Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA

ThiI has been identified as an essential enzyme involved in the biosynthesis of thiamine and the tRNA thionucleoside modification, 4-thiouridine. In Escherichia coli and Salmonella enterica, ThiI acts as a sulfurtransferase, receiving the sulfur donated from the cysteine desulfurase IscS and transferring it to the target molecule or additional sulfur carrier proteins. However, in Bacillus subtilis and most species from the Firmicutes phylum, ThiI lacks the rhodanese domain that contains the site responsible for the sulfurtransferase activity. The lack of the gene encoding for a canonical IscS cysteine desulfurase and the presence of a short sequence of ThiI in these bacteria pointed to mechanistic differences involving sulfur trafficking reactions in both biosynthetic pathways. Here, we have carried out functional analysis of B. subtilis thiI and the adjacent gene, nifZ, encoding for a cysteine desulfurase. Gene inactivation experiments in B. subtilis indicate the requirement of ThiI and NifZ for the biosynthesis of 4-thiouridine, but not thiamine. In vitro synthesis of 4-thiouridine by ThiI and NifZ, along with labeling experiments, suggests the occurrence of an alternate transient site for sulfur transfer, thus obviating the need for a rhodanese domain. In vivo complementation studies in E. coli IscS- or ThiI-deficient strains provide further support for specific interactions between NifZ and ThiI. These results are compatible with the proposal that B. subtilis NifZ and ThiI utilize mechanistically distinct and mutually specific sulfur transfer reactions.