Abstract
The role of the nfuA gene encoding an iron-sulfur ([Fe-S]) cluster-delivery protein in the pathogenic bacterium Pseudomonas aeruginosa was investigated. The analysis of nfuA expression under various stress conditions showed that superoxide generators, a thiol-depleting agent and CuCl2 highly induced nfuA expression. The expression of nfuA was regulated by a global [2Fe-2S] cluster containing the transcription regulator IscR. Increased expression of nfuA in the ΔiscR mutant under uninduced conditions suggests that IscR acts as a transcriptional repressor. In vitro experiments revealed that IscR directly bound to a sequence homologous to the Escherichia coli Type-I IscR-binding motifs on a putative nfuA promoter that overlapped the -35 element. Binding of IscR prevented RNA polymerase from binding to the nfuA promoter, leading to repression of the nfuA transcription. Physiologically, deletion of nfuA reduced the bacterial ability to cope with oxidative stress, iron deprivation conditions and attenuated virulence in the Caenorhabditis elegans infection model. Site-directed mutagenesis analysis revealed that the conserved CXXC motif of the Nfu-type scaffold protein domain at the N-terminus was required for the NfuA functions in conferring the stress resistance phenotype. Furthermore, anaerobic growth of the ΔnfuA mutant in the presence of nitrate was drastically retarded. This phenotype was associated with a reduction in the [Fe-S] cluster containing nitrate reductase enzyme activity. However, NfuA was not required for the maturation of [Fe-S]-containing proteins such as aconitase, succinate dehydrogenase, SoxR and IscR. Taken together, our results indicate that NfuA functions in [Fe-S] cluster delivery to selected target proteins that link to many physiological processes such as anaerobic growth, bacterial virulence and stress responses in P. aeruginosa.
Highlights
Pseudomonas aeruginosa is one of the most common gram-negative bacteria causing nosocomial infections with high mortality worldwide
The ISC and SUF systems contribute to the biogenesis of [Fe-S] clusters that are supplied for the maturation of most proteins in the cell; the NIF system is required for maturation of nitrogenase in nitrogen-fixing bacteria [4, 5]
Nonsignificant induction of the gene expression was observed with H2O2-treated cells
Summary
Pseudomonas aeruginosa is one of the most common gram-negative bacteria causing nosocomial infections with high mortality worldwide. Expression of NfuAC43,47S complemented the phenotypes of increased sensitivity to oxidative stress (PQ, PB, H2O2, CuOOH and NaOCl), metal stress (CuCl2, CdCl2, CoCl3) and iron depletion (2,2’dipyridyl) in the ΔnfuA mutant to levels similar to those attained by a parental strain (Fig 5A and 5B).
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