In an attempt to identify components of a ferric citrate uptake system in Pseudomonas aeruginosa, a mutant library of a siderophore-deficient strain (IA614) was constructed and screened for defects in citrate-promoted growth in an Fe-restricted medium. A mutant disrupted in gene PA3901, encoding a homologue of the outer-membrane ferric citrate receptor, FecA, of Escherichia coli (FecAE.c.), was recovered and shown to be deficient in citrate-promoted growth and citrate-mediated Fe uptake. A mutant disrupted in gene PA4825, encoding a homologue of the MgtA/MgtB Mg2+ transporters in Salmonella enterica, was similarly deficient in citrate-promoted growth, though this was due to a citrate sensitivity of the mutant apparently resulting from citrate-promoted acquisition of Fe2+ and resultant oxidative stress. Consistent with citrate delivering Fe to cells as Fe2+, a P. aeruginosa mutant lacking the FeoB Fe2+ transporter homologue, PA4358, was compromised for citrate-promoted growth in Fe-restricted medium and showed markedly reduced citrate-mediated Fe uptake. Subsequent elimination of two Fe3+ transporter homologues, PA5216 and PA4687, in the feoB mutant failed to further compromise citrate-promoted growth or Fe uptake, though the additional loss of pcoA, encoding a periplasmic ferroxidase implicated in Fe2+ acquisition, completely abrogated citrate-mediated Fe uptake. Fe acquisition mediated by other siderophores (e.g. pyoverdine) was, however, unaffected in the quadruple knockout strain. These data indicate that Fe delivered to P. aeruginosa by citrate is released as Fe2+, probably in the periplasm, prior to its transport into cells via Fe transport components.
AdhikariP.,
BerishS. A.,
NowalkA. J.,
VeraldiK. L.,
MorseS. A.,
MietznerT. A.1996; The fbpABC locus of Neisseria gonorrhoeae functions in the periplasm-to-cytosol transport of iron. J Bacteriol 178:2145–2149
AusubelF. M.,
BrentR.,
KingstonR. E.,
MooreD. D.,
SeidmanJ. G.,
SmithJ. A.,
StruhlK.1992Short Protocols in Molecular Biology, 2nd edn. New York: Wiley;
CaoL.,
SrikumarR.,
PooleK.2004; MexAB-OprM hyperexpression in NalC type multidrug resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720–PA3719. Mol Microbiol 53:1423–1436
ChoiK. H.,
KumarA.,
SchweizerH. P.2005; A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64:391–397
CornelisP.,
MoguilevskyN.,
JacquesJ. F.,
MassonP. L.1987; Study of the siderophores and receptors in different clinical isolates of Pseudomonas aeruginosa
. Antibiot Chemother 39:290–306
CuivP. O.,
ClarkeP.,
O'ConnellM.2006; Identification and characterization of an iron-regulated gene, chtA, required for the utilization of the xenosiderophores aerobactin, rhizobactin 1021 and schizokinen by Pseudomonas aeruginosa
. Microbiology 152:945–954
CuivP. O.,
KeoghD.,
ClarkeP.,
O'ConnellM.2007; FoxB of Pseudomonas aeruginosa functions in the utilization of the xenosiderophores ferrichrome, ferrioxamine B, and schizokinen: evidence for transport redundancy at the inner membrane. J Bacteriol 189:284–287
de LorenzoV.,
HerreroM.,
JakubzikU.,
TimmisK. N.1990; Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J Bacteriol 172:6568–6572
FaguyD. M.,
BayleyD. P.,
KostyukovaA. S.,
ThomasN. A.,
JarrellK. F.1996; Isolation and characterization of flagella and flagellin proteins from the thermoacidophilic archaea Thermoplasma volcanium and Sulfolobus shibatae
. J Bacteriol 178:902–905
GhyselsB.,
OchsnerU.,
MollmanU.,
HeinischL.,
VasilM.,
CornelisP.,
MatthijsS.2005; The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues. FEMS Microbiol Lett 246:167–174
GreenwaldJ.,
HoegyF.,
NaderM.,
JournetL.,
MislinG. L.,
GraumannP. L.,
SchalkI. J.2007; Real time fluorescent resonance energy transfer visualization of ferric pyoverdine uptake in Pseudomonas aeruginosa. A role for ferrous iron. J Biol Chem 282:2987–2995
HeinrichsD. E.,
YoungL.,
PooleK.1991; Pyochelin-mediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein. Infect Immun 59:3680–3684
HoangT. T.,
Karkhoff-SchweizerR. R.,
KutchmaA. J.,
SchweizerH. P.1998; A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86
HustonW. M.,
JenningsM. P.,
