Combined sacB-Based Negative Selection and cre-lox Antibiotic Marker Recycling for Efficient Gene Deletion in Pseudomonas Aeruginosa

Figures & data

Table 1. Bacterial Strains and Plasmids Used in this Study

Strain or Plasmid	Genotype or Relevant Characteristics	Reference or Source
P. aeruginosa
CHA	Wild-type strain, mucoid CF isolate	(10)
CHAΔSGmlox	CHAΔexoS:: Gmlox Gm^R	This study
CHAΔSlox	CHAΔexoS::lox	This study
CHAΔTGmlox	CHAΔ exoT:: Gmlox Gm^R	This study
CHAΔTlox	CHAΔexoT::lox	This study
CHAΔSTGmlox	CHAΔexoS:: loxΔ exoT:: Gmlox Gm^R	This study
CHAΔSTlox	CHAΔexoS:: loxΔ exoT::lox	This study
E. coli
S17.1	RecApro (RP4-2Tet:: Mu Kan::Tn7)	(12)
Plasmids
PUCGm	Ap^R, Gm^R vector containing the aacC1 gene	(11)
pUC18Gm	Ap^R, Gm^R, pUC18-based vector containing the aacC1 gene	This study
pUCGmlox	Ap^R, Gm^R, pUC18-based vector containing the lox flanked aacC1 gene	This study
pEX100T	Ap^R, pUC19-based gene replacement vector	(5)
pEX100Tlink	Ap^R, pEX100TwithaMCS	This study
pCM351	Ap^R, Gm^R, vector containing the aacC1lox gene (Gmlox)	(6)
pCM157	Tc^R, cre expression vector	(6)
pLQ34	Ap^R, pEX100T containing 5′ and 3′flanking sequence of exoS	This study
pLQ35	Ap^R, pEX100T containing 5′ and 3′ flanking sequence of exoT	This study
pLQ36	Ap^R, Gm^R, pEX100T containing 5′and 3′flanking sequence of exoS:: Gmlox	This study
pLQ37	Ap^R, Gm^R, pEX100T containing 5′and 3′flanking sequence of exoT:: Gmlox	This study

Antibiotic resistances are indicated as follows: Ap^R, ampicillin; Gm^R, gentamicin; TcR, tetracycline. P. aeruginosa, Pseudomonas aeruginosa; E. coli, Escherichia coli.

Figure 1. Plasmid maps.

(A) Plasmid map of the allelic exchange vector pEX100Tlink. pEX100Tlink is derived from the pEX100T suicide vector (GenBank accession no. U17500) with the addition of a multicloning site (MCS). Antibiotic resistance is encoded by bla (ampicillin). The sacB gene confers sucrose sensitivity on the Pseudomonas aeruginosa strain when grown on LB medium supplemented with 5% sucrose. oriT is the origin of transfer for conjugation-mediating transfer, and ori is the pMB1-based origin of replication (5). (B) Plasmid map of the pUCGmlox vector. pUCGmlox is derived from pUCGm (GenBank accession no. U04610). Antibiotic resistances are encoded by bla (ampicillin) and by aacC1 (gentamicin). The aacC1 gene, flanked by the lox sequences, was amplified from pCM351 (GenBank accession no. AY093430). The entire aacC1lox sequence is flanked by a palindromic MCS.

Table 2. PCR Primers

Primer	Sequence	Restriction Site	Insertion of PCR Fragments
GmloxSacS	5′-TAGAGCTCTTCAGCTGTACAATTGG-3′	SacI	pUC
GmloxSacF	5′-ATGTGAGCTCACCGGTTAACACGCG-3′	SacI	-SacI aacC1lox SacI-
ES5′ BamS	5′-CGCGGATCCGCCAGGAGGGATTGCAAC-3′	BamHI
ES5′HindF ES3′HindS	5′-CGCAAGCTTGCCTTCTCCTGATGTTTC-3′ 5′-CG G A AGCTTCAG GCG AG GTCCAG G-3′	HindIII HindIII	inpEX100Tlink-BamHI exoS 3′ HindIII exoS 5′ EcoRI-
ES3′ EcoF	5′-GCCGAATTCCAATCGCCACCTGGGA-3′	EcoRI
ET5′ EcoS	5′-CGAATTCTTGACCAGGCTGTTGCTCAG-3′	EcoRI
ET5′HindF	5′-CAAGCTTGGAGAACCCGATGGGATATT-3′	HindIII	inpEX100Tlink-EcoRI exoT3′ HindIII exoT5′ BamHI-
ET3′HindS	5′-CAAGCTTGCACGATAACCACCCTGTTC-3′	HindIII
ET3′ BamF	5′-CGGATCCGATTATTGGTTCTGTGGCTT-3′	BamHI

Restriction sites are in bold.

Figure 2. Strategy and validation of the antibiotic recycling method inPseudomonas aeruginosa.

(A) Strategy for allelic exchange and antibiotic marker recycling. Allelic exchange leads to an aacC1lox deletion/insertion (1 kb) mutant that can then be unmarked through the introduction of the cre expression plasmid pCM157. The process can then be repeated with a second target gene to generate double mutations. Sizes correspond to PCR amplification between primers (black arrows). (B) PCR analysis of allelic exchange and subsequent marker removal for exoS and exoT loci. The forward primer for amplification of the upstream flank and the reverse primer for the downstream flank were used together to amplify across the entire locus. The molecular weight standard in the first lane is a 1-kb DNA Ladder (Promega France, Charbonnieres, France). (C) Secretion profiles of wild-type (WT) and mutant strains. Coomassie blue stained 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of supernatant fraction in type III secretion system (TTSS) induced condition (+) or non-induced conditions (−). Type III-related exoproducts and molecular weight standard (LMW-SDS Marker kit; Amersham Biosciences Europe GmbH, Orsay, France) are indicated on the left side of the gel.

Schweizer, H.P. and T.T.Hoang. 1995. An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene158:15–22.

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