. 2009 Jun 5;75(15):4936–4949. doi: 10.1128/AEM.02564-08

TABLE 2.

Plasmids used in this work

Plasmid	Vector	Description	Source and/or reference(s)
pCOS5	pCOS5	Cloning vector containing cat, oriT, ColE1, and oriV; Ap^r Cm^r	10
pCR2.1-TOPO	pCR2.1	TOPO cloning vector for capturing PCR products using TA cloning	Invitrogen
pCVD442	pCVD442	R6K ori mob sacB bla; suicide vector for allelic exchange; Ap^r	13
pNEB206A	pNEB206A	USER friendly cloning vector; Ap^r	New England Biolabs; 3
pRK437	pRK437	Cloning vector containing tetAR and lacZα; deletion derivative of pRK404	38
pUC19	pUC19	Cloning vector; lacZα Ap^r	51
pUC4K	pUC4K	Cloning vector containing aph and multiple-cloning site of M13mp7	43, 44
pGTR265	pCVD442	ΔrtxC; rtxC upstream and downstream PCR amplicons digested with NotI and then USER cloned into pGTR1113; Ap^r	This work
pGTR272	pCVD442	ΔrtxA1; rtxA1 upstream and downstream PCR amplicons USER cloned into pGTR1129; Ap^r	This work
pGTR274	pCVD442	ΔrtxA1::aph; aph-NotI cassette from pGTR1114 made blunt ended with Klenow fragment and inserted into the SmaI site between the rtxA1 upstream and downstream fragments of pGTR272; Ap^r Km^r	This work
pGTR1090	pCR2.1-TOPO	flaCDE upstream sequences PCR amplified with primers flaCDE-up5′ and flaCDE-up3′ TOPO TA cloned into pCR2.1-TOPO; Ap^r	This work
pGTR1091	pCR2.1-TOPO	flaCDE downstream sequences PCR amplified with primers flaCDE-down5′ and flaCDE-down3′ TOPO TA cloned into pCR2.1-TOPO; Ap^r	This work
pGTR1092	pCR2.1-TOPO	flaFBA upstream sequences PCR amplified with primers flaFBA-up5′ and flaFBA-up3′ TOPO TA cloned into pCR2.1-TOPO; Ap^r	This work
pGTR1093	pCR2.1-TOPO	flaFBA downstream sequences PCR amplified with primers flaFBA-down5′ and flaFBA-down3′ TOPO TA cloned into pCR2.1-TOPO; Ap^r	This work
pGTR1106	pCR2.1-TOPO	ΔflaFBA; pGTR1093 digested with XhoI and NotI and ligated with flaFBA upstream sequences from pGTR1092 removed with SalI and NotI; Ap^r	This work
pGTR1107	pCR2.1-TOPO	ΔflaCDE; pGTR1091 digested with XhoI and NotI and ligated with flaCDE upstream sequences from pGTR1090 removed with SalI and NotI; Ap^r	This work
pGTR1108	pCVD442	ΔflaFBA sequences from pGTR1106 removed with SstI and XbaI inserted into pCVD442 digested with XbaI and SstI; Ap^r	This work
pGTR1111	pCVD442	pCVD442 XbaI-SmaIΩlacZα; lacZα from HaeII fragment from pUC19; Ap^r	This work
pGTR1112	pCVD442	ΔflaCDE from pGTR1107 removed with SstI and XbaI inserted into pGTR1111 digested with XbaI and SstI; Ap^r	This work
pGTR1113	pCVD442	pGTR1111lacZα::USER; pGTR1111 digested with SphI and SstI and ligated with USER friendly cloning fragment created with oligonucleotides pCVD442-USER Linker top and pCVD442-USER Linker bottom; Ap^r	This work
pGTR1114	pCVD442	aph gene PCR amplified from pUC4K with primers aph5′USER and aph3′USER and USER cloned into pGTR1113; Ap^r Km^r	This work
pGTR1116	pCR2.1	cat gene PCR amplified from pCOS5 with primers cat-5′ and cat-3′ and TOPO-TA cloned into pCR2.1-TOPO; Ap^r Km^r Cm^r	This work
