Isolation of single-site Escherichia coli mutants deficient in thiamine and 4-thiouridine syntheses: identification of a nuvC mutant

Abstract

A method is described to rapidly select and classify many independent near-UV irradiation-resistant Escherichia coli mutants, which include tRNA modification and RNA synthesis control mutants. One class of these mutants was found to be simultaneously deficient in thiamine biosynthesis and in the ability to modify uridine in tRNA to 4-thiouridine, known to be the target for near-UV irradiation. These mutants were found to be unable to make thiazole, a thiamine precursor. The addition of thiazole restores the thiamine deficiency but does not render the cells near-UV irradiation sensitive. In vitro studies on one of these mutants indicated a deficiency in protein factor C (nuvC), required for the 4-thiouridine modification of tRNA. In Pl transduction, the thiazole marker cotransduced with the histidine marker, which places the thiazole marker between 42 and 46 min on the E. coli chromosome map. Both thiamine production and 4-thiouridine production were resumed by 87% of the spontaneous reversions, suggesting a single-point mutation. Our results indicate that we have isolated nuvC mutants and that the nuvC polypeptide is involved in two functions, tRNA modification and thiazole biosynthesis.

Thiouridine residues in tRNAs are responsible for a synergistic effect of UVA and UVB light in photoinactivation of Escherichia coli

“…Second, thiI and iscS are the only genes common to the biosynthetic pathway of thiamine and s 4 U. Both were first characterized in screenings for minimalized UV-sensitivity (as nuvA and nuvC) (Ramabhadran and Jagger, 1976;Ryals et al, 1982;Taylor et al, 1998); to our knowledge, no other enzyme of the biosynthetic pathway of thiamine has been reported to influence the UV sensitivity of E. coli. Finally, thiamine is not a UVA chromophore and is therefore not a likely locus of UVA photosensitivity.…”

Near ultraviolet DNA damage induces the SOS responses in Escherichia coli.

“…To check whether the near u.v. light is able to trigger the SOS response, we have determined this response in a pair of strains isogenic except at the nuvA locus, the structural gene for the thiolase which post-transcriptionally converts uridine 8 of tRNAs into 4-thiouridine (Ryals et al, 1982). nuvA cells lack 4-thiouridine in their tRNAs and do not exhibit the near u.v.induced growth delay (Thomas and Favre, 1980).…”

“…The two strains carry in common the following markers: thr, leu, proA, his, arg, lac, gal, xyl, mtl, ara, tsx, str. They differ at the nuvA locus, the structural gene of the thiolase specifically involved in 4-thiouridine biosynthesis (Ryals et al, 1982). The PM2 strain is nuvA-: it lacks 4-thiouridine and exhibits no growth delay after near u.v.…”

A mutation in Escherichia coli K-12 results in a requirement for thiamine and a decrease in L-serine deaminase activity

“…Genetic comparison with previously described thiamine-requiring mutants. Several thiamine-requiring mutants have been described (1,3,7,9,19). These map at four loci: 9.7, 24.6, ca.…”