantly hyd rolyzed cefpirome, Once its gene was cloned and expressed in E, coli (pDEL- 1), ACC-2 conferred resistance to ceftazidime and cefotaxime but also an un common reduced susceptibility to cefpirome, A divergently transcribed ampR gene with an overlapping promoter compared with ampC (bla(ACC-2)) was ident ified in H. alvei 1, encoding an AmpR protein that shared 64% amino acid id entity with the closest AmpR protein from P. stuartii, beta-Lactamase induc tion experiments showed that the ampC gene was repressed in the absence of ampR and was activated when cefoxitin or imipenem was added as an inducer. From H, alvei 1 cultures that expressed an inducible-cephalosporinase pheno type, several ceftazidime- and cefpirome-cross-resistant H. alvei 1 mutants were obtained upon selection on cefpirome- or ceftazidime-containing plate s, and H. alvei 1 DER, a ceftazidime-resistant mutant, stably overproduced cephalosporinase, Transformation of H. alvei 1 DER or E. coli JRG582 (ampDE mutant) harboring ampC and ampR from H. alt ei 1 with a recombinant plasmi d containing ampD from E. coli resulted in a decrease in the MIC of beta-la ctam and recovery of an inducible phenotype for H. alvei 1 DER Thus, AmpR a nd AmpD proteins mag regulate biosynthesis of the H, alvei cephalosporinase similarly to other enterobacterial cephalosporinases.