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

Abstract

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.

Mycobacterium tuberculosis thymidylate synthase gene thyX is essential and potentially bifunctional, while thyA deletion confers resistance to p-aminosalicylic acid

“…Earlier studies have indeed questioned whether the true biological substrates of ThyX have been identified, as many of these enzymes (including ThyX from M. tuberculosis and C. glutamicum ) have very low TS catalytic activity (Agrawal et al , 2004; Hunter et al , 2008; Kan et al , 2010). It should be noted that thyX from C. glutamicum is only thought to be essential upon reaching stationary phase (Park et al , 2010), and so the biological roles of ThyX appear to differ between these two relatively closely related species. Importantly, we have shown that this enzyme is a plausible tuberculosis drug target, and our collaborators have recently published their findings that compounds specifically designed to inhibit M. tuberculosis ThyX are effective, with no activity against ThyA (Kögler et al , 2011).…”

“…It is known that both thyA and thyX from M. tuberculosis (Rengarajan et al , 2004; Sampathkumar et al , 2005) and Corynebacterium glutamicum (Park et al , 2010) can functionally complement an E. coli thyA deletion strain. ThyA and ThyX from these two species exhibit 72 and 63 % sequence similarity at the amino acid level, respectively (Kan et al , 2010; Park et al , 2010). Both M. tuberculosis ThyA and ThyX proteins have been shown to have TS activity in vitro , with ThyA being substantially more efficient than ThyX (Hunter et al , 2008).…”

“…However, purified C. glutamicum ThyX does not exhibit any detectable TS activity (Kan et al , 2010). Mutational studies of C. glutamicum show that thyX is not essential for in vitro growth, but is thought to play a role in survival during stationary phase (Park et al , 2010). M. tuberculosis thyA is not thought to be essential, as thyA transposon mutants are viable (Sassetti et al , 2003), and some clinical M. tuberculosis p -aminosalicylic acid (PAS)-resistant strains contain point mutations within thyA (Leung et al , 2010; Mathys et al , 2009; Rengarajan et al , 2004; Zhang et al , 2007).…”

Mechanistic and structural basis for inhibition of thymidylate synthase ThyX

“…Mycobacterium strains are peculiar due to the presence of both thyX and thyA genes, but even in this case, thyX has been shown to code for essential cellular function(s) [24]. Moreover, in other Corynebacteriaceae, ThyX proteins have been specifically implicated in survival during the stationary growth phase [25]. …”

The gene encoding the alternative thymidylate synthase ThyX is regulated by sigma factor SigB in Corynebacterium glutamicum ATCC 13032

“…This could be because both ThyA and ThyX contribute to the synthesis of the one‐carbon unit for the biosynthesis of thymidine in C. glutamicum . Moreover, because only the ΔthyX strain exhibited poor survival during the stationary growth phase, it has been suggested that the expression levels of thyA and thyX differ in response to different growth conditions (Fivian‐Hughes et al ., ; Park et al ., ).…”