TRANSCRIPTION AND EXPRESSION ANALYSIS, USING LACZ AND PHOA GENE FUSIONS, OF MYCOBACTERIUM-FORTUITUM BETA-LACTAMASE GENES CLONED FROM A NATURAL ISOLATE AND A HIGH-LEVEL BETA-LACTAMASE PRODUCER

The gene encoding a class A beta-lactamase was cloned from a natural i solate of Mycobacterium fortuitum (blaF) and from a high-level amoxici l lin-resistant mutant that produces large amounts of beta-lactamase ( blaF). The nucleotide sequences of the two genes differ at 11 positio ns, including two in the region upstream from the coding sequence. Gen e fusions to Escherichia coli lacZ and transcription and expression an alysis of the cloned genes in Mycobacterium smegmatis indicated that h igh-level production of the beta-lactamase in the mutant is mainly or wholly due to a single base pair difference in the promoter. These ana lyses also showed that transcription and translation start at the same position. A comparison of the. amino acid sequence of BlaF, as predic ted from the nucleotide sequence, with the determined N-terminal amino acid sequence indicated the presence of a typical signal peptide. The fusion of blaF (or blaF) to the E. coli gene phoA resulted in the pr oduction of BlaF-PhoA hybrid proteins that had alkaline phosphatase ac tivity. These results demonstrate that phoA can be used as a reporter gene for studying protein export in mycobacteria.