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. 2006 Jan;74(1):682-93.
doi: 10.1128/IAI.74.1.682-693.2006.

Genome Engineering in Bacillus Anthracis Using Cre Recombinase

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Free PMC article

Genome Engineering in Bacillus Anthracis Using Cre Recombinase

Andrei P Pomerantsev et al. Infect Immun. .
Free PMC article

Abstract

Genome engineering is a powerful method for the study of bacterial virulence. With the availability of the complete genomic sequence of Bacillus anthracis, it is now possible to inactivate or delete selected genes of interest. However, many current methods for disrupting or deleting more than one gene require use of multiple antibiotic resistance determinants. In this report we used an approach that temporarily inserts an antibiotic resistance marker into a selected region of the genome and subsequently removes it, leaving the target region (a single gene or a larger genomic segment) permanently mutated. For this purpose, a spectinomycin resistance cassette flanked by bacteriophage P1 loxP sites oriented as direct repeats was inserted within a selected gene. After identification of strains having the spectinomycin cassette inserted by a double-crossover event, a thermo-sensitive plasmid expressing Cre recombinase was introduced at the permissive temperature. Cre recombinase action at the loxP sites excised the spectinomycin marker, leaving a single loxP site within the targeted gene or genomic segment. The Cre-expressing plasmid was then removed by growth at the restrictive temperature. The procedure could then be repeated to mutate additional genes. In this way, we sequentially mutated two pairs of genes: pepM and spo0A, and mcrB and mrr. Furthermore, loxP sites introduced at distant genes could be recombined by Cre recombinase to cause deletion of large intervening regions. In this way, we deleted the capBCAD region of the pXO2 plasmid and the entire 30 kb of chromosomal DNA between the mcrB and mrr genes, and in the latter case we found that the 32 intervening open reading frames were not essential to growth.

