Marker removal in staphylococci via Cre recombinase and different lox sites

Abstract

Allelic replacement in staphylococci is frequently aided by antibiotic resistance markers that replace the gene(s) of interest. In multiply modified strains, the number of mutated genes usually correlates with the number of selection markers in the strain's chromosome. Site-specific recombination systems are capable of eliminating such markers, if they are flanked by recombinase recognition sites. In this study, a Cre-lox setting was established that allowed the efficient removal of resistance genes from the genomes of Staphylococcus carnosus and S. aureus. Two cassettes conferring resistance to erythromycin or kanamycin were flanked with wild-type or mutant lox sites, respectively, and used to delete single genes and an entire operon. After transformation of the cells with a newly constructed cre expression plasmid (pRAB1), genomic eviction of the resistance genes was observed in approximately one out of ten candidates analyzed and subsequently verified by PCR. Due to its thermosensitive origin of replication, the plasmid was then easily eliminated at nonpermissive temperatures. We anticipate that the system presented here will prove useful for generating markerless deletion mutants in staphylococci.

FIG. 1.

Comparison between different lox sites. The upper strands of loxP, lox66, lox71, and lox72 are shown in a 5′-to-3′ direction. loxP constitutes the wild-type sequence with a 13-bp palindrome flanking the 8-bp asymmetric core region (boxed), which confers directionality of the sites. The 5-bp exchanges compared to loxP at the 5′ end of lox66, at the 3′ end of lox71, or on both sides (lox72) are underlined.

FIG. 2.

Schematic representation of the 8.35-kbp cre expression vector pRAB1. The singular restriction sites for SacI and PstI were used for cloning of a P_pagA-cre fragment (P_pagA is symbolized by a bent arrow). The features for plasmid propagation in E. coli are the β-lactamase encoding the bla gene for ampicillin selection and the pMB1 derived oriE region. The Staphylococcus specific regions are repF(Ts), which denotes a thermosensitive ori for plasmid maintenance, and the cat gene encoding Cm acetyltransferase for Cm resistance.

FIG. 3.

(A) Genetic situation of the wild-type S. carnosus TM300 srtA region (upper panel), after srtA disruption by loxP-ermB-loxP (middle panel), and after Cre-mediated eviction of ermB (lower panel). Depicted are three genes (white arrows) upstream and one gene downstream of srtA (grey arrow). loxP sites are indicated as black triangles, and the ermB cassette is symbolized by a black arrow. Dotted lines between the upper and the middle panel indicate the boundaries of homologous regions as used for the allelic replacement vector. Dotted lines between the middle and the lower panel indicate the region that was removed by Cre. Except for the primers (depicted as small black arrows), the representations are drawn to scale, with a 500-bp ruler given at the bottom. Primers: I, delso-1neu; II, Erm1; III, Erm2; IV, delso-B. (B) Gel electrophoresis result of PCR products obtained with primers I and IV using chromosomal DNA of different S. carnosus strains, as indicated. Lanes: 1, molecular weight marker (relevant sizes are given in bps); 2, wild type; 3, srtA::loxP-ermB-loxP; 4, srtA::loxP, clone 1; 5, srtA::loxP, clone 2. Analogous PCR analyses with the primers II and III or the primers I and III yielded specific products only when srtA::loxP-ermB-loxP DNA was used as a template (not shown).

FIG. 4.

(A) Genetic situation of the wild-type S. aureus SA113 arcA region (upper panel), after arcA deletion by lox66-aphAIII-lox71 (middle panel), and after Cre-mediated eviction of aphAIII (lower panel). Depicted are two genes (white arrows) upstream and two genes downstream of arcA (grey arrow). lox sites are indicated as black triangles, and the aphAIII cassette is symbolized by a black arrow. Dotted lines between the upper and the middle panel indicate the boundaries of homologous regions as used for the allelic replacement vector. Dotted lines between the middle and the lower panel indicate the region that was removed upon Cre treatment. Except for the primers (depicted as small black arrows), the representations are drawn to scale, with a 500-bp ruler given at the bottom. Primers: I, Kontr_2969_fw; II, Km1; III, Km2; IV, Kontr_2969_rw. (B) Gel electrophoresis result of PCR products obtained with primers I and IV using chromosomal DNA of different S. aureus strains, as indicated. Lanes: 1, molecular weight marker (relevant sizes are given in base pairs); 2, wild type; 3, arcA::lox66-aphAIII-lox71; 4, arcA::lox72, clone 1; 5, arcA::lox72, clone 2. Analogous PCR analyses with the primers II and III or the primers I and III yielded specific products only when arcA::lox66-aphAIII-lox71 DNA was used as a template (not shown).

FIG. 5.

(A) Genetic situation of the wild-type S. aureus SA113 arc operon (upper panel), after operon deletion by lox66-aphAIII-lox71 (middle panel), and after Cre-mediated eviction of aphAIII (lower panel). Depicted are two genes (white arrows) upstream and one gene downstream of the arcABDCR genes (grey arrows). lox sites are indicated as black triangles, and the aphAIII cassette is symbolized by a black arrow. Dotted lines between the upper and the middle panel indicate the boundaries of homologous regions as used for the allelic replacement vector. Dotted lines between the middle and the lower panel indicate the region that was removed upon Cre treatment. Except for the primers (depicted as small black arrows), the representations are drawn to scale, with a 500-bp ruler given at the bottom. Primers: I, Kontr_2969_fw; II, Komp_02969_fw; III, Km1.2; IV, Km2.2 (for one not depicted control PCR Km2 instead of Km2.2 was used, whose binding sites within aphAIII differ by 24 bp); V, Komp_arc_rw. (B) Gel electrophoresis result of PCR products obtained with primers II and V using chromosomal DNA of different S. aureus strains, as indicated. Lanes: 1, molecular weight marker (relevant sizes are given in base pairs); 2, wild type; 3, arcABDCR::lox66-aphAIII-lox71; 4, arcABDCR::lox72. Analogous PCR analyses with the primers III and IV or the primers I and IV yielded specific products only when arcA::lox66-aphAIII-lox71 DNA was used as a template (not shown).