Abstract:
The genome of Mycobacterium smegmatis was probed for B12 riboswitches using sequence conserved between the two different B12 riboswitch domains of M. tuberculosis. A putative B12 element was identified upstream of an ABC-type transporter system comprising an operonic arrangement of genes encoding a lipoprotein, permease and ATPase. A mutant of M. smegmatis dependent on external supplementation with vitamin B12 (or methionine) was constructed by elimination of both de novo cobalamin synthesis (cobK) and the cobalamin-independent methionine synthase (metE). This background was used for the inactivation of the lipoprotein by homologous recombination. The resulting triple mutant was unable to grow on external B12 in liquid medium, but was unimpaired for growth on solid medium containing B12, suggesting that M. smegmatis might employ two mutually exclusive systems for B12 uptake. Complementation of this phenotype is in progress.
