Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster

Rella, Manuela; Mercenier, Annick; Haas, Dieter

doi:10.1016/0378-1119(85)90237-9

Cited by 103 publications

(73 citation statements)

References 43 publications

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“…One clone was an isogenic transposon-bearing PA01 mutant (Jacobs et al, 2003) with an insertion in pvdF, a gene required for synthesis of the primary siderophore pyoverdine (Visca et al, 2007). The remaining clones evolved from the methionine auxotroph PA06049 (a transposon mutant of PA01: Rella et al, 1985)) under selection for siderophore cheats (Harrison and Buckling, 2005; Supplementary information). Biofilms were grown using a microplate/peg lid system (Moskowitz et al, 2004), in iron-limited or iron-supplemented medium (Full methods in Supplementary information).…”

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Siderophore production and biofilm formation as linked social traits

Harrison

Buckling

2009

The ISME Journal

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The virulence of pathogenic microbes can depend on individual cells cooperating in the concerted production of molecules that facilitate host colonization or exploitation. However, cooperating groups can be exploited by social defectors or ‘cheats’. Understanding the ecology and evolution of cooperation is therefore relevant to clinical microbiology. We studied two genetically linked cooperative traits involved in host exploitation by the opportunistic human pathogen Pseudomonas aeruginosa. Clones that defected from cooperative production of iron-scavenging siderophores were deficient in biofilm formation. The presence of such clones in mixed biofilms with a wild-type clone led to reduced biofilm mass. The fitness advantage of siderophore-deficient mutants in the presence of wild-type bacteria was no greater in biofilm than in planktonic culture, suggesting that these mutants did not gain an additional advantage by exploiting wild-type biofilm polymer. Reduced biofilm formation therefore represents a pleiotropic cost of defection from siderophore production.

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Siderophore production and biofilm formation as linked social traits

Harrison

Buckling

2009

The ISME Journal

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“…1). Plasmid pME3585 was mobilized from an exponential culture of transfer-proficient E. coli S17-1 (28) into P. aeruginosa PAO1 grown to stationary phase at 43ЊC; the mating conditions were done according to the method of Rella et al (22). Selection for tetracycline (TC) resistance (on minimal medium E containing 50 g of TC per ml) at 37ЊC produced PAO1 transconjugants carrying a chromosomally integrated copy of pME3585 at about 10 Ϫ7 per donor.…”

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Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr

et al. 1995

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The Pseudomonas aeruginosa gene anr, which encodes a structural and functional analog of the anaerobic regulator Fnr in Escherichia coli, was mapped to the SpeI fragment R, which is at about 59 min on the genomic map of P. aeruginosa PAO1. Wild-type P. aeruginosa PAO1 grew under anaerobic conditions with nitrate, nitrite, and nitrous oxide as alternative electron acceptors. An anr deletion mutant, PAO6261, was constructed. It was unable to grow with these alternative electron acceptors; however, its ability to denitrify was restored upon the introduction of the wild-type anr gene. In addition, the activities of two enzymes in the denitrification pathway, nitrite reductase and nitric oxide reductase, were not detectable under oxygen-limiting conditions in strain PAO6261 but were restored when complemented with the anr ؉ gene. These results indicate that the anr gene product plays a key role in anaerobically activating the entire denitrification pathway.

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“…These enzymes are encoded by the arcA, arcB, and arcC genes, respectively (34). The arcABC genes have been cloned and sequenced (2,3) and are organized in an operon (24,30). The arc operon is under control of a positive regulatory protein, ANR, an FNR-like protein of P. aenrginosa (16,18,37), which is thought to activate transcription of the arc genes during oxygen limitation (26).…”

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arcD, the first gene of the arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa, encodes an arginine-ornithine exchanger

et al. 1992

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In the absence of oxygen and nitrate, Pseudomonas aeruginosa metabolizes arginine via the arginine deiminase pathwayt, which allows slow growth on rich media. The conversion of arginine to ornithine, C02, and NH3 is coupled to the production of ATP from ADP. The enzymes of the arginine deiminase pathway are organized in the arcDABC operon. The arcD gene encodes a hydrophobic polytopic membrane protein.Translocation of arginine and ornithine in membrane vesicles derived from an Escherichia coli strain harboring a recombinant plasmid carrying the arcD gene was studied. Arginine and ornithine uptake was coupled to the proton motive force with a bias toward the transmembrane electrical potential. Accumulated ornithine was readily exchangeable for external arginine or lysine. The exchange was several orders of magnitude faster than proton motive force-driven transport. The ArcD protein was reconstituted in proteoliposomes after detergent solubilization of membrane vesicles. These proteoliposomes mediate a stoichiometric exchange between arginine and ornithine. It is concluded that the ArcD protein is a transport system that catalyzes an electroneutral exchange between arginine and ornithine to allow high-efficiency energy conversion in the arginine deiminase pathway.To survive anaerobiosis in the absence of nitrate as a terminal electron acceptor, Pseudomonas aeruginosa uses arginine as an energy source for viability, motility, and slow growth on rich media (31, 34). Arginine is metabolized in the arginine deiminase pathway (for a review, see reference 6). This pathway includes three cytoplasmic enzymes: (i) arginine deiminase, which catalyzes the conversion of arginine into citrulline and amnmonia (in an essentially irreversible reaction); (ii) catabolic ornithine carbamoyl transferase, which catalyzes the reaction of citrullinen with Pi to yield ornithine and carbamoyl phosphate; and (iii) carbamate kinase, which converts carbamoyl phosphate and ADP to ATP, CO2. and ammonia. These enzymes are encoded by the arcA, arcB, and arcC genes, respectively (34). The arcABC genes have been cloned and sequenced (2, 3) and are organized in an operon (24,30). The arc operon is under control of a positive regulatory protein, ANR, an FNR-like protein of P. aenrginosa (16,18,37)

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Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster

Cited by 103 publications

References 43 publications

Siderophore production and biofilm formation as linked social traits

Siderophore production and biofilm formation as linked social traits

Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr

arcD, the first gene of the arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa, encodes an arginine-ornithine exchanger

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