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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.


Characterization of the lp_0640-41-42 operon. A Layout and genetic context of the lp_0640-41-42 operon. The numbers in parentheses indicate the genome locus. B Schematic diagram showing in-frame gnp deletion and marker elimination. Lp_0640-41-42 mediated allelic replacement resulted in disruption of the gnp gene and insertion of the cat marker, while Cre subsequently eliminated the marker. C dsDNA recombination assay with different protein combinations. 1 μg dsDNA substrate was electroporated into L. plantarum JDM1 expressing various combinations of proteins, and the respective resultant chloramphenicol resistant colony number were shown. pSIP411 was used as the control. Results are the averages from at least three independent experiments, with standard deviations indicated by error bars. D Inspection of potential Δgnp::cat mutants by PCR testing using primers gnp-testA (a) and gnp-testB (b), as shown in B. Lanes 1 and 24 show DNA ladders, lanes 2 and 23 the wild-type strains expected to generate amplicons of ∼3.9 kb. Lanes 3–22 were tested colonies, with correct mutants expected to generate amplicons of ∼4.3 kb
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Fig1: Characterization of the lp_0640-41-42 operon. A Layout and genetic context of the lp_0640-41-42 operon. The numbers in parentheses indicate the genome locus. B Schematic diagram showing in-frame gnp deletion and marker elimination. Lp_0640-41-42 mediated allelic replacement resulted in disruption of the gnp gene and insertion of the cat marker, while Cre subsequently eliminated the marker. C dsDNA recombination assay with different protein combinations. 1 μg dsDNA substrate was electroporated into L. plantarum JDM1 expressing various combinations of proteins, and the respective resultant chloramphenicol resistant colony number were shown. pSIP411 was used as the control. Results are the averages from at least three independent experiments, with standard deviations indicated by error bars. D Inspection of potential Δgnp::cat mutants by PCR testing using primers gnp-testA (a) and gnp-testB (b), as shown in B. Lanes 1 and 24 show DNA ladders, lanes 2 and 23 the wild-type strains expected to generate amplicons of ∼3.9 kb. Lanes 3–22 were tested colonies, with correct mutants expected to generate amplicons of ∼4.3 kb

Mentions: In a previous study, a RecT/Beta analogue was identified in L. plantarum WCFS1, which has 46 % identity with the known L. reuteri RecT [29]. The coding gene of this protein, lp_0641, is an ORF in prophage P1 (a 44-kb temperate pac-site phage) [30]. Most recombinase proteins are encoded by bacterial phages, prophages or their remnants, as is the case for Red and RecET in E. coli, and SSAP–exonuclease pairs always lie next to each other. After further inspection of prophage P1, we found lp_0641, lp_0640 and lp_0642 could transcript as an operon (Fig. 1A), as indicated in the MetaCyc Metabolic Pathway Database (http://metacyc.org/). lp_0642 overlapped by 77 nucleotides (nt) with lp_0641 and encodes a 286-residue protein. BLAST search of this protein showed specific hits to the DUF3799 conserved domain (proteins of this family are likely to be nucleases) over an 80 % query coverage [31]. lp_0640 encodes a 128-residue protein similar to Gam (138 residues) in size.Fig. 1


Prophage recombinases-mediated genome engineering in Lactobacillus plantarum.

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

Characterization of the lp_0640-41-42 operon. A Layout and genetic context of the lp_0640-41-42 operon. The numbers in parentheses indicate the genome locus. B Schematic diagram showing in-frame gnp deletion and marker elimination. Lp_0640-41-42 mediated allelic replacement resulted in disruption of the gnp gene and insertion of the cat marker, while Cre subsequently eliminated the marker. C dsDNA recombination assay with different protein combinations. 1 μg dsDNA substrate was electroporated into L. plantarum JDM1 expressing various combinations of proteins, and the respective resultant chloramphenicol resistant colony number were shown. pSIP411 was used as the control. Results are the averages from at least three independent experiments, with standard deviations indicated by error bars. D Inspection of potential Δgnp::cat mutants by PCR testing using primers gnp-testA (a) and gnp-testB (b), as shown in B. Lanes 1 and 24 show DNA ladders, lanes 2 and 23 the wild-type strains expected to generate amplicons of ∼3.9 kb. Lanes 3–22 were tested colonies, with correct mutants expected to generate amplicons of ∼4.3 kb
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig1: Characterization of the lp_0640-41-42 operon. A Layout and genetic context of the lp_0640-41-42 operon. The numbers in parentheses indicate the genome locus. B Schematic diagram showing in-frame gnp deletion and marker elimination. Lp_0640-41-42 mediated allelic replacement resulted in disruption of the gnp gene and insertion of the cat marker, while Cre subsequently eliminated the marker. C dsDNA recombination assay with different protein combinations. 1 μg dsDNA substrate was electroporated into L. plantarum JDM1 expressing various combinations of proteins, and the respective resultant chloramphenicol resistant colony number were shown. pSIP411 was used as the control. Results are the averages from at least three independent experiments, with standard deviations indicated by error bars. D Inspection of potential Δgnp::cat mutants by PCR testing using primers gnp-testA (a) and gnp-testB (b), as shown in B. Lanes 1 and 24 show DNA ladders, lanes 2 and 23 the wild-type strains expected to generate amplicons of ∼3.9 kb. Lanes 3–22 were tested colonies, with correct mutants expected to generate amplicons of ∼4.3 kb
Mentions: In a previous study, a RecT/Beta analogue was identified in L. plantarum WCFS1, which has 46 % identity with the known L. reuteri RecT [29]. The coding gene of this protein, lp_0641, is an ORF in prophage P1 (a 44-kb temperate pac-site phage) [30]. Most recombinase proteins are encoded by bacterial phages, prophages or their remnants, as is the case for Red and RecET in E. coli, and SSAP–exonuclease pairs always lie next to each other. After further inspection of prophage P1, we found lp_0641, lp_0640 and lp_0642 could transcript as an operon (Fig. 1A), as indicated in the MetaCyc Metabolic Pathway Database (http://metacyc.org/). lp_0642 overlapped by 77 nucleotides (nt) with lp_0641 and encodes a 286-residue protein. BLAST search of this protein showed specific hits to the DUF3799 conserved domain (proteins of this family are likely to be nucleases) over an 80 % query coverage [31]. lp_0640 encodes a 128-residue protein similar to Gam (138 residues) in size.Fig. 1

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.