Mob/oriT, a mobilizable site-specific recombination system for unmarked genetic manipulation in Bacillus thuringiensis and Bacillus cereus

Abstract

Background: Bacillus thuringiensis and Bacillus cereus are two important species in B. cereus group. The intensive study of these strains at the molecular level and construction of genetically modified bacteria requires the development of efficient genetic tools. To insert genes into or delete genes from bacterial chromosomes, marker-less manipulation methods were employed.

Results: We present a novel genetic manipulation method for B. thuringiensis and B. cereus strains that does not leave selection markers. Our approach takes advantage of the relaxase Mob02281 encoded by plasmid pBMB0228 from Bacillus thuringiensis. In addition to its mobilization function, this Mob protein can mediate recombination between oriT sites. The Mob02281 mobilization module was associated with a spectinomycin-resistance gene to form a Mob-Spc cassette, which was flanked by the core 24-bp oriT sequences from pBMB0228. A strain in which the wild-type chromosome was replaced with the modified copy containing the Mob-Spc cassette at the target locus was obtained via homologous recombination. Thus, the spectinomycin-resistance gene can be used to screen for Mob-Spc cassette integration mutants. Recombination between the two oriT sequences mediated by Mob02281, encoded by the Mob-Spc cassette, resulted in the excision of the Mob-Spc cassette, producing the desired chromosomal alteration without introducing unwanted selection markers. We used this system to generate an in-frame deletion of a target gene in B. thuringiensis as well as a gene located in an operon of B. cereus. Moreover, we demonstrated that this system can be used to introduce a single gene or an expression cassette of interest in B. thuringiensis.

Conclusion: The Mob/oriT recombination system provides an efficient method for unmarked genetic manipulation and for constructing genetically modified bacteria of B. thuringiensis and B. cereus. Our method extends the available genetic tools for B. thuringiensis and B. cereus strains.

Keywords: Bacillus cereus; Bacillus thuringiensis; Conjugation; Mob protein; Relaxase; Site-specific recombination.

Fig. 1

Parasporal crystals and protein expression of cry6Aa in transconjugants. a Phase-contrast microscopy image of parasporal crystals and spores produced in transconjugants. The red arrow indicates the rice-shaped crystals encoded by cry6Aa. The yellow arrow indicates the diamond-shaped crystals encoded by the endogenous crystal genes for YBT1520Str [5] and CT43::Kan [40]. b Protein analysis of the transconjugants by SDS-PAGE. The red arrow indicates the 54-kDa protein produced by cry6Aa. 1 BMB171Str/cry6Aa-pHTMob02281. 2 BMB171Str/pHTMob02281. 3 YBT1520Str/cry6Aa-pHTMob02281. 4 YBT-1520Str/pHTMob02281. 5 CT-43::Kan/cry6Aa-pHTMob02281. 6 CT-43::Kan/pHTMob02281. 7 UW85R/cry6Aa-pHTMob02281. 8 UW85R/pHTMob02281. 9 ATCC 10987Str/cry6Aa-pHTMob02281. 10 ATCC 10987Str/pHTMob02281. M Protein molecular weight marker

Fig. 2

Effects of oriT deletions on Mob02281-mediated recombination. The 1–352 bp oriT1 and 1–370 bp oriT2 segments are numbered. The structures were shown in [37]. Arrows indicate the presence of inverted repeats (IRs). The nic site is indicated as a vertical arrowhead. The arrangements of the two oriT sequences with or without deletion mutations in each substrate plasmid are listed. Deleted oriT was cloned at the corresponding site into pBMBT10. pBMBmob1 was used as a helper plasmid containing the gene encoding a mobilization protein. The frequency of oriT-specific recombination for each substrate plasmid was estimated following the methods described in [37]

Fig. 3

Recombination frequency of pBMBTmini. a Structure of the recombination cassette and the resulting product after recombination. Arrows indicate the direction of transcription; spc spectinomycin-resistance gene, Pgfp the promoter of the kanamycin-resistance gene, gfp gene encoding green fluorescent protein. b Observation of BMB171 (pBMBTmini + pBMBmob1) before and after recombination. PC phase-contrast microscopy; GFP fluorescence microscopy. Bar indicates 10 μm. c Restriction enzyme digestion of substrate plasmid before and after recombination; M1 λDNA/HindIII marker; M2 Trans2 K Plus DNA marker.1, before recombination, substrate plasmid pBMBTmini; 2–3, from two single Spc^S colonies after recombination; 4, before recombination, substrate plasmid pBMBTmini, digested with BamHI; 5–6, from two single Spc^S colonies after recombination, digested with BamHI; v vector, 6.5 kb; nr non-recombined cassette, 2.6 kb. r recombined cassette, 1.3 kb. d Increase of the percentage of antibiotic-resistance excision colonies over the number of generations

Fig. 4

Map of the temperature-sensitive-mobilizable recombination vector pRec-mob1-Ts. Arrows indicate the direction of transcription or replication. The Mob-Spc cassette is shown in red, including the spectinomycin-resistance gene (spc), the mob02281 gene, and the two mini-oriT sites (24-bp). The orientations of the replication of E. coli (ori.Ec) and the ampicillin-resistance gene (amp) are shown in light green; the orientations of the replication of B. thuringiensis and the erythromycin-resistance gene (erm) are shown in purple. The purple box flanking ori.Bt (Ts) indicates that the replication orientation of the temperature-sensitive replication region of B. thuringiensis is not known

Fig. 5

Design and confirmation of amyE disruption in B. thuringiensis BMB171. a Depiction of amyE disruption. b PCR detection with primers amyE-wS and amyE-wA. DNA templates were from: 1 pBMB0260 (negative control); 2 BMB171; 3 amyE gene-disruption strain BMB0260; M Trans2 K Plus II DNA marker; c Sequence analysis of the deletion region of BMB0260

Fig. 6

Construction of crystal protein gene insertion mutants BMB0261 and BMB0262. Depiction of cry5Ba (a) and cry2Aa (b) insertion. PCR detection with primers amyE-wS and amyE-wA (c) and 5B-S and 5B-A (lanes 1–4) or 2A-S and 2A-A (lanes 5–8) (d); DNA templates were from: 1, pBMB0261 (negative control); 2 and 6, BMB171; 3–4, mutant BMB0261; 5, pBMB0262 (negative control); 7–8, mutant BMB0262; M Trans2 K Plus II DNA marker. e Scanning electron microscopy image of parasporal crystals in unmarked cry integrate mutants. 1, BMB171/cry5Ba-pHT304; 2–3, BMB0261; 4, YBT-1518; 5, BMB171/cry2Aa-pHT304; 6–7, BMB0262; 8, CT-43; 9, BMB171. The arrows indicate the diamond-shaped crystals encoded by cry5Ba and the round-shaped crystals encoded by cry2Aa. Bar indicates 1 μm

Fig. 7

Design and confirmation of zmaJ disruption in B. cereus UW85R. a Depiction of zmaJ disruption. b PCR detection with primers zmaJ-wS and zmaJ-wA. DNA templates were from: 1, UW85R; 2, zmaJ gene-disrupted strain BMB0263; 3, pBMB0263 (negative control); M, DNA Marker III. c LC–MS detection of a molecular weight corresponding to ZmA in the culture supernatants. The molecular weight corresponding to ZmA is indicated by a rectangular frame