TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Genotype and relevant characteristicsa | Source or reference |
|---|---|---|
| Strains | ||
| P. aeruginosa | ||
| PAO1 | Wild type | |
| PAO1ΔchpA | In-frame deletion of chpA | 61 |
| PAO1ΔchpA(comp) | PAO1ΔchpA complemented at the chpA locus | This study |
| PAO1ΔfliC | In-frame deletion of fliC | This study |
| PAO1ΔfliC(comp) | PAO1ΔfliC complemented at the fliC locus | This study |
| PAO1ΔpilA | In-frame deletion of pilA | This study |
| PAO1ΔpilA(comp) | PAO1ΔpilA complemented at the pilA locus | This study |
| PAO1ΔpilB | In-frame deletion of pilB | This study |
| PAO1ΔpilA(comp) | PAO1ΔpilB complemented at the pilB locus | This study |
| PAO1ΔpilG | In-frame deletion of pilG | This study |
| PAO1ΔpilG(comp) | PAO1ΔpilG complemented at the pilG locus | This study |
| PAO1ΔpilH | In-frame deletion of pilH | 3 |
| PAO1ΔpilH(comp) | PAO1ΔpilH complemented at the pilH locus | This study |
| PAO1ΔpilR | In-frame deletion of pilR | This study |
| PAO1ΔpilR(comp) | PAO1ΔpilR complemented at the pilR locus | This study |
| PAO1ΔpilTCTX-pilU | In-frame deletion of pilT; pilU and 1 kb upstream sequence at attB site | This study |
| PAO1pilTTrunc | pilT truncation mutant, deletes 429 bp of pilT including the start codon; stop codons in all three reading frames | This study |
| PAO1ΔpilT(comp) | PAO1ΔpilT complemented at the pilT locus | This study |
| PAO1ΔpilU | In-frame deletion of pilU | This study |
| PAO1ΔpilU(comp) | PAO1ΔpilU complemented at the pilU locus | This study |
| PAO1ΔchpAΔpilH | In-frame deletion of chpA and pilH | This study |
| PAO1ΔchpΔpilTCTX-pilU | In-frame deletion of chpA and pilT; pilU and 1 kb upstream sequence at attB site | This study |
| PAO1ΔchpAΔpilU | In-frame deletion of chpA and pilU | This study |
| PAO1ΔchpAΔpilTΔpilU | In-frame deletion of chpA, pilT, and pilU | This study |
| PAO1ΔpilBΔpilH | In-frame deletion of pilB and pilH | This study |
| PAO1ΔpilBΔpilTCTX-pilU | In-frame deletion of pilB and pilT; pilU and 1 kb upstream sequence at attB site | This study |
| PAO1ΔpilBΔpilU | In-frame deletion of pilB and pilU | This study |
| PAO1ΔpilBΔpilTΔpilU | In-frame deletion of pilB, pilT, and pilU | This study |
| PAO1ΔpilGΔpilH | In-frame deletion of pilG and pilH | This study |
| PAO1ΔpilGΔpilT-gent | PAO1ΔpilG with a Gmr-tagged insertion in pilT | This study |
| PAO1ΔpilGΔpilTCTX-pilU | In-frame deletion of pilG and pilT; pilU and 1 kb upstream sequence at attB site | This study |
| PAO1ΔpilGΔpilU-gent | PAO1ΔpilG with a Gmr-tagged insertion in pilU | This study |
| PAO1ΔpilGΔpilU | In-frame deletion of pilG and pilU | This study |
| PAO1ΔpilGΔpilTΔpilU | In-frame deletion of pilG, pilT, and pilU | This study |
| PAO1ΔpilHΔpilTCTX-pilU | In-frame deletion of pilH and pilT; pilU and 1 kb upstream sequence at attB site | This study |
| PAO1ΔpilTΔpilU | In-frame deletion of pilT and pilU | This study |
| PAO1ΔpilHΔpilU | In-frame deletion of pilH and pilU | This study |
| PAO1ΔpilHΔpilTΔpilU | In-frame deletion of pilH, pilT, and pilU | This study |
| PAO1chpA-FLAG | chpA with a double FLAG tag inserted into the NotI/KpnI sites of chpA at the chpA locus | This study |
| PAO1chpA(AAA)-FLAG | chpA-FLAG with ChpA D2091A, D2092A, and G2093A | This study |
| PAO1pilG-His | pilG with a C-terminal His6 tag at the pilG locus | This study |
| PAO1pilG(D58A)-His | pilG-His with PilG D58A | This study |
| PAO1pilH-His | pilH with a C-terminal His6 tag at the pilH locus | This study |
| PAO1pilH(D52A)-His | pilH-His with PilH D52A | This study |
| PAO1::CTX-PpilA-lacZ | PAO1; pilA promoter fused to lacZ at attB site | |
