Genome engineering in Bacillus anthracis using Cre recombinase

Bacterial Toxins and Therapeutics Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-4349, USA.
Infection and Immunity (Impact Factor: 4.16). 02/2006; 74(1):682-93. DOI: 10.1128/IAI.74.1.682-693.2006
Source: PubMed

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.

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Available from: Andrei P Pomerantsev, Aug 24, 2015
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    • "The Cre–lox system combines the advantages of both techniques and has been applied in several microorganisms (Campo et al., 2002; Pomerantsev et al., 2006; Banerjee and Biswas, 2008). This strategy uses the Cre recombinase of the bacteriophage P1 that recognizes specific DNA sequences, the so called loxP sites, and subsequently removes chromosomal DNA fragments such as antibiotic resistance genes that are flanked (floxed) by two loxP sites (Sternberg and Hamilton, 1981; Sternberg et al., 1981). "
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    • "An improvement relevant to this discussion is to render the expression host unable to sporulate. Strains that are sporulation-deficient can be isolated as spontaneous mutants using the simple morphological screens mentioned above and then validated by sequencing [9] [8], or can be engineered by intentional deletion of specific sequences [30]. Thus, strains derived by these methods and specifically deleted in the spo0A gene are being used to produce candidate anthrax vaccine proteins [8,31e33], although whether mutations in spo0A or other sporulation-related genes enhance toxin production has not yet been determined. "
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    • "To determine the role of lipoproteins in B. anthracis, we constructed an lgt-deficient mutant. The lgt gene (BA5391 in the Ames strain) was disrupted without polar effects using the CreloxP system (Pomerantsev et al., 2006) as shown in Fig. 1A. We confirmed by PCR and sequencing that the lgt gene was deleted (Fig. 1B). "
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