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

Elsen S, Efthymiou G, Peteinatos P, Diallinas G, Kyritsis P, Moulis JM - BMC Microbiol. (2010)

Bottom Line: A sub-group binds two [4Fe-4S] clusters with unequal and extremely low values of the reduction potentials.These unusual properties are associated with two specific fragments of sequence.These data identify a new potential antimicrobial target in this and other pathogenic Proteobacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, iRTSV, CEA, Grenoble, France.

ABSTRACT

Background: Ferredoxins are small iron-sulfur proteins belonging to all domains of life. A sub-group binds two [4Fe-4S] clusters with unequal and extremely low values of the reduction potentials. These unusual properties are associated with two specific fragments of sequence. The functional importance of the very low potential ferredoxins is unknown.

Results: A bioinformatic screening of the sequence features defining very low potential 2[4Fe-4S] ferredoxins has revealed the almost exclusive presence of the corresponding fdx gene in the Proteobacteria phylum, without occurrence in Archaea and Eukaryota. The transcript was found to be monocistronic in Pseudomonas aeruginosa, and not part of an operon in most bacteria. Only fdx genes of bacteria which anaerobically degrade aromatic compounds belong to operons. As this pathway is not present in all bacteria having very low potential 2[4Fe-4S] ferredoxins, these proteins cannot exclusively be reductants of benzoyl CoA reductases. Expression of the ferredoxin gene did not change in response to varying growth conditions, including upon macrophage infection or aerobic growth with 4-hydroxy benzoate as carbon source. However, it increased along the growth curve in Pseudomonas aeruginosa and in Escherichia coli. The sequence immediately 5' upstream of the coding sequence contributed to the promotor activity. Deleting the fdx gene in Pseudomonas aeruginosa abolished growth, unless a plasmid copy of the gene was provided to the deleted strain.

Conclusions: The gene of the very low potential 2[4Fe-4S] ferredoxin displays characteristics of a housekeeping gene, and it belongs to the minority of genes that are essential in Pseudomonas aeruginosa. These data identify a new potential antimicrobial target in this and other pathogenic Proteobacteria.

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β-Galactosidase activities in P. aeruginosa strains containing chromosomal lacZ fusions to the fdx1 5' sequence. (A) Scheme of the two constructs used to monitor transcriptional activities of the fdx1 promoter. The -529 and -200 positions are relative to the +1 start of translation. (B) Relative β-galactosidase activities triggered by the constructs in (A) under normal conditions (white bars), for calcium depleted (for T3SS induction) cells (black bars), and for cells grown under semi-aerobic conditions with KNO3 (gray bars). (C) β-galactosidase activities were measured in pFdx1Z and pFdx1shortZ strains grown in LB medium at the indicated OD600. The reported activity values are the average of at least two independent experiments performed in duplicate or triplicate. Error bars indicate standard deviations.
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Figure 4: β-Galactosidase activities in P. aeruginosa strains containing chromosomal lacZ fusions to the fdx1 5' sequence. (A) Scheme of the two constructs used to monitor transcriptional activities of the fdx1 promoter. The -529 and -200 positions are relative to the +1 start of translation. (B) Relative β-galactosidase activities triggered by the constructs in (A) under normal conditions (white bars), for calcium depleted (for T3SS induction) cells (black bars), and for cells grown under semi-aerobic conditions with KNO3 (gray bars). (C) β-galactosidase activities were measured in pFdx1Z and pFdx1shortZ strains grown in LB medium at the indicated OD600. The reported activity values are the average of at least two independent experiments performed in duplicate or triplicate. Error bars indicate standard deviations.

Mentions: To look for regulation, measurements of the P. aeruginosa fdx1 mRNA levels have been carried out under different conditions. It was found that the relative expression of fdx increased along the growth phase (Figure 3B, see also below Figure 4C). Since P. aeruginosa is an opportunistic pathogen, we wondered whether fdx1 expression was also triggered during host-bacterium interaction or co-regulated with other virulence factors. Calcium depletion by EGTA to chemically induce synthesis of the Type 3 Secretion System (T3SS) [24], a major virulence factor of P. aeruginosa, did not change the expression of fdx1 (Figure 3B). T3SS is naturally induced when bacteria contact host cells [25]. Yet, P. aeruginosa cells in the presence of macrophages showed similar amounts of fdx1 mRNA, relative to rRNA, from the time of contact up to 2 hours later (Figure 3C).


