Vanadium has an antibacterial activity against Pseudomonas aeruginosa, especially under conditions of iron limitation. Some degree of resistance to V is inducible by prior exposure to the metal. One mutant (VS1) with a higher sensitivity to V was obtained by transposon mutagenesis of P. aeruginosa PA 59.20, a clinical isolate. This mutant had an insertion in a non-coding region, upstream of a cluster of four genes. Three of them show similarities to genes corresponding to known P. aeruginosa antibiotic efflux systems, including an efflux protein, a membrane fusion protein and an outer-membrane porin. This cluster was named mexGHI-opmD. By allelic exchange, three mutants, ncr (for non-coding region), mexI and opmD were constructed in P. aeruginosa PAO1. Next to V sensitivity, the ncr, mexI and opmD mutants also showed reduced production of elastase, rhamnolipids, pyocyanine, pyoverdine and had reduced swarming motility, phenotypes that are known to be regulated by quorum sensing. All wild-type phenotypes, including growth in the presence of V, were restored by complementation with the complete cluster. The production of N-acyl-homoserine lactones (AHLs) was detected using the Chromobacter violaceum bioassay. Total extracts from the three mutants failed to induce the production of violacein by C. violaceum, although AHLs were detected by TLC and C. violaceum overlay. Violacein production was restored by complementation with mexGHI-opmD. The opmD mutant grew very slowly in LB or CAA medium, indicating that OpmD has an important physiological function for the cell. In conclusion, it is believed that the MexGHI-OpmD pump is probably involved in AHL homeostasis in P. aeruginosa.
BaysseC.,
De VosD.,
NaudetY.,
VandermondeA.,
OchsnerU.,
MeyerJ. M.,
BudzikiewiczH.,
FuchsR.,
CornelisP.
2000; Vanadium interferes with siderophore-mediated iron uptake in Pseudomonas aeruginosa
. Microbiology 146:2425–2434
BolivarF.
1978; Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules. Gene 4:121–136[CrossRef]
CornelisP.,
AnjaiahV.,
KoedamN.,
DelfosseP.,
JacquesP.,
ThonartP.,
NeirinckxL.
1992; Stability, frequency and multiplicity of transposon insertions in the pyoverdine region in the chromosomes of different fluorescent pseudomonads. J Gen Microbiol 138:1337–1343[CrossRef]
de LorenzoV.,
HerreroM.,
JakubzikU.,
TimmisK. N.
1990; Mini-Tn 5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative Eubacteria
. J Bacteriol 172:6568–6572
DiggleS. P.,
WinzerK.,
LazdunskiA.,
WilliamsP.,
CámaraM.
2002; Advancing the quorum in Pseudomonas aeruginosa : MvaT and the regulation of N-acylhomoserine lactone production and virulence gene expression. J Bacteriol 184:2576–2586[CrossRef]
EvansK.,
PassadorL.,
SrikumarR.,
TsangE.,
NezezonJ.,
PooleK.
1998; Influence of the MexAB-OprM multidrug efflux system on quorum sensing in Pseudomonas aeruginosa
. J Bacteriol 180:5443–5447
FukudaN.,
YamaseT.
1997; In vitro antibacterial activity of vanadate and vanadyl compounds against Streptococcus pneumoniae
. Biol Pharm Bull 20:927–930[CrossRef]
HassanM. T.,
van der LelieD.,
SpringaelD.,
RömlingU.,
AhmedN.,
MergeayM.
1999; Identification of a gene cluster, czr , involved in cadmium and zinc resistance in Pseudomonas aeruginosa
. Gene 238:417–425[CrossRef]
HernándezA.,
MelladoR. P.,
Martı́nezJ. L.
1998; Metal accumulation and vanadium-induced multidrug resistance by environmental isolates of Escherichia hermannii and Enterobacter cloacae
. Appl Environ Microbiol 64:4317–4320
HöfteM.,
BuysensS.,
KoedamN.,
CornelisP.
1993; Zinc affects siderophore-mediated high-affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2. BioMetals 6:85–91
KöhlerT.,
KokM.,
Michea-HamzehpourM.,
HenzeU.,
GotohN.,
CurtyL. K.,
PechèreJ. C.
1997; Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa
. Mol Microbiol 23:345–354[CrossRef]
KöhlerT.,
CurtyL. K.,
BarjaF.,
van DeldenC.,
PechèreJ. C.
2000; Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J Bacteriol 182:5990–5996[CrossRef]
KöhlerT.,
van DeldenC.,
CurtyL. K.,
HamzehpourM. M.,
PechèreJ. C.
2001; Overexpression of the MexEF-OprN multidrug efflux system affects cell-to-cell signaling in Pseudomonas aeruginosa
. J Bacteriol 183:5213–5222[CrossRef]
LatifiA.,
FoglinoM.,
TanakaK.,
WilliamsP.,
LazdunskiA.