| PAO1ΔchpA::CTX-PpilA-lacZ | ΔchpA; pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔchpA(comp)::CTX-PpilA-lacZ | ΔchpA(comp); pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔfliC::CTX-PpilA-lacZ | ΔfliC; pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔfliC(comp)::CTX-PpilA-lacZ | ΔfliC(comp); pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilA::CTX-PpilA-lacZ | ΔpilA; pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilA(comp)::CTX-PpilA-lacZ | ΔpilA(comp); pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilB::CTX-PpilA-lacZ | ΔpilB; pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilA(comp)::CTX-PpilA-lacZ | ΔpilB(comp); pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilG::CTX-PpilA-lacZ | ΔpilG; pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilG(comp)::CTX-PpilA-lacZ | ΔpilG(comp); pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilH::CTX-PpilA-lacZ | ΔpilH; pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilH(comp)::CTX-PpilA-lacZ | ΔpilH(comp); pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilR::CTX-PpilA-lacZ | ΔpilR; pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilR(comp)::CTX-PpilA-lacZ | ΔpilR(comp); pilA promoter fused to lacZ at attB site | This study |
| PAO1pilTTrunc::CTX-PpilA-lacZ | pilT-Trunc; pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilT(comp)::CTX-PpilA-lacZ | ΔpilT(comp); pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilU::CTX-PpilA-lacZ | ΔpilU; pilA promoter fused to lacZ at attB site | This study |
| PAO1ΔpilU(comp)::CTX-PpilA-lacZ | ΔpilU(comp); pilA promoter fused to lacZ at attB site | This study |
| E. coli | ||
| DH5α | hsdR rec lacZYA φ80 lacZΔM15 | Invitrogen |
| S17.1λpir | Thi pro hsdR recA RP4-2(Tc::Mu)(Km::Tn7) | Stratagene |
| SM10 | thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu Km λpir | 15 |
| Plasmids | ||
| pOK12 | E. coli cloning vector; Kmr | 54 |
| pUCPSK | P. aeruginosa-E. coli shuttle vector; Apr | 56 |
| pGEM-T | E. coli cloning vector; Apr | Promega |
| pEX100T | Allelic replacement suicide plasmid; Apr (Cbr) | 49 |
| pX1918GT | Source of Gmr cassette; Apr | 49 |
| mCTX-lacZ | Contains promoterless lacZ for constructing transcriptional fusions at the attB site; Tcr | 4 |
| mCTX2 | Construct for introduction of exogenous DNA fragments at the attB site | 24 |
| pFLP2 | Source of Flp recombinase; Apr | 23 |
| pAL64 | pUCPSK carrying chpA with an internal double FLAG tag inserted between the native NotI/NruI sites | 39 |
| pEN34 | Allelic replacement suicide plasmid; Tcr | 61 |
| pJEN34PAO1KO3 | pJEN34 carrying ΔpilA on SpeI fragment | This study |
| pJB4 | pOK12 carrying ΔchpA on EcoRI/XbaI fragment | 61 |
| pJB43 | pGEM-T containing 1-kb region 5′ of pilG | This study |
| pJB44 | pGEM-T containing 1-kb region 3′ of pilG | This study |
| pJB48 | pGEM-T containing 1-kb region 3′ of pilH | 3 |
| pJB60 | XhoI/HindIII fragment from pJB43 and HindIII/XbaI from pJB44 concatamerized and cloned into XhoI/XbaI sites of pOK12 | This study |
| pJB62 | pOK12 carrying ΔpilH on KpnI/XbaI fragment | 3 |
| pJB87 | pJEN34 carrying ΔpilG on SpeI fragment | This study |
| pJB96 | XhoI/HindIII fragment from pJB43 and HindIII/XbaI from pJB48 concatamerized and cloned into XhoI/XbaI sites of pOK12 | This study |
| pJB97 | pJEN34 carrying ΔpilGΔpilH on SpeI fragment | This study |
| pJB100 | pEX100T derivative replacing SmaI site with the SpeI site | |
| pJB109 | pJB100 carrying ΔpilT on SpeI fragment | This study |
| pJB110 | Gmr-tagged ΔpilT in pJB100; pX1918-derived Gmr cassette cloned as HindIII fragment into pJB109 | This study |
| pJB111 | pJB100 carrying ΔpilU on SpeI fragment | This study |
