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Prophage recombinases-mediated genome engineering in Lactobacillus plantarum.

Yang P, Wang J, Qi Q - Microb. Cell Fact. (2015)

Bottom Line: Based on this, we developed a method for marker-free genetic manipulation of the chromosome in L. plantarum.This Lp_0640-41-42-mediated recombination allowed easy screening of mutants and could serve as an alternative to other genetic manipulation methods.We expect that this method can help for understanding the probiotic functionality and physiology of LAB.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, People's Republic of China. fwjt63298@126.com.

ABSTRACT

Background: Lactobacillus plantarum is a food-grade microorganism with industrial and medical relevance belonging to the group of lactic acid bacteria (LAB). Traditional strategies for obtaining gene deletion variants in this organism are mainly vector-based double-crossover methods, which are inefficient and laborious. A feasible possibility to solve this problem is the recombineering, which greatly expands the possibilities for engineering DNA molecules in vivo in various organisms.

Results: In this work, a double-stranded DNA (dsDNA) recombineering system was established in L. plantarum. An exonuclease encoded by lp_0642 and a potential host-nuclease inhibitor encoded by lp_0640 involved in dsDNA recombination were identified from a prophage P1 locus in L. plantarum WCFS1. These two proteins, combined with the previously characterized single strand annealing protein encoded by lp_0641, can perform homologous recombination between a heterologous dsDNA substrate and host genomic DNA. Based on this, we developed a method for marker-free genetic manipulation of the chromosome in L. plantarum.

Conclusions: This Lp_0640-41-42-mediated recombination allowed easy screening of mutants and could serve as an alternative to other genetic manipulation methods. We expect that this method can help for understanding the probiotic functionality and physiology of LAB.

No MeSH data available.


Related in: MedlinePlus

Cre-mediated excision of the selectable marker. After Cre induction and incubation, potential Δgnp::lox72 mutants were inspected by PCR testing using primers gnp-testA and gnp-testB. Lane 1 shows DNA ladder, lane 2 was the Δgnp::cat strain. Lanes 3–11 were tested colonies, with marker-free mutants expected to generate amplicons of ∼3.2 kb
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Fig4: Cre-mediated excision of the selectable marker. After Cre induction and incubation, potential Δgnp::lox72 mutants were inspected by PCR testing using primers gnp-testA and gnp-testB. Lane 1 shows DNA ladder, lane 2 was the Δgnp::cat strain. Lanes 3–11 were tested colonies, with marker-free mutants expected to generate amplicons of ∼3.2 kb

Mentions: To eliminate the cat selection marker after recombination, the loxP/Cre system was employed. The phage protein Cre catalyzes site-specific recombination between two of its recognition sites, loxP. DNA sequence flanked by loxP sites would be excised when the loxP sites are convergently oriented and inverted when the loxP sites are divergently oriented [35]. To minimize genetic instability, convergently oriented lox66/lox71 sites were used in our experiments. The recombinase-expressing plasmid pLP-gba was eliminated by culturing a Δgnp::cat mutant in the absence of erythromycin selection for 24 h. Then the Cre helper plasmid pLP-cre was introduced. After Cre induction and incubation, single colonies were checked by PCR using primers gnp-testA and gnp-testB. Recombination between lox66 and lox71 would lead to excision of the cat marker gene and form a lox72 site that is poorly recognized by Cre (Fig. 1B). On analysis, all of the nine tested colonies showed the mutant genotype and could not grow on chloramphenicol-containing media (Fig. 4). Finally, the Cre helper plasmid was cured by culturing a marker-free mutant for 24 h in the absence of erythromycin selection.Fig. 4


Prophage recombinases-mediated genome engineering in Lactobacillus plantarum.

Yang P, Wang J, Qi Q - Microb. Cell Fact. (2015)

Cre-mediated excision of the selectable marker. After Cre induction and incubation, potential Δgnp::lox72 mutants were inspected by PCR testing using primers gnp-testA and gnp-testB. Lane 1 shows DNA ladder, lane 2 was the Δgnp::cat strain. Lanes 3–11 were tested colonies, with marker-free mutants expected to generate amplicons of ∼3.2 kb
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4595204&req=5

Fig4: Cre-mediated excision of the selectable marker. After Cre induction and incubation, potential Δgnp::lox72 mutants were inspected by PCR testing using primers gnp-testA and gnp-testB. Lane 1 shows DNA ladder, lane 2 was the Δgnp::cat strain. Lanes 3–11 were tested colonies, with marker-free mutants expected to generate amplicons of ∼3.2 kb
Mentions: To eliminate the cat selection marker after recombination, the loxP/Cre system was employed. The phage protein Cre catalyzes site-specific recombination between two of its recognition sites, loxP. DNA sequence flanked by loxP sites would be excised when the loxP sites are convergently oriented and inverted when the loxP sites are divergently oriented [35]. To minimize genetic instability, convergently oriented lox66/lox71 sites were used in our experiments. The recombinase-expressing plasmid pLP-gba was eliminated by culturing a Δgnp::cat mutant in the absence of erythromycin selection for 24 h. Then the Cre helper plasmid pLP-cre was introduced. After Cre induction and incubation, single colonies were checked by PCR using primers gnp-testA and gnp-testB. Recombination between lox66 and lox71 would lead to excision of the cat marker gene and form a lox72 site that is poorly recognized by Cre (Fig. 1B). On analysis, all of the nine tested colonies showed the mutant genotype and could not grow on chloramphenicol-containing media (Fig. 4). Finally, the Cre helper plasmid was cured by culturing a marker-free mutant for 24 h in the absence of erythromycin selection.Fig. 4

Bottom Line: Based on this, we developed a method for marker-free genetic manipulation of the chromosome in L. plantarum.This Lp_0640-41-42-mediated recombination allowed easy screening of mutants and could serve as an alternative to other genetic manipulation methods.We expect that this method can help for understanding the probiotic functionality and physiology of LAB.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, People's Republic of China. fwjt63298@126.com.

ABSTRACT

Background: Lactobacillus plantarum is a food-grade microorganism with industrial and medical relevance belonging to the group of lactic acid bacteria (LAB). Traditional strategies for obtaining gene deletion variants in this organism are mainly vector-based double-crossover methods, which are inefficient and laborious. A feasible possibility to solve this problem is the recombineering, which greatly expands the possibilities for engineering DNA molecules in vivo in various organisms.

Results: In this work, a double-stranded DNA (dsDNA) recombineering system was established in L. plantarum. An exonuclease encoded by lp_0642 and a potential host-nuclease inhibitor encoded by lp_0640 involved in dsDNA recombination were identified from a prophage P1 locus in L. plantarum WCFS1. These two proteins, combined with the previously characterized single strand annealing protein encoded by lp_0641, can perform homologous recombination between a heterologous dsDNA substrate and host genomic DNA. Based on this, we developed a method for marker-free genetic manipulation of the chromosome in L. plantarum.

Conclusions: This Lp_0640-41-42-mediated recombination allowed easy screening of mutants and could serve as an alternative to other genetic manipulation methods. We expect that this method can help for understanding the probiotic functionality and physiology of LAB.

No MeSH data available.


Related in: MedlinePlus