1996; A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhlR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol Microbiol 21:1137–1146[CrossRef]
LiX.-Z.,
BarréN.,
PooleK.
2000; Influence of MexA-MexB-OprM multidrug efflux system on expression of the MexC-MexD-Opr J and MexE-MexF-OprN multidrug efflux systems in Pseudomonas aeruginosa
. J Antimicrob Chemother 46:885–893[CrossRef]
MavrodiD. V.,
BonsallR. F.,
DelaneyS. M.,
SouleM. J.,
PhillipsG.,
ThomashowL. S.
2001; Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. J Bacteriol 183:6454–6465[CrossRef]
McCleanK. H.,
KinsonM. K.,
FishL.9 other authors1997; Quorum sensing and Chromobacterium violaceum : exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143:3703–3711[CrossRef]
McKnightS. L.,
IglewskiB. H.,
PesciE. C.
2000; The Pseudomonas quinolone signal regulates rhl quorum sensing in Pseudomonas aeruginosa
. J Bacteriol 182:2702–2708[CrossRef]
MeyerJ. M.,
StintziA.,
De VosD.,
CornelisP.,
TappeR.,
TarazK.,
BudzikiewiczH.
1997; Use of siderophores to type pseudomonads: the three Pseudomonas aeruginosa pyoverdine systems. Microbiology 143:35–43[CrossRef]
PatteryT.,
HernalsteensJ. P.,
De GreveH.
1999; Identification and molecular characterization of a novel Salmonella enteritidis pathogenicity islet encoding an ABC transporter. Mol Microbiol 33:791–805[CrossRef]
PatteryT.,
MondtK.,
AudenaertK.,
HöfteM.,
CornelisP.
2001; Identification of phzM, a new phenazine biosynthesis gene necessary for the production of pyocyanine by Pseudomonas aeruginosa. Abstract presented at the Pseudomonas 2001 meetingBrussels, Belgium17–21 September 2001
PearsonJ. P.,
van DeldenC.,
IglewskiB. H.
1999; Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J Bacteriol 181:1203–1210
PesciE. C.,
MilbankJ. B.,
PearsonJ. P.,
McKnightS.,
KendeA. S.,
GreenbergE. P.,
IglewskiB. H.
1999; Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa
. Proc Natl Acad Sci USA 96:11229–11234[CrossRef]
PooleK.
2001b; Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aerugin osa and related organisms. J Mol Microbiol Biotechnol 3:255–264
SiegmundI.,
WagnerF.
1991; New methods for detecting rhamnolipids excreted by Pseudomonas species during growth in mineral agar. BioTechniques 5:265–268
SimonR.,
PrieferU.,
PühlerA.
1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1:784–791[CrossRef]
StoverC. K.,
PhamX. Q.,
ErwinA. L.
28 other authors2000; Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964[CrossRef]
TsengT. T.,
GratwickK. S.,
KollmanJ.,
ParkD.,
NiesD. H.,
GoffeauA.,
SaierM. H.
1999; The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human diseases and development proteins. J Mol Microbiol Biotechnol 1:107–125
van den EedeG.,
DeblaereR.,
GoethalsK.,
van MontaguM.,
HolstersM.
1992; Broad-host-range and promoter selection vectors for bacteria that interact with plants. Mol Plant–Microbe Interact 5:228–234[CrossRef]
Van HauteE.,
JoosH.,
MaesM.,
WarrenM.,
Van MontaguM.
1983; Intergenic transfer and recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmid of Agrobacterium tumefaciens
. EMBO J 2:411–417
WhiteleyM.,
LeeK. M.,
GreenbergE. P.
1999; Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa
. Proc Natl Acad Sci USA 96:13904–13909[CrossRef]
WhiteleyM.,
GreenbergE. P.
2001; Promoter specificity elements in Pseudomonas aeruginosa quorum-sensing-controlled genes. J Bacteriol 183:5529–5534[CrossRef]
WilsonK.
1990; Preparation of genomic DNA from bacteria. In Current Protocols in Molecular Biology pp 24.1–24.5 Edited by
AusubelF. M.,
BrentR.,
KingstonR. E.,
MooreD. D.,
SeidmanJ. D.,
SmithJ. A.,
StruhlK.
New York: Wiley;
WinzerK.,
FalconerC.,
GarberN. C.,
DiggleS. P.,
CámaraM.,
WilliamsP.
2000; The Pseudomonas aeruginosa lectins PA-IL and PA-IIL are controlled by quorum sensing and by RpoS. J Bacteriol 182:6401–6411[CrossRef]
ZhaiY.,
SaierM. H.
2001; A web-based program (WHAT) for the simultaneous prediction of hydropathy, amphipathicity, secondary structure and transmembrane topology of a single protein sequence. J Mol Microbiol Biotechnol 3:501–502