Identification of Fur-regulated genes in Actinobacillus actinomycetemcomitans

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Abstract
Actinobacillus actinomycetemcomitans is an oral pathogen that causes aggressive periodontitis as well as sometimes life-threatening, extra-oral infections. Iron regulation is thought to be important in the pathogenesis of A. actinomycetemcomitans infections and, consistent with this hypothesis, the fur gene has recently been identified and characterized in A. actinomycetemcomitans. In this study, 14 putatively Fur-regulated genes were identified by Fur titration assay (Furta) in A. actinomycetemcomitans, including afuA, dgt, eno, hemA, tbpA, recO and yfe – some of which are known to be Fur regulated in other species. A fur mutant A. actinomycetemcomitans strain was created by selecting for manganese resistance in order to study the Fur regulon. Comparisons between the fur gene sequences revealed that nucleotide 66 changed from C in the wild-type to T in the mutant strain, changing leucine to isoleucine. The fur mutant strain expressed a nonfunctional Fur protein as determined by Escherichia coli-based ferric uptake assays and Western blotting. It was also more sensitive to acid stress and expressed higher levels of minC than the wild-type strain. minC, which inhibits cell division in other bacterial species and whose regulation by iron has not been previously described, was found to be Fur regulated in A. actinomycetemcomitans by Furta, by gel shift assays, and by RT-qPCR assays for gene expression.