pGTR1117	pCR2.1	tetAR genes PCR amplified from pRK437 with primers tetAR5′ and tetAR3′ and TOPO-TA cloned into pCR2.1-TOPO; Ap^r Km^r Tc^r	This work
pGTR1120	pCVD442	ΔflaFBA::cat; cat gene from pGTR1116 inserted into the NotI site between the flaFBA upstream and downstream sequences of pGTR1108; Ap^r Cm^r	This work
pGTR1128	pCVD442	pGTR1111 EcoRVΩtetAR; tetAR genes from pGTR1117 removed with NotI, made blunt ended with Klenow fragment, and cloned into pGTR1111 at EcoRV; Ap^r Tc^r	This work
pGTR1129	pCVD442	pGTR1113 EcoRVΩcat; cat gene from pGTR1116 removed with NotI, made blunt ended with Klenow fragment, and cloned into pGTR1113 at EcoRV; Ap^r Cm^r	This work
pGTR1130	pCVD442	pGTR1113 EcoRVΩtetAR; tetAR genes from pGTR1117 removed with NotI, made blunt ended with Klenow fragment, and cloned into pGTR1113 at EcoRV; Ap^r Tc^r	This work
pGTR1132	pCVD442	pGTR1111 EcoRVΩcat; cat gene from pGTR1116 removed with NotI, made blunt ended with Klenow fragment, and cloned into pGTR1111 at EcoRV; Ap^r Cm^r	This work
pGTR1133	pCVD442	pGTR1111 NdeIΩaph; aph gene removed from pUC4K with BamHI, made blunt ended with Klenow fragment, and inserted into pGTR1111 digested with NdeI and made blunt ended with Klenow fragment	This work
pGTR1136	pCVD442	ΔflaCDE::aph; aph-NotI cassette from pGTR1114 inserted at the NotI site between the flaCDE upstream and downstream sequences of pGTR1112; Ap^r Km^r	This work
pGTR1137	pCVD442	ΔmotAB crossover PCR product USER cloned into pGTR1113; Ap^r	This work
pGTR1141	pCVD442	ΔmotAB::aph; aph gene removed from pGTR1114 with NotI and inserted at the NotI site of pGTR1137; Ap^r Km^r	This work
pGTR1144	pCVD442	pGTR1113 EcoRVΩaph; aph gene from pGTR1114 removed with NotI, made blunt ended with Klenow fragment, and cloned into pGTR1113 at EcoRV; Ap^r Km^r	This work
pGTR1147	pNEB206A	ΔcheY-3 crossover PCR product USER cloned into pNEB206A; Ap^r	This work
pGTR1157	pNEB206A	ΔcheY-3::aph; aph gene from pGTR1114 inserted into the NotI site between the cheY-3 upstream and downstream sequences of pGTR1147	This work
pGTR1160	pRK437	pRK437-USER; pRK437 digested with BamHI and HindIII and ligated with a USER friendly cloning fragment created with oligonucleotides pRK437-USER Linker top and pRK437-USER Linker bottom; Tc^r	This work
pGTR1171	pCVD442	ΔcheY-3::aph; ΔcheY-3::aph sequences removed from pGTR1157 with EcoRI, made blunt ended with Klenow fragment, and cloned into pGTR1111 digested with SmaI; Ap^r Km^r	This work
pGTR1200	pCOS5	pCOS5 digested with HindIII and ClaI, made blunt ended with Klenow fragment, and religated; Cm^r	This work
pGTR1202	pCOS5	pCOS5::lacZα; lacZα HaeII fragment from pUC19 made blunt ended with Klenow fragment and inserted in a reverse direction relative to those of bla and cat of pGTR1200, digested with BamHI and XbaI, and made blunt ended with Klenow fragment; Cm^r	This work
pGTR1204	pCOS5	pCOS5::lacZα::USER; 47-bp USER friendly sequences from pGTR1113 removed by digestion with HindIII and SstI and ligated into pGTR1202 digested with HindIII and SstI; Cm^r	This work