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

Elsen S, Efthymiou G, Peteinatos P, Diallinas G, Kyritsis P, Moulis JM - BMC Microbiol. (2010)

β-Galactosidase activities in P. aeruginosa strains containing chromosomal lacZ fusions to the fdx1 5' sequence. (A) Scheme of the two constructs used to monitor transcriptional activities of the fdx1 promoter. The -529 and -200 positions are relative to the +1 start of translation. (B) Relative β-galactosidase activities triggered by the constructs in (A) under normal conditions (white bars), for calcium depleted (for T3SS induction) cells (black bars), and for cells grown under semi-aerobic conditions with KNO3 (gray bars). (C) β-galactosidase activities were measured in pFdx1Z and pFdx1shortZ strains grown in LB medium at the indicated OD600. The reported activity values are the average of at least two independent experiments performed in duplicate or triplicate. Error bars indicate standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984482&req=5

Figure 4: β-Galactosidase activities in P. aeruginosa strains containing chromosomal lacZ fusions to the fdx1 5' sequence. (A) Scheme of the two constructs used to monitor transcriptional activities of the fdx1 promoter. The -529 and -200 positions are relative to the +1 start of translation. (B) Relative β-galactosidase activities triggered by the constructs in (A) under normal conditions (white bars), for calcium depleted (for T3SS induction) cells (black bars), and for cells grown under semi-aerobic conditions with KNO3 (gray bars). (C) β-galactosidase activities were measured in pFdx1Z and pFdx1shortZ strains grown in LB medium at the indicated OD600. The reported activity values are the average of at least two independent experiments performed in duplicate or triplicate. Error bars indicate standard deviations.
Mentions: To look for regulation, measurements of the P. aeruginosa fdx1 mRNA levels have been carried out under different conditions. It was found that the relative expression of fdx increased along the growth phase (Figure 3B, see also below Figure 4C). Since P. aeruginosa is an opportunistic pathogen, we wondered whether fdx1 expression was also triggered during host-bacterium interaction or co-regulated with other virulence factors. Calcium depletion by EGTA to chemically induce synthesis of the Type 3 Secretion System (T3SS) [24], a major virulence factor of P. aeruginosa, did not change the expression of fdx1 (Figure 3B). T3SS is naturally induced when bacteria contact host cells [25]. Yet, P. aeruginosa cells in the presence of macrophages showed similar amounts of fdx1 mRNA, relative to rRNA, from the time of contact up to 2 hours later (Figure 3C).

Bottom Line: A sub-group binds two [4Fe-4S] clusters with unequal and extremely low values of the reduction potentials.These unusual properties are associated with two specific fragments of sequence.These data identify a new potential antimicrobial target in this and other pathogenic Proteobacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, iRTSV, CEA, Grenoble, France.

ABSTRACT

Background: Ferredoxins are small iron-sulfur proteins belonging to all domains of life. A sub-group binds two [4Fe-4S] clusters with unequal and extremely low values of the reduction potentials. These unusual properties are associated with two specific fragments of sequence. The functional importance of the very low potential ferredoxins is unknown.

Results: A bioinformatic screening of the sequence features defining very low potential 2[4Fe-4S] ferredoxins has revealed the almost exclusive presence of the corresponding fdx gene in the Proteobacteria phylum, without occurrence in Archaea and Eukaryota. The transcript was found to be monocistronic in Pseudomonas aeruginosa, and not part of an operon in most bacteria. Only fdx genes of bacteria which anaerobically degrade aromatic compounds belong to operons. As this pathway is not present in all bacteria having very low potential 2[4Fe-4S] ferredoxins, these proteins cannot exclusively be reductants of benzoyl CoA reductases. Expression of the ferredoxin gene did not change in response to varying growth conditions, including upon macrophage infection or aerobic growth with 4-hydroxy benzoate as carbon source. However, it increased along the growth curve in Pseudomonas aeruginosa and in Escherichia coli. The sequence immediately 5' upstream of the coding sequence contributed to the promotor activity. Deleting the fdx gene in Pseudomonas aeruginosa abolished growth, unless a plasmid copy of the gene was provided to the deleted strain.

Conclusions: The gene of the very low potential 2[4Fe-4S] ferredoxin displays characteristics of a housekeeping gene, and it belongs to the minority of genes that are essential in Pseudomonas aeruginosa. These data identify a new potential antimicrobial target in this and other pathogenic Proteobacteria.

Show MeSH
Related in: MedlinePlus