Molecular analysis of Mycobacterium tuberculosis phosphate specific transport system in Mycobacterium smegmatis.: Characterization of recombinant 38 kDa (PstS-1)

The functionality of the putative Mycobacterium tuberculosis phosphate transport operon was studied by operon-lacZ promoterless fusions in mycobacterium smegmatis. The expression of the operon genes was evaluated in transformed M. smegmatis growing in medium with low and high phosphate concentration. Although the gene fusions expressed P-galactosidase in medium with phosphate, a higher activity was detected in bacteria growing in medium with low phosphate. In contrast, alkaline phosphatase activity from M, smegmatis was detected only in bacteria growing in medium with low phosphate. The expression of the operon genes was driven by a promoter located 5 ' upstream from the start codon of the pstB gene. A second putative internal promoter 5 ' upstream of the pstS-1 gene was also detected. Furthermore, comparative analysis between the native and recombinant PstS-1 proteins showed that they were very similar. Like the native protein, the recombinant protein was also secreted to the culture medium as a glycosylated band. The results show that M. smegmatis recognized phosphate regulatory signals of the M. tuberculosis phosphate transport operon genes, and open the possibility to study gene phosphate regulation in mycobacteria. (C) 2001 Academic Press.