Identification of Protein Lipoylation Pathways in Bacillus subtilis

MANSILLA, MC; MARTIN, N.; DE MENDOZA D

Tirrenia, Pisa. Italia

Conferencia; 4th Conference on Functional Genomics of Gram-Positive Microorganisms - 14th International Conference on Bacilli; 2007

Lipoic acid (6,8-thioctic acid), a covalently bound cofactor, is essential for function of several key enzymes involved in oxidative metabolism in most prokaryotic and eukaryotic organisms. The lipoylated proteins include pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, branched-chain 2-oxoacid dehydrogenase, and the glycine cleavage system. In many organisms lipoylation is catalized by two separate enzymes, lipoyl protein ligase A (LplA) or octanoyl-[acyl carrier protein]-protein transferase (LipB). While LplA uses exogenous lipoic acid, LipB transfers endogenous octanoic acid to the target proteins. These octanoylated domains are converted into lipoylated derivatives by the S-adenosyl-L-methionine dependent enzyme, lipoyl synthase (LipA), which catalyzes the insertion of sulfur atoms into the six- and eight-carbon positions of the corresponding fatty acids. This process bypasses the requirement for an exogenous supply of lipoic acid. In bacteria, enzymes involved in lipoylation have gained increasing attention because of their implication in pathogenicity.  Analysis of the Bacillus subtilis genome sequence revealed that this microorganism possess two ORFs with homology with LplAs (yhfJ and yqhM), but we could not find any ORF with significant similarity with LipB from either Gram-negative or Gram-positive bacteria. As we have previously determined that B. subtilis is able to synthesize lipoic acid, we whished to identify the enzyme/s involved in the endogenous lipoylation pathway. One of the candidates was ywfL, a gene with unknown function located in a putative operon related to sulfur assimilation, who showed moderate similarity to yhfJ. We constructed a knock-out ywfL mutant, NM51. This strain is impaired to grow in minimal media, but its growth was restored with addition of acetate plus succinate and branched-chain fatty acid precursors, or lipoic acid. NM51 was not able to sporulate in SM medium but formed spores in SM supplemented with lipoic acid. In addition, this strain showed an strong induction of the transcription of the desaturase gene. These results suggest that YwfL is the only enzyme responsible for the attachment of  octanoic acid to the apoenzymes in B. subtilis, so we renamed ywfL as lipB. The phenotypes of single, double and triple mutants in the B. subtilis putative ligases of the endogenous and exogenous lipoylation  pathways will be presented.