Pseudomonas aeruginosa nfuA: Gene regulation and its physiological roles in sustaining growth under stress and anaerobic conditions and maintaining bacterial virulence

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.

Pseudomonas aeruginosa glutathione biosynthesis genes play multiple roles in stress protection, bacterial virulence and biofilm formation

“…Total RNA isolation, agarose-formaldehyde gel electrophoresis, blotting, and hybridization were performed as previously described [ 7 , 55 ]. Purified total RNA (20 μg) was loaded into the gel.…”

Transcriptional regulation of the Pseudomonas aeruginosa iron-sulfur cluster assembly pathway by binding of IscR to multiple sites

“…Gel mobility shift assays were performed by using a labeled probe containing either native or mutagenic iscR -promoters that were amplified using 32 P-labeled BT3612 and BT3613 primers and native or mutagenized iscR -promoters as DNA templates as previously described [4, 25, 34]. Binding reactions were conducted using 3 fmol of labeled probe in 25 μl of reaction buffer containing 20 mM Tris-HCl (pH 8.0), 50 mM KCl, 4 mM MgCl 2 , 0.5 mM EDTA, 0.02 mg ml -1 bovine serum albumin (BSA), 5 mM dithiothreitol (DTT), 10% (v/v) glycerol, and 200 ng of poly(dI-dC).…”

“…Site-directed mutagenesis was performed to mutate the putative IscR binding sites on the iscR promoter from for 3’-end site B mutagenesis (MU3) by using PCR-based mutagenesis as previously described [25]. The sequences of mutagenic forward and reverse primers for site-directed mutagenesis at IscR binding site A, BT4303 and BT4304, at the 5’-end of IscR binding site B, BT4305 and BT4306, and at the 3’-end of IscR binding site B, EBI369 and EBI370, are shown in Table 2.…”

“…P . aeruginosa IscR was described, through an in vitro binding assay and gene expression analysis, as a repressor of tpx , a gene encoding thiol peroxidase, which plays a role in hydrogen peroxide resistance [5] and of nfuA , in which its gene product functions in [Fe-S] cluster delivery and is required for sustaining growth under stress and anaerobic conditions and maintaining bacterial virulence [25]. The C92, C98, C104, and H107 residues in P .…”

La protéine Fe-S NfuA, un nouvel acteur essentiel dans la virulence de Pseudomonas aeruginosa

“…Chez les bactéries, comme dans tous les organismes, les protéines Fe-S sont impliquées dans de nombreuses voies cellulaires essentielles, dont la respiration ou le maintien de l'intégrité génomique. Récemment, Romsang et al [4] se sont intéressés à la protéine d'échafaudage NfuA nécessaire à la maturation des centres Fe-S des protéines en condition de stress oxydatif ou de carence en fer. Sa fonction chez P. aeruginosa est peu connue à ce jour.…”