1 2 3 Functional analysis of Bacillus subtilis genes involved 4 in the biosynthesis of 4 - thiouridine in tRNA 5

19 ThiI has been identified as an essential enzyme involved in the biosynthesis of thiamine and the 20 tRNA thionucleoside modification, 4-thiouridine. In Escherichia coli and Salmonella enterica, 21 ThiI acts as a sulfurtransferase, receiving the sulfur donated from the cysteine desulfurase IscS 22 and transferring it to the target molecule or additional sulfur carrier proteins. However, in 23 Bacillus subtilis and most species from the Firmicute phylum, ThiI lacks the rhodanese domain 24 that contains the site responsible for the sulfurtransferase activity. The lack of the gene 25 encoding for a canonical IscS cysteine desulfurase and the presence of a short sequence of 26 ThiI in these bacteria pointed to mechanistic differences involving sulfur trafficking reactions in 27 both biosynthetic pathways. Here, we have carried out functional analysis of B. subtilis thiI and 28 the adjacent gene, nifZ, encoding for a cysteine desulfurase. Gene inactivation experiments in 29 B. subtilis indicate the requirement of ThiI and NifZ for the biosynthesis of 4-thiouridine, but not 30 thiamine. In vitro synthesis of 4-thiouridine by ThiI and NifZ, along with labeling experiments 31 suggest the occurrence of an alternate transient site for sulfur transfer, thus obviating the need 32 for a rhodanese domain. In vivo complementation studies in E. coli IscS or ThiI deficient strains 33 provide further support for specific interactions between NifZ and ThiI. These results are 34 compatible with the proposal that B. subtilis NifZ and ThiI utilize mechanistically distinct and 35 mutually specific sulfur transfer reactions. 36 37 on A uust 5, 2017 by gest http/jb.asm .rg/ D ow nladed fom