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Abstract
Tuberculosis (TB) is the primary cause of death in humans by a single infectious agent (Mycobacterium tuberculosis). Bacteria utilize P-type ATPases to transport specific metal cations across the cytoplasmic membrane. The CtpB amino acid sequence from M. tuberculosis has homology to P-type ATPases, but its role in metal transport has not been elucidated. Herein, we report that this protein is highly conserved among pathogenic mycobacteria. Among the P-type ATPases in M. tuberculosis, CtpB is most similar to copper(I)-efflux protein CtpA. Induced expression of the ctpB gene from a tetracycline-controlled promoter reduces the growth rates of Mycobacterium smegmatis and M. tuberculosis in copper-containing media. In contrast, growth of M. tuberculosis and Mycobacterium bovis BCG mutants deleted for ctpB was similar to wild type in copper-containing medium, but impaired in copper-restricted medium. Together, these data support CtpB as a copper-import protein. Discerning the function of mycobacterial genes is accelerated by the abilities to delete and conditionally express genes of interest. Multiple molecular genetic vectors, tools, and methods for mycobacteria have greatly advanced efforts to characterize the functions of many genes in M. tuberculosis and in other mycobacteria. However, difficulties arise when attempting to delete or clone specific genes of unknown function. We have taken various approaches to circumvent challenges encountered. In this report, we focus on genetic elements used in vectors designed to facilitate targeted genomic deletions or for conditional expression of genes in mycobacteria. Results suggest that problems may arise from low expression of genes after introduction into the chromosome in single copy or from expression that is too high on multi-copy plasmids.