Alternative thymidylate synthase, ThyX, involved in Corynebacterium glutamicum ATCC 13032 survival during stationary...

A BLASTP search has shown the presence of a gene homologous to an alternative thymidylate synthase (TS), thyX, in Corynebacterium glutamicum ATCC 13032. To determine if thyX is functionally analogous to thyA, thyX was cloned in a plasmid and the resulting construct was transferred by transformation into a thyA mutant of Escherichia coli. The ThyX from C. glutamicum compensated for the defect in TS-deficient E. coli. A functional knockout of the thyX gene was constructed by allelic replacement using a sucrose counter-selectable suicide plasmid and confirmed by PCR and reverse transcriptase-PCR analyses. This mutant was viable without thymidine supplementation, suggesting that thyX is not an essential gene in C. glutamicum. Growth of the thyX mutant was dependent upon coupling activity of dihydrofolate reductase (DHFR) with ThyA for the synthesis of thymidine, and thus showed sensitivity to the inhibition of DHFR by the experimental inhibitor, WR99210. This indicates that thymidine synthesis was at least partially dependent on thyX expression. As it approached stationary phase, the thyX mutant lost viability much more rapidly than the parental wild type and the mutant complemented the thyX gene, suggesting that the activity of the ThyX enzyme is important in that phase of the growth cycle.