TABLE 1.

Plasmids and phages

Plasmid or phage	Descriptiona	Relevant genotype	Selective markerb	Replicon	Source or reference
pACYC-RIL		argU ileY leuW	Cm	p15A	Stratagene
pCR6	pCR21Δ[NdeI-SacI]Ω[pTiA6 NdeI-SacI 152,657-155,086]c	virB4+	Ap	pMB1	This study
pCR21	pMS119HEΔ[HindIII-EcoR1]Ω[pKW21Δ1 HindIII-EcoRI 2,670-bp fragment, RP4 20,545-23,127], Ptac/lacIq	trbE+	Ap	pMB1	This study
pCR21HN	pCR21Δ[XbaI-NdeI]Ω[XbaI-NdeI adaptor]d	his6-trbE+	Ap	pMB1	This study
pCR21Δ1e	pCR21Δ[RP4 20,548-20,607]	trbEΔ1+	Ap	pMB1	This study
pCR21Δ2e	pCR21Δ[RP4 20,548-20,625]	trbEΔ2+	Ap	pMB1	This study
pCR21HNΔ2e	pCR21HNΔ[RP4 20,548-20,625]	his6-trbEΔ2+	Ap	pMB1	This study
pCR21Δ3e	pCR21Δ[RP4 20,620-20,661]	trbEΔ3+	Ap	pMB1	This study
pCR21Δ4e	pCR21Δ[RP4 20,548-20,706]	trbEΔ4+	Ap	pMB1	This study
pCR21HNΔ5e	pCR21HNΔ[RP4 20,548-21,039]	his6-trbEΔ5+	Ap	pMB1	This study
pCR21HNΔ6e	pCR21HNΔ[RP4 20,548-21,519]	his6-trbEΔ6+	Ap	pMB1	This study
pCR21Δ7e	pCR21Δ[RP4 23,086-23,127]	trbEΔ7+	Ap	pMB1	This study
pCR21Δ8e	pCR21Δ[RP4 23,068-23,127]	trbEΔ8+	Ap	pMB1	This study
pCR21Δ9e	pCR21Δ[RP4 23,053-23,127]	trbEΔ9+	Ap	pMB1	This study
pCR21Δ10e	pCR21Δ[RP4 23,014-23,127]	trbEΔ10+	Ap	pMB1	This study
pCR21Δ11e	pCR21Δ[RP4 22,945-23,127]	trbEΔ11+	Ap	pMB1	This study
pCR38	pCR21Δ[NdeI-EcoRI]Ω[R388 1,025-3,496]f	trwK+	Ap	pMB1	This study
pCR2000	pMS470Δ8Δ[EcoRI-HindIII]Ω[adaptor EcoRI-RsrII-SacI-AgeI-KpnI-NdeI-HindIII], cloning vector, Ptac/lacIqg		Ap	pMB1	This study
pCR2001	pCR2000Δ[RsrII-NdeI]Ω[pML123 RsrII-NdeI 4,370-bp RP4 24,301-19,932]	(trbD to trbF)+, antitranscriptive orientation	Ap	pMB1	This study
pCR2001ΔtrbE	pCR2001Δ[RP4 23,088-20,557]	trbE0	Ap	pMB1	This study
pCRA6	pMS119HEΔ[XmaI-SacI]Ω[pTiA6 XmaI-SacI 152,568-155,086]h	Original RBS, virB4+	Ap	pMB1	This study
pCR21X000Ze	Amino acid substitutions	trbE+, mutation indicated	Ap	pMB1	This study
pDB126	pML123Ω[BamHI; RP4 BfaI 45,893-53,462 bp]	(trbB to trbM)+ (traF to traM)+oriT+	Cm	ColD	2
pDB126ΔtrbE	pDB126Δ[RP4 20,557-23,088 bp]	(trbB to trbD, trbF to trbM)+ (traF to traM)+oriT+trbE0	Cm	ColD	This study
pGZ119EH, HE	Cloning vector, Ptac/lacIq		Cm	ColD	37
pKW21Δ1	pMS67HEΩ[mKW21trbF:BglII, 2.6 kb]	trbE+	Ap	RSF1010	27
pML123	pGZ119EHΔ[EcoRI-BamHI]Ω[EcoRI-XmnI adaptor, RP4 XmnI-NotI, 18,841-30,042 bp]	(trbB to trbM)+	Cm	ColD	39
pML123ΔtrbE	pMS123Δ[RP4 20,557-23,088 bp]	(trbB to trbD, trbF to trbM)+trbE0	Cm	ColD	This study
pMS119EH, HE	Cloning vector, Ptac/lacIq		Ap	pMB1	56
pMS123	pML123Δ[BamHI-HindIII]Ω[BamHI-XbaI adaptor, pWP471 XbaI-HindIII; RP4 18,841-30,042 and 45,909-46,577 bp]i	(trbB to trbM)+ (traF)+	Cm	ColD	This study
pMS123ΔtrbE	pMS123Δ[RP4 20,557-23,088 bp]	(trbB to trbD, trbF to trbM)+ (traF)+trbE0	Cm	ColD	This study
pMS470Δ8	pMS119EHΔ[XbaI-PstI]Ω[pT7-7 XbaI-NdeI, 40-bp fragment, R751 traC AvaI-SphI, 1.4 kb], cloning vector, Ptac/lacIq	traC+	Ap	pMB1	3
pSU4054	Plasmid encoding R388 TraW	trwK+ (among others)	Ap	pMB8	7
M13 mp18	Filamentous ssDNA phage; IncF specific				62
Pf3	Filamentous ssDNA phage; IncP specific				55
PRD1	Lipid-containing dsDNA phage; IncP, IncW, IncN specific				4
PRR1	RNA phage; IncP specific				42

^aThe accession numbers of the RP4, R388, and pTiA6 sequences are L27758, X81123, and AF242881, respectively.

^bAp, ampicillin; Cm, chloramphenicol.

^cThe following primers were used (nucleotides derived from the original sequence are in italics, and restriction sites are underlined): ATATATATCATATGCTCGGCGCGAGTGGAACG and TATATAGAGCTCGAAAGCTGCAGGTCAAAACCGTTGC.

^dThe adaptor was as follows: CTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACACATGCACCATCACCATCACCA TTTATTAAAACAAATTGAAATTCTTCCTCTATATGTGTACGTGGTAGTGGTAGTGGTAT

^eFor the mutagenesis strategy used, see Materials and Methods.

^fThe following primers were used: ATATATATCATATGGGGGCAATTGAATCCCGCAAGCTCC and ATATATATGAATTCTCATACGTCGCTCCTTTCGGCTTTCACACGG.

^gThe adaptor was as follows: AATTCCGGTCCGGAGCTCACCGGTGGTACCCATATGA GGCCAGGCCTCGAGTGGCCACCATGGGTATACTTCGA

^hThe following primers were used: TATATACCCGGGGGAAGAAGCATTGATAGTGCAGTTTGGGG and TATATAGAGCTCGAAAGCTGCAGGTCAAAACCGTTGC.

ⁱThe adaptor was as follows: GATCCGCGGT GCGCCAGATC