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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.


Optimization of dsDNA recombination parameters. a The effect of the induction time on selection efficiency. The culture was incubated for 3–8 h before addition of the inducing peptides. 0.5 μg dsDNA substrates with 1.4-kb homologies were used for electroporation and the recovery time was 3 h. OD600 of the culture on induction is also shown. b The effect of the quantity of dsDNA substrates on selection efficiency. The culture was incubated for 4 h before addition of the inducing peptides. 0.5–5 μg dsDNA substrates with 1.4-kb homologies were used for electroporation and the recovery time was 3 h. c The effect of the recovery time on selection efficiency. The culture was incubated for 4 h before addition of the inducing peptides. 1 μg dsDNA substrates were used for electroporation and the recovery time was 1–5 h. d The effect of the homology length of dsDNA substrates on selection efficiency. PCR products with various homology lengths were generated by PCR and added at constant molarity. The culture was incubated for 4 h before addition of the inducing peptides. 1 μg dsDNA substrates was used for electroporation and the recovery time was 1 h. Results are the averages from at least three independent experiments, with standard deviations indicated by error bars
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Fig2: Optimization of dsDNA recombination parameters. a The effect of the induction time on selection efficiency. The culture was incubated for 3–8 h before addition of the inducing peptides. 0.5 μg dsDNA substrates with 1.4-kb homologies were used for electroporation and the recovery time was 3 h. OD600 of the culture on induction is also shown. b The effect of the quantity of dsDNA substrates on selection efficiency. The culture was incubated for 4 h before addition of the inducing peptides. 0.5–5 μg dsDNA substrates with 1.4-kb homologies were used for electroporation and the recovery time was 3 h. c The effect of the recovery time on selection efficiency. The culture was incubated for 4 h before addition of the inducing peptides. 1 μg dsDNA substrates were used for electroporation and the recovery time was 1–5 h. d The effect of the homology length of dsDNA substrates on selection efficiency. PCR products with various homology lengths were generated by PCR and added at constant molarity. The culture was incubated for 4 h before addition of the inducing peptides. 1 μg dsDNA substrates was used for electroporation and the recovery time was 1 h. Results are the averages from at least three independent experiments, with standard deviations indicated by error bars

Mentions: To enhance the mutants selection efficiency, we examined induction time of the recombinases from 3 to 8 h. 0.5 μg dsDNA substrates with 1.4-kb homologies were used for electroporation and the recovery time was 3 h. As a result, induction at 4 h, when the OD600 was 0.56, yielded the most recombinants (Fig. 2a). This OD600 is consistent with the optima for electrocompetent-cell preparation. Then, we determined the effect of substrate concentration on the selection efficiency by varying the amount of dsDNA (0.5–5 μg) added to the electroporation mix. Higher frequency was observed when the substrate concentration was increased from 0.5 to 1 μg, but further increase did not improve the efficiency (Fig. 2b), so 1 μg substrate concentration was used further. Essentially, allele replacement occurs during recovery cultivation. Therefore, extending the recovery cultivation time may increase the selection efficiency. However, fewer recombinants appeared as the incubation time was extended from 1 to 2 or 3 h (Fig. 2c). For 5 h, more CmR colonies appeared due to an increase in viable cells (the ratio between CmR colonies and viable cells did not increase, at about 35 ± 4 CmR colonies/108 viable cells), but to save time, 1 h was applicable. The 1 h optimum recovery was short compared with that following plasmid-electroporation, which is generally 2–3 h.Fig. 2


Prophage recombinases-mediated genome engineering in Lactobacillus plantarum.

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

Optimization of dsDNA recombination parameters. a The effect of the induction time on selection efficiency. The culture was incubated for 3–8 h before addition of the inducing peptides. 0.5 μg dsDNA substrates with 1.4-kb homologies were used for electroporation and the recovery time was 3 h. OD600 of the culture on induction is also shown. b The effect of the quantity of dsDNA substrates on selection efficiency. The culture was incubated for 4 h before addition of the inducing peptides. 0.5–5 μg dsDNA substrates with 1.4-kb homologies were used for electroporation and the recovery time was 3 h. c The effect of the recovery time on selection efficiency. The culture was incubated for 4 h before addition of the inducing peptides. 1 μg dsDNA substrates were used for electroporation and the recovery time was 1–5 h. d The effect of the homology length of dsDNA substrates on selection efficiency. PCR products with various homology lengths were generated by PCR and added at constant molarity. The culture was incubated for 4 h before addition of the inducing peptides. 1 μg dsDNA substrates was used for electroporation and the recovery time was 1 h. Results are the averages from at least three independent experiments, with standard deviations indicated by error bars
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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Fig2: Optimization of dsDNA recombination parameters. a The effect of the induction time on selection efficiency. The culture was incubated for 3–8 h before addition of the inducing peptides. 0.5 μg dsDNA substrates with 1.4-kb homologies were used for electroporation and the recovery time was 3 h. OD600 of the culture on induction is also shown. b The effect of the quantity of dsDNA substrates on selection efficiency. The culture was incubated for 4 h before addition of the inducing peptides. 0.5–5 μg dsDNA substrates with 1.4-kb homologies were used for electroporation and the recovery time was 3 h. c The effect of the recovery time on selection efficiency. The culture was incubated for 4 h before addition of the inducing peptides. 1 μg dsDNA substrates were used for electroporation and the recovery time was 1–5 h. d The effect of the homology length of dsDNA substrates on selection efficiency. PCR products with various homology lengths were generated by PCR and added at constant molarity. The culture was incubated for 4 h before addition of the inducing peptides. 1 μg dsDNA substrates was used for electroporation and the recovery time was 1 h. Results are the averages from at least three independent experiments, with standard deviations indicated by error bars
Mentions: To enhance the mutants selection efficiency, we examined induction time of the recombinases from 3 to 8 h. 0.5 μg dsDNA substrates with 1.4-kb homologies were used for electroporation and the recovery time was 3 h. As a result, induction at 4 h, when the OD600 was 0.56, yielded the most recombinants (Fig. 2a). This OD600 is consistent with the optima for electrocompetent-cell preparation. Then, we determined the effect of substrate concentration on the selection efficiency by varying the amount of dsDNA (0.5–5 μg) added to the electroporation mix. Higher frequency was observed when the substrate concentration was increased from 0.5 to 1 μg, but further increase did not improve the efficiency (Fig. 2b), so 1 μg substrate concentration was used further. Essentially, allele replacement occurs during recovery cultivation. Therefore, extending the recovery cultivation time may increase the selection efficiency. However, fewer recombinants appeared as the incubation time was extended from 1 to 2 or 3 h (Fig. 2c). For 5 h, more CmR colonies appeared due to an increase in viable cells (the ratio between CmR colonies and viable cells did not increase, at about 35 ± 4 CmR colonies/108 viable cells), but to save time, 1 h was applicable. The 1 h optimum recovery was short compared with that following plasmid-electroporation, which is generally 2–3 h.Fig. 2

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.