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

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 P1 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.

Enzymes producing 4-thiouridine in Escherichia coli tRNA: approximate chromosomal locations of the genes and enzyme activities in a 4-thiouridine-deficient mutant.

A previously described mutant of Escherichia coli which lacks 4-thiouridine in its tRNA was here shown to be deficient in factor A, one of the two proteins responsible for this thiolation of uridine. Addition of exogenous factor A restored the thiolating ability of extracts prepared from the mutant. The activities of the two thiolation proteins were governed by genes at two widely separated positions on the chromosome, as determined with F-prime merodiploids. The site governing factor A activity lay roughly in the region of the recently reported position of nuv, a gene controlling the production of 4-thiouridine in tRNA.

Zeatin ribonucleosides in the transfer ribonucleic acid of Rhizobium leguminosarum, Agrobacterium tumefaciens, Corynebacterium fascians, and Erwinia amylovora.

Until recently, the presence in transfer ribonucleic acid (tRNA) of the hydroxylated cytokinin ribosylzeatin [N6-(4-hydroxy-3-methylbut-2-enyl)adenosine]was thought to be unique to higher plants. This extension of work from several laboratories indicates the presence of 2-methylthioribosylzeatin in the tRNA of the plant-associated bacteria Rhizobium leguminosarum, Agrobacterium tumefaciens, and Corynebacterium fascians, but not in that of Erwinia amylovora. This cytokinin has the cis configuration, as is normally found in the tRNA's of plants. The tRNA thionucleotide patterns in these bacteria are different from those of Escherichia coli, Bacillus subtilis, and Salmonella typhimurium, which contain the unhydroxylated analogs of ribosylzeatin or 2-methylthioribosylzeatin.