Genome engineering in Bacillus anthracis using tyrosine site-specific recombinases

Abstract

Tyrosine site-specific recombinases (T-SSR) are polynucleotidyltransferases that catalyze cutting and joining reactions between short specific DNA sequences. We developed three systems for performing genetic modifications in Bacillus anthracis that use T-SSR and their cognate target sequences, namely Escherichia coli bacteriophage P1 Cre-loxP, Saccharomyces cerevisiae Flp-FRT, and a newly discovered IntXO-PSL system from B. anthracis plasmid pXO1. All three tyrosine recombinase systems were used for creation of a B. anthracis sporulation-deficient, plasmid-free strain deleted for ten proteases which had been identified by proteomic analysis as being present in the B. anthracis secretome. This strain was used successfully for production of various recombinant proteins, including several that are candidates for inclusion in improved anthrax vaccines. These genetic tools developed for DNA manipulation in B. anthracis were also used for construction of strains having chromosomal insertions of 1, 2, or 3 adjacent atxA genes. AtxA is a B. anthracis global transcriptional regulator required for the response of B. anthracis virulence factor genes to bicarbonate. We found a positive correlation between the atxA copy number and the expression level of the pagA gene encoding B. anthracis protective antigen, when strains were grown in a carbon dioxide atmosphere. These results demonstrate that the three T-SSR systems described here provide effective tools for B. anthracis genome editing. These T-SSR systems may also be applicable to other prokaryotes and to eukaryotes.

Fig 1. T-SSR system used for deletion/insertion of DNA fragments in B. anthracis genome.

(A) Scheme of the T-SSR application. White and black rectangles are fragments flanking Δ, the DNA fragment to be deleted, while the striped rectangle is the DNA fragment to be inserted in place of Δ. Black triangles represent target sequences loxP, FRT or PSL for Cre, Flp or IntXO T-SSR, respectively. The detailed description of the application of Cre-loxP, Flp-FRT and IntXO-PSL were described previously [–8] and in the Results section. (B) PCR verification of deletions in cysP1, nprC, vpR and s41 protease genes with corresponding primer pairs 1995seqF/R, 2183 seqF/R, 4584 seqF/R and 5414 seqF/R. GenBank accession numbers for proteases and strains used to verify the retention of specific segments are indicated at the top of the gel. Mr, GeneRuler DNA ladder mix for size determination (e.g., 3,000 bp).

Fig 2. SDS-PAGE analysis of the PA, LF, EF and HtrA proteins (Coomassie stained).

Proteins were purified from BH500 strains containing pYS5, pSJ115, pSJ136EFOS and pUTE29-htrA plasmids, respectively. The strains were grown in FA medium with 20 μg/ml of kanamycin (or 10 μg/ml tetracycline for pUTE29-htrA) at 37°C for 14 h. Protein purification was performed following procedures described in the Materials and Methods section. Mr—PageRuler unstained protein ladder mix for size determination (e.g., 85 kDa).

Fig 3. Creation of B. anthracis strains containing variable numbers of atxA genes replacing the nprC gene.

(A) Plasmids used for sequential atxA gene insertions. (B) Scheme indicating sequential atxA gene insertions: nprC gene was replaced by the first atxA gene using Flp-FRT system with pSCF-2183LA plasmid as a donor of atxA; the second atxA was added from pSC-A plasmid with Cre-loxP system; and the third atxA copy was inserted with IntXO-PSL system from pSCP-A plasmid. (C) PCR confirmation of atxA gene insertions into genome of B. anthracis (primers 2183seqF/2183seqR). Strains with one, two, and three copies of atxA are indicated on the top of the gel. (D) PCR confirmation of atxA intergenic regions in strains with double and triple atxA genes (primers seqALAF/ALARseqR).

Fig 4. Increased atxA copy number enhances pagA gene transcription and PA content in the B. anthracis secretome.

(A) Genetic structure of pPAGK plasmid. TT—T4 phage transcription terminator. (B) Active transcription of atxA and pagA in BH490 derivatives containing the pPAGK plasmid. Two-step qPCR results of atxA and pagA transcription respectively from cultures grown in NBY broth with 0.8% NaHCO₃ in 15% CO₂ (top panels) and in air (bottom panels). Relative expression represents the 2^-ΔΔCt (RQ) value normalized to Ames 35 and evaluated with three reference genes: rpoB, gyrB, and dnaJ. Values reported are the mean relative expression ± standard error of the mean calculated from ExpressionSuite and GraphPad Prism software. An asterisk indicates a significant difference (p<0.05) from an unpaired t-test compared to Ames 35. (C) Western blot analysis of PA production in strains grown in the same conditions as in panel B.