Pseudomonas aeruginosa IscR-Regulated Ferredoxin NADP(+) Reductase Gene (fprB) Functions in Iron-Sulfur Cluster Biogenesis and Multiple Stress Response.

P. aeruginosa (PAO1) has two putative genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. Here, the regulation of fprB expression and the protein's physiological roles in [4Fe-4S] cluster biogenesis and stress protection are characterized. The fprB mutant has defects in [4Fe-4S] cluster biogenesis, as shown by reduced activities of [4Fe-4S] cluster-containing enzymes. Inactivation of the gene resulted in increased sensitivity to oxidative, thiol, osmotic and metal stresses compared with the PAO1 wild type. The increased sensitivity could be partially or completely suppressed by high expression of genes from the isc operon, which are involved in [Fe-S] cluster biogenesis, indicating that stress sensitivity in the fprB mutant is partially caused by a reduction in levels of [4Fe-4S] clusters. The pattern and regulation of fprB expression are in agreement with the gene physiological roles; fprB expression was highly induced by redox cycling drugs and diamide and was moderately induced by peroxides, an iron chelator and salt stress. The stress-induced expression of fprB was abolished by a deletion of the iscR gene. An IscR DNA-binding site close to fprB promoter elements was identified and confirmed by specific binding of purified IscR. Analysis of the regulation of fprB expression supports the role of IscR in directly regulating fprB transcription as a transcription activator. The combination of IscR-regulated expression of fprB and the fprB roles in response to multiple stressors emphasizes the importance of [Fe-S] cluster homeostasis in both gene regulation and stress protection.

The selectable antibiotic marker, tetA(C), increases Pseudomonas aeruginosa susceptibility to the herbicide/superoxide generator, paraquat.

The presence of the widely used selectable antibiotic marker, tetA(C), unexpectedly increased the sensitivity of Pseudomonas aeruginosa PAO1 to the superoxide-generating herbicide, paraquat. A DNA fragment spanning the first 99 amino acids of TetA(C) was sufficient to confer paraquat sensitivity. The TetA(C)-induced paraquat sensitive phenotype was observed in other Gram-negative bacteria such as Agrobacterium tumefaciens, Salmonella enterica ser. Typhimurium and Xanthomonas campestris suggesting that this is a general property of tetA(C). This finding serves as a cautionary note for those using tetA(C) as a selectable marker for genetic manipulations in studies using paraquat either as a superoxide stress generator or a redox cycling drug.