TABLE 1.

Plasmids used in this study

Plasmid	Descriptiona	Relevant genotype	Selective markerb	Replicon	Reference or source
pDB126	pML123Ω[BamHI; RP4 BfaI 45,893–53,462 bp]	(trbB-trbM)+ (traF-traM)+oriT+	Cm	ColD	2
pDB126ΔtrbC	pDB126Δ[RP4 19,803–20,226 bp]	(trbB trbD-trbM)+ (traF-traM)+oriT+trbC0	Cm	ColD	This work
pDB129	pDB126Δ[RP4 45,893–46,583 bp]	(trbB-trbM)+ (traG-traM)+oriT+ traF0	Cm	ColD	19
pDJ100c	pRE100Δ[BsrGI-AatII]Ω[RP4 BsrGI-AatII, 18,190–19,441 bp]		Ap	pMB1	This work
pDJ100trbCX00Zd	pRE100Δ[BsrGI-AatII]Ω[RP4 BsrGI-AatII, 18,190–19,441 bp]	Mutation indicated	Ap	pMB1	This work
pGZ119EH, HE	Cloning vector; Ptac/lacIq		Cm	ColD	27
pJH472	pGZ119EHΔ[KpnI-EcoRI]Ω[RP4 46,060–46,591 bp]	traF+	Cm	ColD	13
pJH472X00Zd	pGZ119EHΔ[KpnI-EcoRI]Ω[RP4 46,060–46,591 bp]	traF+, mutation indicated	Cm	ColD	This work
pJJ178	pMS119EHΔ[NdeI-HindIII]Ω[R751 18,511–19,288 bp]	trbC+ R751	Ap	pMB1	This work
pML123	pGZ119EHΔ[EcoRI-BamHI]Ω[EcoRI-XmnI adapter, RP4 XmnI-NotI, 18,841–30,042 bp]	(trbB-trbM)+	Cm	ColD	28
pML123mtrbC45	pML123Ω[dCTAGTCTAGACTAG at position RP4 19,938]	(trbB trbD-trbM)+trbC0	Cm	ColD	20
pML123ΔtrbC	pML123Δ[RP4 19,803–20,226 bp]	(trbB, trbD-trbM)+trbC0	Cm	ColD	This work
pML123trbCX00Zd	pGZ119EHΔ[EcoRI-BamHI]Ω[EcoRI-XmnI adapter, RP4 XmnI-NotI, 18,841–30,042 bp]	(trbB trbC+, mutation indicated, trbD-trbM)+	Cm	ColD	This work
pMS119EH	Cloning vector; Ptac/lacIq		Ap	pMB1	49
pMS470Δ8	pMS119EHΔ[XbaI-PstI]Ω[pT7-7 XbaI-NdeI 40-bp fragment, R751 traC AvaI-SphI 1.4 kb] cloning vector; Ptac/lacIq		Ap	pMB1	3
pPL178	pMS119EHΔ[NdeI-EcoRI]Ω[tra 1,755–2,156 bp]	trbC+ pTiC58	Ap	pMB1	This work
pPLtraF	pKK38ASHΔ[NcoI-PstI]Ω[tra 2,255–2,523 bp]	traF+ pTiC58	Tc		This work
pRE100	pMS119HEΔ[HindIII-EcoRI]Ω[HindIII-BsrGI-XbaI-AatII-EcoRI adapter] cloning vector; Ptac/lacIq		Ap	pMB1	This work
pRE178	pMS119EHΔ[EcoRI-HindIII]Ω[RP4 19,797–20,244 bp]	trbC+	Ap	pMB1	13
pRE178X00Zd	pMS119EHΔ[EcoRI-HindIII]Ω[RP4 19,797–20,244 bp]	trbC+, mutation indicated	Ap	pMB1	This work
pRE178Δ3	pMS119EHΔ[EcoRI-HindIII]Ω[RP4 19,797–20,163 bp]	trbCΔ3+	Ap	pMB1	This work
pRE178Δ3.05	pMS119EHΔ[EcoRI-HindIII]Ω[RP4 19,797–20,160 bp]	trbCΔ3.05+	Ap	pMB1	This work
pRE178Δ3.1	pMS119EHΔ[EcoRI-HindIII]Ω[RP4 19,797–20,157 bp]	trbCΔ3.1+	Ap	pMB1	This work
pRE178Δ4	pMS119EHΔ[EcoRI-HindIII]Ω[RP4 19,797–20,151 bp]	trbCΔ4+	Ap	pMB1	This work
pWP471	pJF119EHΩ[RP4 NspI-HaeII 45,909–46,577 bp]	traF+	Ap	pMB1	54
pWP471X00Zd	pJF119EHΩ[RP4 NspI-HaeII 45,909–46,577 bp]	traF+, mutation indicated	Ap	pMB1	This work

^aRP4 (M93696), R751 (U67194), and pTiC58 (AF057718, trbC; U40389 traF; X53264, virB2) sequence coordinates of inserted fragments are given according to the published sequence data; GenBank accession numbers are in parentheses. 

^bAp, ampicillin resistance; Cm, chloramphenicol resistance; Tc, tetracycline resistance. 

^cpDJ100 contains the indicated Tra2 fragment in an antitranscriptive orientation. The fragment starts inside the trbB gene and ends within the first third of trbE. 

^dFor mutagenesis strategy, see details in Materials and Methods.