RECOMBINANT EXPRESSION AND CHARACTERIZATION OF THE MAJOR BETA-LACTAMASE OF MYCOBACTERIUM-TUBERCULOSIS

New antibiotic regimens are needed for the treatment of multidrug-resi stant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglyc an layer, and the penicillin-binding proteins involved in its biosynth esis are inhibited by clinically relevant concentrations of beta-lacta m antibiotics. beta-Lactamase production appears to be the major mecha nism by which M. tuberculosis expresses beta-lactam resistance. beta-L actamases from the broth supernatant of 3- to 4-week-old cultures of h . tuberculosis H37Ra were partially purified by sequential gel filtrat ion chromatography and chromatofocusing. Three peaks of beta-lactamase activity with pi values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing beta- lactamase activity, respectively, were identified. The beta-lactamases with pi values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the beta-la ctamase with a pi value of 4.5 showed relatively greater cephalosporin ase activity. An open reading frame in cosmid Y49 of the DNA library o f M. tuberculosis H37Rv with homolog to known class A beta-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli. The recombinant enzyme was kin etically similar to the pi 5.1 and 4.9 enzymes purified directly from M. tuberculosis, It exhibited predominant penicillinase activity and w as especially active against azlocillin. It was inhibited by clavulani c acid and m-aminophenylboronic acid but not by EDTA. We conclude that the major beta-lactamase of M. tuberculosis is a class A beta-lactama se with predominant penicillinase activity. A second, minor beta-lacta mase with relatively greater cephalosporinase activity is also present .