| pJB112 | Gmr-tagged ΔpilU in pJB100; pX1918-derived Gmr-cassette cloned as HindIII fragment into pJB109 | This study |
| pJB113 | pJB100 carrying ΔpilT complementation construct on SpeI fragment | This study |
| pJB114 | pJB100 carrying ΔpilU complementation construct on SpeI fragment | This study |
| pJB116 | pJB100 carrying ΔchpA on SpeI fragment; subcloned from pJB4 | This study |
| pJB118 | pJB100 carrying ΔpilG on SpeI fragment; subcloned from pJB60 | This study |
| pJB119 | pJB100 carrying ΔpilH on SpeI fragment; subcloned from pJB62 | This study |
| pJB122 | pJB100 carrying ΔpilB on SpeI fragment | This study |
| pJB124 | pJB100 carrying pilH-His allelic exchange construct on SpeI fragment | This study |
| pJB126 | pJB100 carrying ΔpilG complementation construct on SpeI fragment | This study |
| pJB127 | pJB100 carrying pilG-His allelic exchange construct on SpeI fragment | This study |
| pJB163 | pJB100 carrying ΔpilTΔpilU on the SpeI fragment | This study |
| pJB202 | FLAG-tagged chpA histidine kinase domain in pOK12; subcloned as KpnI/ClaI fragment from pAL64 | This study |
| pJB203 | pJB100 carrying a pilT truncation construct on SpeI fragment | This study |
| pJB207 | chpA(AAA) histidine kinase domain in pOK12 | This study |
| pJB212 | pilU and 1-kb 5′ sequence cloned as SpeI/HindIII fragment into mCTX2 | This study |
| pJB215 | pJB100 carrying ΔfliC on SpeI fragment | This study |
| pJB219 | Modified pOK12 lacking KpnI and ClaI sites; BglII/SacI fragment removed from pOK12 MCS | This study |
| pJB220 | 3′ chpA fragment including 1-kb 3′ flanking sequence in pJB219 | This study |
| pJB221 | chpA including 1 kb of 5′ and 3′ flanking sequence in pJB219 | This study |
| pJB230 | pJB100 carrying ΔfliC complementation construct on SpeI fragment | This study |
| pJB231 | pJB100 carrying ΔchpA complementation construct on SpeI fragment; subcloned from pJB221 | This study |
| pJB232 | chpA-FLAG, including 1 kb of 5′ and 3′ flanking sequence in pJB219; KpnI/ClaI fragment from pJB202 subcloned into pJB221 from which the corresponding KpnI/ClaI fragment had been removed | This study |
| pJB233 | chpA(AAA)-FLAG including 1 kb of 5′ and 3′ flanking sequence in pJB219; KpnI/ClaI fragment from pJB207 subcloned into pJB221 from which the corresponding KpnI/ClaI fragment had been removed | This study |
| pJB234 | pJB100 carrying chpA-FLAG allelic exchange on SpeI fragment; subcloned from pJB232 | This study |
| pJB235 | pJB100 carrying chpA(AAA)-FLAG allelic exchange on SpeI fragment; subcloned from pJB233 | This study |
| pJB241 | pJB100 carrying ΔpilA complementation construct on SpeI fragment | This study |
| pJB246 | pJB100 carrying ΔpilB complementation construct on SpeI fragment | This study |
| pJB247 | pOK12 carrying pilG-His allelic-exchange construct on SpeI fragment | This study |
| pJB248 | pOK12 carrying pilH-His allelic-exchange construct on SpeI fragment | This study |
| pJB249 | pilA reporter construct cloned as XhoI/BamHI fragment into mCTX-lacZ | This study |
| pJB250 | pJB100 carrying ΔpilR on SpeI fragment | This study |
| pJB251 | pJB100 carrying ΔpilR complementation construct on SpeI fragment | This study |
| pJB252 | pOK12 carrying pilG(D58A)-His allelic-exchange construct on SpeI fragment | This study |
| pJB254 | pOK12 carrying pilH(D582)-His allelic-exchange construct on SpeI fragment | This study |
| pJB256 | pJB100 carrying pilG(D58A)-His allelic-exchange construct on SpeI fragment | This study |
| pJB258 | pJB100 carrying pilH(D582)-His allelic-exchange construct on SpeI fragment | This study |
a
Cbr, carbenicillin resistance; Tcr, tetracycline resistance; Apr, ampicillin resistance; Gmr, gentamicin resistance; Kmr, kanamycin resistance.