A bacteria-specific 2[4Fe-4S] ferredoxin is essential in Pseudomonas aeruginosa

Pseudomonas aeruginosa has two genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. We show here that P. aeruginosa fprA is an essential gene. However, the ΔfprA mutant could only be successfully constructed in PAO1 strains containing an extra copy of fprA on a mini-Tn7 vector integrated into the chromosome or carrying it on a temperature-sensitive plasmid. The strain containing an extra copy of the ferredoxin gene (fdx1) could suppress the essentiality of FprA. Other ferredoxin genes could not suppress the requirement for FprA, suggesting that Fdx1 mediates the essentiality of FprA. The expression of fprA was highly induced in response to treatments with a superoxide generator, paraquat, or sodium hypochlorite (NaOCl). The induction of fprA by these treatments depended on FinR, a LysR-family transcription regulator. In vivo and in vitro analysis suggested that oxidized FinR acted as a transcriptional activator of fprA expression by binding to its regulatory box, located 20 bases upstream of the fprA -35 promoter motif. This location of the FinR box also placed it between the -35 and -10 motifs of the finR promoter, where the reduced regulator functions as a repressor. Under uninduced conditions, binding of FinR repressed its own transcription but had no effect on fprA expression. Exposure to paraquat or NaOCl converted FinR to a transcriptional activator, leading to the expression of both fprA and finR. The ΔfinR mutant showed an increased paraquat sensitivity phenotype and attenuated virulence in the Drosophila melanogaster host model. These phenotypes could be complemented by high expression of fprA, indicating that the observed phenotypes of the ΔfinR mutant arose from the inability to up-regulate fprA expression. In addition, increased expression of fprB was unable to rescue essentiality of fprA or the superoxide-sensitive phenotype of the ΔfinR mutant, suggesting distinct mechanisms of the FprA and FprB enzymes.

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“…aeruginosa . Fdx1 has been shown to be essential for the viability of PAO1 [ 40 ]. The physiological role of Fdx1 in P .…”

Section: Resultsmentioning

confidence: 99%

“…aeruginosa . An fdx1 encoding putative reaction partner of FprA is also an essential gene in PAO1 [ 40 ]. Thus, the link between FprA and Fdx1 is important to PAO1 physiology.…”

Section: Resultsmentioning

confidence: 99%

The FinR-regulated essential gene fprA, encoding ferredoxin NADP+ reductase: Roles in superoxide-mediated stress protection and virulence of Pseudomonas aeruginosa

et al. 2017

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“…These novel proteins contain two [4Fe-4S] clusters of widely differing and very low redox potential (E 0 1 ϳ Ϫ650 mV; E 0 2 ϳ Ϫ450 mV) due to unique structural motifs (36). Elsen et al found evidence that the essential Alvin ferredoxin in Pseudomonas aeruginosa was likely not involved in the catabolism of aromatic compounds but instead that its gene had the constitutive expression characteristics of a housekeeping gene (37). It's essentiality in that organism showed that other redox paralogs could not substitute for its essential function.…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

Lauhon

2019

J Bacteriol

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In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo 5 U) or 5-methoxycarbonylmethoxyuridine (mcmo 5 U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho 5 U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho 5 U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho 5 U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho 5 C synthesis in E. coli. Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo 5 U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho 5 U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo 5 U as that observed for mo 5 U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho 5 U synthesis in E. coli to the same extent as yegQ. Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho 5 U biosynthesis in bacteria is similar to that of ho 5 C, but additional genes and substrates are required for complete modification. IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli. These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

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“…Cells with higher levels of IscA protein might be more efficient in repairing and assembling damaged Fe-S proteins. Ferredoxins play an important role in a metabolism and are involved in electron transfer processes [5]. Oxidative stress down-regulates ferredoxin, and its overexpression can counteract this effect [8].…”

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confidence: 99%

Hsp20, a Small Heat Shock Protein of Deinococcus radiodurans, Confers Tolerance to Hydrogen Peroxide in Escherichia coli

Singh

Appukuttan²,

Lim³

2014

Journal of Microbiology and Biotechnology

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Heat shock proteins (HSPs) are molecular chaperones that maintain cellular homeostasis. They bind to and assist in the proper folding of misfolded cellular proteins, thereby preventing protein aggregation [6, 10, 25]. These processes play an important role in protecting cells against various stresses, such as radiation, heat, cold, and oxidative stress [9]. Among the various HSPs that have been identifed, small HSPs (sHSPs) are proteins of low molecular mass (12-30 kDa) that are present in organisms from all domains of life [4, 17]. Deinococcus radiodurans is a Gram-positive coccus that is highly resistant to a variety of stresses, including gamma radiation (γ-radiation), UV light, desiccation, heat, hydrogen peroxide (H 2 O 2), and other DNA-damaging agents [18]. D. radiodurans contains two sHSPs, Hsp17.7 (DR1691) and Hsp20.2 (DR1114). The two proteins differ in sequence, structure, and function. Hsp17 forms a dimer and unstable substrate complexes [2], whereas Hsp20 shows constitutive and stable expression, typical chaperone activity, and forms spherical homo-oligomers and stable substrate complexes. The expression of hsp20 (dr1114) has been found to be up-regulated under the conditions of stress [2, 24].

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A bacteria-specific 2[4Fe-4S] ferredoxin is essential in Pseudomonas aeruginosa

Cited by 10 publications

References 49 publications

The FinR-regulated essential gene fprA, encoding ferredoxin NADP+ reductase: Roles in superoxide-mediated stress protection and virulence of Pseudomonas aeruginosa

The FinR-regulated essential gene fprA, encoding ferredoxin NADP+ reductase: Roles in superoxide-mediated stress protection and virulence of Pseudomonas aeruginosa

Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

Hsp20, a Small Heat Shock Protein of Deinococcus radiodurans, Confers Tolerance to Hydrogen Peroxide in Escherichia coli

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