DETERMINATION OF THE PATHWAY FOR RHAMNOSE BIOSYNTHESIS IN MYCOBACTERIA - CLONING, SEQUENCING AND EXPRESSION OF THE MYCOBACTERIUM-TUBERCULOSIS GENE ENCODING ALPHA-D-GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE

The mycobacterial cell wall core consists of an outer lipid layer of m ycolic acids connected, via arabinogalactan polysaccharide, to an inne r peptidoglycan layer. An alpha-L-rhamnopyranosyl residue has been sho wn to be a key component linking the mycolated arabinogalactan to the peptidoglycan and, therefore, the biosynthesis of L-rhamnose (Rha) in mycobacteria was investigated as the first step of developing inhibito rs of its biosynthesis. Biochemical assays were used to show that dTDP -Rha was synthesized in Mycobacterium smegmatis from alpha-D-glucose 1 -phosphate (alpha-D-Glc-1-P) and dTTP by the same four enzymic steps u sed by Escherichia coli and other bacteria. PCR primers based on conse nsus regions of known sequences of the first enzyme in this series, al pha-D-Glc-1-P thymidylyltransferase (RfbA) were used to amplify rfbA D NA from M. tuberculosis. The entire rfbA gene was then cloned and sequ enced. The deduced amino acid sequence revealed a 31362 Da putative pr otein product which showed similarity to RfbA proteins of other bacter ia (59% identity to that found in E. coli). Sequencing of DNA flanking the rfbA gene did not reveal any of the other rib genes required for dTDP-Rha biosynthesis. Therefore, the four Rha biosynthetic genes are not clustered in M. tuberculosis. The enzymic activity of the sequence d gene product was confirmed by transformation of E. coil with pBluesc ript KS(-) containing the rfbA gene from M. tuberculosis. Analysis of enzyme extracts prepared from this transformant revealed an 11-fold in crease in alpha-D-Glc-1-P thymidylyltransferase activity.