Figures

FIG. 1.
FIG. 1.
Cre-loxP system for gene knockout in B. anthracis. (I) The gene targeted for knockout is cloned into a ts plasmid and interrupted by insertion of the loxP-sp-loxP (Spr) cassette. The plasmid is transformed into B. anthracis, which is then grown at the restrictive temperature. (II) The allelic exchange event is selected by the Spr phenotype, accompanied by loss of the pDC plasmid (and erythromycin resistance). (III) Removal of the Spr cassette from the chromosome is achieved by Cre-mediated recombination (excision) after transforming the strain with pCrePA at 30°C. Cre recombinase expression plasmid pCrePA contains the cre gene of bacteriophage P1 under control of the B. anthracis protective antigen (pagA) gene promoter from pAE5 (27). The signal peptide of protective antigen was eliminated in order to retain Cre inside the cell. pCrePA also contains the Emr gene as a selectable marker and the strongly ts replicon from pHY304 (30). (IV) Growth at 37°C eliminates the Cre recombinase-producing ts vector pCrePA. (V) The result is replacement of a portion of the targeted gene by a single 34-bp loxP site.
FIG. 2.
FIG. 2.
Knockout of pepM and spo0A genes. For each gene, the diagrams show the native gene (a), the gene with an inserted Ω-sp cassette (b), and the gene after deletion of the Ω-sp cassette (c). Locations of the primers used for PCR analysis are shown by arrows. Gray areas indicate the Ω-sp cassette flanked by two loxP sites (b) and residual loxP sequences resulting from the Ω-sp cassette deletion (c).
FIG. 3.
FIG. 3.
DNA analysis of modified B. anthracis strains. (a) pepM gene amplified with M4S/M4E primers from Ames 33 (lane 1), strain MΩL33, containing the Ω-sp cassette (lane 2), and strain MSLL33, containing the deleted gene (lane 3). (b) spoOA gene amplified with Spo1/Spo2 primers from Ames 33 (lane 1), strain SΩL33, containing the Ω-sp cassette (lane 2), and strain MSLL33, containing the deleted gene (lane 3). (c) pagA gene amplified with PAF/PAR primers from Ames 35 (lane 1) and MSLL35 (lane 2) and pXO2 fragments amplified with pXO2-AT-f1/pXO2-AT-r1 primers from Ames 34 (lane 3) and MSLL34 (lane 4). (d) Virulence plasmid content of Ames 35 (lane 1), MSLL35 (lane 2), Ames 34 (lane 3), and MSLL34 (lane 4). The arrows indicate pXO1, pXO2, and chromosomal DNA bands, and the Mr lane is a GeneRuler DNA ladder for comparison.
FIG. 4.
FIG. 4.
Schematic diagram of Cre-mediated excision of loxP-Ω-sp-loxP from the mutated B. anthracis spo0A gene. Cre recognizes and binds to the 13-bp recombinase binding elements (RBEs) within the loxP site, which are arranged as inverted repeats surrounding a central 8-bp spacer (shown in bold lowercase). The central 8 bp are asymmetric with respect to sequence and define the directionality of the site. Vertical arrows indicate the cleavage sites for Cre-mediated recombination. The mutated base (A) in the right RBE of loxPR is shown in larger type and in boldface. The corresponding, nonmutated base (G) in the right RBE of loxPL is shown in larger type. The mutated, right RBE from loxPR replaced the normal right RBE of loxPR in the single loxP site remaining in the spo0A gene as a result of Cre-mediated recombination, accompanied by the excision of Ω-sp, which contains the single loxP and cannot be replicated.
FIG. 5.
FIG. 5.
Phenotypic consequences of pepM and spo0A disruption in B. anthracis. (a) Proteolysis of casein induced by B. anthracis MSLL33 (right side) is weak compared to the parent Ames 33 strain (left side). For the test, 5 μl of MSLL33 or Ames 33 overnight culture was spotted on casein agar and grown for 12 h. (b) Congo red agar distinguishes the parental B. anthracis strain (left side; Ames 33 strain) from the B. anthracis spo0A mutant (right side; strain MSLL33). Ames 33 or MSLL33 was streaked on the Congo red agar and grown for 24 h. Light micrographs demonstrate either Ames 33 spores (left side) or remains of nonsporulating MSLL33 vegetative cells (right side). Both strains were grown at 30°C for 5 days on NBY-Mn agar.
FIG. 6.
FIG. 6.
Comparison of PA and capsule production in parent and mutated B. anthracis strains. (a) Immunoprecipitation of PA produced by colonies of B. anthracis Ames 35 (pXO1+; left side) and the isogenic double mutant MSLL35 (pXO1+; center), with Ames 34 (pXO1; right side) as a negative control. The cultures were grown for 18 h on CA agar supplemented with 0.8% (wt/vol) sodium bicarbonate, 5% (vol/vol) horse serum, and 5% (vol/vol) PA antiserum (from sheep) in a 20% CO2 environment at 37°C. (b) B. anthracis double mutant MSLL34 (pXO2+) produces less capsule (right side) than the parent Ames 34 strain (pXO2+; left side). The Ames 35 strain (pXO2) was used as a negative control (center). The cultures were grown for 18 h in bicarbonate agar supplemented with 0.8% (wt/vol) bicarbonate and 10% (vol/vol) horse serum in 20% CO2 at 37°C. Cells were removed from the colonies shown, and capsule was visualized with India ink (bottom panel). Bar, 5 μm.
FIG. 7.
FIG. 7.
Multistep deletion of the capBCAD region from the B. anthracis plasmid pXO2. The mutant strain CΩ1 was obtained as a result of insertion of the loxP-Ω-sp-loxP cassette into the BglII site located between the P1/P2 promoter region and the capB start codon. Strain CΩ1 lost the ability to synthesize capsule in contrast to the parent Ames 34 strain (micrographs on the right show India ink staining for capsule, as in Fig. 6). Cre-mediated excision of the Ω-sp cassette from CΩ1 pXO2 resulted in strain CL1, containing a single loxP site (right-facing arrow) between the promoters and the capB start codon. Capsule formation was restored in this strain. Insertion of the Ω-sp cassette into the capD gene drastically modified the capsule morphology of the resulting CL1DΩ2 strain. Cre treatment of CL1DΩ2 resulted in strain CL1DL2, which contains the pXO2 plasmid with a deleted capBCAD region. As a result of this deletion, the ability of strain CL1DL2 to synthesize capsule was completely lost.
FIG. 8.
FIG. 8.
Verification of 30-kb deletion in B. anthracis strain McrB3P-Mrr-LΔ30. (a) Overlapping PCR products obtained from DNA from the parent strain UM44-1C9 using primer pairs IG1 to IG8 (lanes 1 to 8, respectively). The mcrB-mrr intergenic segment is shown as a shaded bar, and the mcrB and mrr genes are shown as antiparallel shaded arrows. The thin lines represent the overlapping PCR products obtained that collectively span the entire region. The loxP sites inserted into the mcrB and mrr sequences to produce unmarked mutations are shown as open arrows within the genes. Primers Mrr5′ GenP and McrB3P For1 (small antiparallel arrows above the mrr and mcrB genes, respectively) produce no product from UM44-1C9 (lane 9). (b) PCR with primers Mrr 5′ GenP and McrB3P For1 results in a PCR product only when McrB3P-Mrr-LΔ30 genomic DNA is used as a template (lane 2), but not when UM44-1C9 genomic DNA or no DNA is used as template (lanes 1 and 3, respectively). The corresponding arrangement is shown in the schematic below the photograph. Mr is the 1-kb Plus DNA ladder. The schematic diagrams are not drawn to scale.

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