McEwanA. G.2002; The multicopper oxidase of Pseudomonas aeruginosa is a ferroxidase with a central role in iron acquisition. Mol Microbiol 45:1741–1750
KatohH.,
HaginoN.,
GrossmanA. R.,
OgawaT.2001; Genes essential to iron transport in the cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol 183:2779–2784
LlamasM. A.,
SparriusM.,
KloetR.,
JimenezC. R.,
Vandenbroucke-GraulsC.,
BitterW.2006; The heterologous siderophores ferrioxamine B and ferrichrome activate signaling pathways in Pseudomonas aeruginosa
. J Bacteriol 188:1882–1891
LouvelH.,
SaintG. I.,
PicardeauM.2005; Isolation and characterization of FecA- and FeoB-mediated iron acquisition systems of the spirochete Leptospira biflexa by random insertional mutagenesis. J Bacteriol 187:3249–3254
MazoyR.,
LopezE. M.,
FouzB.,
AmaroC.,
LemosM. L.1999; Ferric-reductase activities in Vibrio vulnificus biotypes 1 and 2. FEMS Microbiol Lett 172:205–211
MeyerJ. M.1992; Exogenous siderophore-mediated iron uptake in Pseudomonas aeruginosa: possible involvement of porin OprF in iron translocation. J Gen Microbiol 138:951–958
MeyerJ.-M.,
AbdallahM. A.1978; The fluorescent pigment of Pseudomonas fluorescens: biosynthesis, purification and physiochemical properties. J Gen Microbiol 107:319–328
MeyerJ.-M.,
HornspergerJ. M.1978; Role of pyoverdinePf, the iron-binding fluorescent pigment of Pseudomonas fluorescens, in iron transport. J Gen Microbiol 107:329–331
MeyerJ. M.,
StintziA.,
CoulangesV.,
ShivajiS.,
VossJ. A.,
TarazK.,
BudzikiewiczH.1998; Siderotyping of fluorescent pseudomonads: characterization of pyoverdines of Pseudomonas fluorescens and Pseudomonas putida strains from Antarctica. Microbiology 144:3119–3126
MeyerJ. M.,
StintziA.,
PooleK.1999; The ferripyoverdine receptor FpvA of Pseudomonas aeruginosa PAO1 recognizes the ferripyoverdines of Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens ATCC 13525. FEMS Microbiol Lett 170:145–150
MielczarekE. V.,
RoytP. W.,
Toth-AllenJ.1990; A Mossbauer spectroscopy study of cellular acquisition of iron from pyoverdine by Pseudomonas aeruginosa
. Biol Met 3:34–38
MillerV. L.,
MekalanosJ. J.1988; A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR
. J Bacteriol 170:2575–2583
NaikareH.,
PalyadaK.,
PancieraR.,
MarlowD.,
StintziA.2006; Major role for FeoB in Campylobacter jejuni ferrous iron acquisition, gut colonization, and intracellular survival. Infect Immun 74:5433–5444
NehmeD.,
LiX. Z.,
ElliotR.,
PooleK.2004; Assembly of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa: identification and characterization of mutations in mexA compromising MexA multimerization and interaction with MexB. J Bacteriol 186:2973–2983
OchsnerU. A.,
WildermanP. J.,
VasilA. I.,
VasilM. L.2002; GeneChip® expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes. Mol Microbiol 45:1277–1287
PooleK.,
NeshatS.,
HeinrichsD.1991; Pyoverdine-mediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein. FEMS Microbiol Lett 62:1–5
RedlyG. A.,
PooleK.2003; Pyoverdine-mediated regulation of FpvA synthesis in Pseudomonas aeruginosa: involvement of a probable extracytoplasmic-function sigma factor, FpvI. J Bacteriol 185:1261–1265
SandersJ. D.,
CopeL. D.,
HansenE. J.1994; Identification of a locus involved in the utilization of iron by Haemophilus influenzae
. Infect Immun 62:4515–4525
SchäferA.,
TauchA.,
JägerW.,
KalinowskiJ.,
ThierbachG.,
PühlerA.1994; Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum
. Gene 145:69–73
SimonR.,
PrieferU.,
PuehlerA.1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1:784–791
SobelM. L.,
McKayG. A.,
PooleK.2003; Contribution of the MexXY multidrug transporter to aminoglycoside resistance in Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother 47:3202–3207
VelayudhanJ.,
HughesN. J.,
McColmA. A.,
BagshawJ.,
ClaytonC. L.,
AndrewsS. C.,
KellyD. J.2000; Iron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high-affinity ferrous iron transporter. Mol Microbiol 37:274–286
ViscaP.,
LeoniL.,
WilsonM. J.,
LamontI. L.2002; Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas
. Mol Microbiol 45:1177–1190
WyckoffE. E.,
MeyA. R.,
LeimbachA.,
FisherC. F.,
PayneS. M.2006; Characterization of ferric and ferrous iron transport systems in Vibrio cholerae
. J Bacteriol 188:6515–6523