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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

Insertion of gusA into the genomic ldhD locus. A Schematic diagram illustrating gusA insertion. The substrate contained ~1-kb flanks located next to each other on the genome. Allelic replacement resulted in disruption of the ldhD gene and simultaneous insertion of the cat marker and gusA gene. In theory, any nonessential locus can be targeted. Primers ldhD-testA (c) and gus-testB (d) used for PCR testing are shown. B Inspection of potential mutants by PCR testing using primers c and d, as shown in A. Lane 1 shows DNA ladder and lane 2 the wild-type strain expected to generate an amplicon of ∼2 kb. Lanes 3–22 were tested colonies, with correct mutants expected to generate amplicons of ∼4.7 kb
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Fig3: Insertion of gusA into the genomic ldhD locus. A Schematic diagram illustrating gusA insertion. The substrate contained ~1-kb flanks located next to each other on the genome. Allelic replacement resulted in disruption of the ldhD gene and simultaneous insertion of the cat marker and gusA gene. In theory, any nonessential locus can be targeted. Primers ldhD-testA (c) and gus-testB (d) used for PCR testing are shown. B Inspection of potential mutants by PCR testing using primers c and d, as shown in A. Lane 1 shows DNA ladder and lane 2 the wild-type strain expected to generate an amplicon of ∼2 kb. Lanes 3–22 were tested colonies, with correct mutants expected to generate amplicons of ∼4.7 kb

Mentions: Lp_0640-41-42-mediated gene insertion was also tested. Different from the gene-deletion cases described above, the homology arms are adjacent on the genome for gene-insertion. The gusA gene from E. coli, coding for β-d-glucuronidase, was chosen because there is a lack of such enzymatic activity in L. plantarum. Recombinants with GusA activity should be readily screenable on plates with X-Gluc-containing medium (positive colonies resulting in a white to blue change) [34]. Similarly, a mutagenesis vector for gusA insertion (Additional file 1: Figure S1) was constructed followed by PCR and Lp_0640-41-42-mediated recombination. Allelic replacement would result in disruption of the ldhD gene and simultaneous insertion of the cat marker and gusA gene (Fig. 3A). As a result, we obtained 35 CmR colonies in total in three independent experiments using 1 μg phosphorothioated dsDNA substrate (1-kb flanks). 20 of them were selected and tested by PCR, of which 15 were correct (Fig. 3B).Fig. 3


Prophage recombinases-mediated genome engineering in Lactobacillus plantarum.

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

Insertion of gusA into the genomic ldhD locus. A Schematic diagram illustrating gusA insertion. The substrate contained ~1-kb flanks located next to each other on the genome. Allelic replacement resulted in disruption of the ldhD gene and simultaneous insertion of the cat marker and gusA gene. In theory, any nonessential locus can be targeted. Primers ldhD-testA (c) and gus-testB (d) used for PCR testing are shown. B Inspection of potential mutants by PCR testing using primers c and d, as shown in A. Lane 1 shows DNA ladder and lane 2 the wild-type strain expected to generate an amplicon of ∼2 kb. Lanes 3–22 were tested colonies, with correct mutants expected to generate amplicons of ∼4.7 kb
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Insertion of gusA into the genomic ldhD locus. A Schematic diagram illustrating gusA insertion. The substrate contained ~1-kb flanks located next to each other on the genome. Allelic replacement resulted in disruption of the ldhD gene and simultaneous insertion of the cat marker and gusA gene. In theory, any nonessential locus can be targeted. Primers ldhD-testA (c) and gus-testB (d) used for PCR testing are shown. B Inspection of potential mutants by PCR testing using primers c and d, as shown in A. Lane 1 shows DNA ladder and lane 2 the wild-type strain expected to generate an amplicon of ∼2 kb. Lanes 3–22 were tested colonies, with correct mutants expected to generate amplicons of ∼4.7 kb
Mentions: Lp_0640-41-42-mediated gene insertion was also tested. Different from the gene-deletion cases described above, the homology arms are adjacent on the genome for gene-insertion. The gusA gene from E. coli, coding for β-d-glucuronidase, was chosen because there is a lack of such enzymatic activity in L. plantarum. Recombinants with GusA activity should be readily screenable on plates with X-Gluc-containing medium (positive colonies resulting in a white to blue change) [34]. Similarly, a mutagenesis vector for gusA insertion (Additional file 1: Figure S1) was constructed followed by PCR and Lp_0640-41-42-mediated recombination. Allelic replacement would result in disruption of the ldhD gene and simultaneous insertion of the cat marker and gusA gene (Fig. 3A). As a result, we obtained 35 CmR colonies in total in three independent experiments using 1 μg phosphorothioated dsDNA substrate (1-kb flanks). 20 of them were selected and tested by PCR, of which 15 were correct (Fig. 3B).Fig. 3

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