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 transfe...

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Bibliographic Details
Published in:Journal of Bacteriology 2012-09, Vol.194 (18), p.4933-4940
Main Authors: Rajakovich, Lauren J, Tomlinson, John, Dos Santos, Patricia C
Format: Article
Language:English
Subjects:
Amino acids
Bacillus subtilis
Bacillus subtilis - enzymology
Bacillus subtilis - genetics
Bacillus subtilis - metabolism
Bacteria
Bacterial proteins
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Bacteriology
biochemical pathways
Biological and medical sciences
Biosynthesis
Biosynthetic Pathways - genetics
Carbon-Sulfur Lyases - genetics
Carbon-Sulfur Lyases - metabolism
cysteine
Enzymes
Escherichia coli
Escherichia coli - genetics
Escherichia coli - metabolism
Fundamental and applied biological sciences. Psychology
Gene Knockout Techniques
Genes
Genetic Complementation Test
Microbiology
Miscellaneous
RNA, Transfer - metabolism
Salmonella enterica
sulfur
Sulfurtransferases - genetics
Sulfurtransferases - metabolism
thiamin
thiosulfate sulfurtransferase
Thiouridine - metabolism
Transfer RNA
transport proteins
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Summary: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.
ISSN:0021-9193
1098-5530
1067-8832
DOI:10.1128/jb.00842-12