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Antimicrobial Agents and Chemotherapy, June 1998, p. 1375-1381, Vol. 42, No. 6
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Recombinant Expression and Characterization of the Major beta -Lactamase of Mycobacterium tuberculosis

Rama Kishan R. Voladri,1 David L. Lakey,1,2 Steven H. Hennigan,1 Barbara E. Menzies,1,3 Kathryn M. Edwards,2 and Douglas S. Kernodle1,3,*

Divisions of Infectious Disease, Department of Medicine,1 and Division of Infectious Diseases, Department of Pediatrics,2 Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2605, and Department of Veterans Affairs Medical Center, Nashville, Tennessee 372123

Received 22 August 1997/Returned for modification 5 December 1997/Accepted 15 March 1997

New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of beta -lactam antibiotics. beta -Lactamase production appears to be the major mechanism by which M. tuberculosis expresses beta -lactam resistance. beta -Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration 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 -lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology 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 kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA. We conclude that the major beta -lactamase of M. tuberculosis is a class A beta -lactamase with predominant penicillinase activity. A second, minor beta -lactamase with relatively greater cephalosporinase activity is also present.

* Corresponding author. A-3310 MCN, Vanderbilt University, 1161 21st Ave. South, Nashville, TN 37212-2605. Phone: (615) 327-4751, ext. 5486. Fax: (615) 343-6160. E-mail: doug.kernodle{at}vanderbilt.edu.

Antimicrobial Agents and Chemotherapy, June 1998, p. 1375-1381, Vol. 42, No. 6
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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