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: 3.73). 02/2006; 74(1):682-93. DOI: 10.1128/IAI.74.1.682-693.2006
Source: PubMed


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
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    • "Thus, the system can be used repeatedly to delete multiple genes in a given strain (Banerjee & Biswas, 2008). Although the Cre/loxP system has not yet been adapted for A. baumannii, a thermosensitive derivative of the pWV01 plasmid carrying Cre recombinase (pCrePA; Pomerantsev et al., 2006) is available and can be used directly in A. baumannii without the need for further modification. "
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    ABSTRACT: Acinetobacter baumannii is an emerging nosocomial pathogen involved in a variety of infections ranging from minor soft-tissue infections to more severe infections such as ventilator-associated pneumonia and bacteremia. A. baumannii has become resistant to most of the commonly used antibiotics and multidrug resistant isolates are becoming a severe problem in the healthcare setting. In the past few years, whole genome sequences of over 200 A. baumannii isolates have been generated. Several methods and molecular tools have been used for genetic manipulation of various Acinetobacter spp. Here we review recent developments of various genetic tools used for modification of A. baumannii genome including various ways to inactivate gene function, chromosomal integration and transposon mutagenesis.
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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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    ABSTRACT: The lack of knowledge about pathogenicity mechanisms of Streptococcus (S.) suis is, at least partially, attributed to limited methods for its genetic manipulation. In the presented study we established a Cre-lox based recombination system for markerless gene deletion which allows genetic manipulation of S. suis with high selective pressure and without undesired side effects.
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    • "The PlcR isogenic mutant [29] was grown in the presence of erythromycin (10 µg/ml). The Hbl isogenic mutant strain was generated by a Cre-loxP based mutagenesis approach [30], [31]. Briefly, two fragments representing the upstream and downstream region of the Hbl-1 locus were amplified using primers Hbl-LF1 and Hbl-LF2, and Hbl-RF1 in combination with Hbl-RF2, respectively (Table S1), and separately cloned into plasmid pSC containing two loxP recognition sites, resulting in pSC_HblF1 and pSC_HblF2, respectively. "
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    ABSTRACT: Bacillus cereus is a spore-forming, Gram-positive bacterium commonly associated with outbreaks of food poisoning. It is also known as an opportunistic pathogen causing clinical infections such as bacteremia, meningitis, pneumonia, and gas gangrene-like cutaneous infections, mostly in immunocompromised patients. B. cereus secretes a plethora of toxins of which four are associated with the symptoms of food poisoning. Two of these, the non-hemolytic enterotoxin Nhe and the hemolysin BL (Hbl) toxin, are predicted to be structurally similar and are unique in that they require the combined action of three toxin proteins to induce cell lysis. Despite their dominant role in disease, the molecular mechanism of their toxic function is still poorly understood. We report here that B. cereus strain ATCC 10876 harbors not only genes encoding Nhe, but also two copies of the hbl genes. We identified Hbl as the major secreted toxin responsible for inducing rapid cell lysis both in cultured cells and in an intraperitoneal mouse toxicity model. Antibody neutralization and deletion of Hbl-encoding genes resulted in significant reductions of cytotoxic activity. Microscopy studies with Chinese Hamster Ovary cells furthermore showed that pore formation by both Hbl and Nhe occurs through a stepwise, sequential binding of toxin components to the cell surface and to each other. This begins with binding of Hbl-B or NheC to the eukaryotic membrane, and is followed by the recruitment of Hbl-L1 or NheB, respectively, followed by the corresponding third protein. Lastly, toxin component complementation studies indicate that although Hbl and Nhe can be expressed simultaneously and are predicted to be structurally similar, they are incompatible and cannot complement each other.
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