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


CONCLUSIONS
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.
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China.
Pengxia
P
Wang
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China.
Yiguang
Y
Zhu
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China.
Yuyang
Y
Zhang
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China.
Chunyi
C
Zhang
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China.
Jianyi
J
Xu
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China.
Yun
Y
Deng
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China.
Donghai
D
Peng
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China.
Lifang
L
Ruan
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, People's Republic of China. m98sun@mail.hzau.edu.cn.
Ming
M
Sun
D000262:Adenylyl Cyclases / Q000235:genetics; D000681:Amylases / Q000235:genetics; D001409:Bacillus cereus / Q000235:genetics* / Q000378:metabolism; D001413:Bacillus thuringiensis / Q000235:genetics* / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism; D006460:Hemolysin Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis; D011995:Recombination, Genetic
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D010455:Peptides; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C088290:zwittermicin A; D004706:Endodeoxyribonucleases; C120220:MobM protein, bacteria; D000681:Amylases; D000262:Adenylyl Cyclases
Bacillus cereus; Bacillus thuringiensis; Conjugation; Mob protein; Relaxase; Site-specific recombination