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High efficiency recombineering in lactic acid bacteria.

van Pijkeren JP, Britton RA - Nucleic Acids Res. (2012)

Bottom Line: To highlight the utility of ssDNA recombineering we reduced the intrinsic vancomymycin resistance of L. reuteri >100-fold.By creating a single amino acid change in the D-Ala-D-Ala ligase enzyme we reduced the minimum inhibitory concentration for vancomycin from >256 to 1.5 µg/ml, well below the clinically relevant minimum inhibitory concentration.Recombineering thus allows high efficiency mutagenesis in lactobacilli and lactococci, and may be used to further enhance beneficial properties and safety of strains used in medicine and industry.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.

ABSTRACT
The ability to efficiently generate targeted point mutations in the chromosome without the need for antibiotics, or other means of selection, is a powerful strategy for genome engineering. Although oligonucleotide-mediated recombineering (ssDNA recombineering) has been utilized in Escherichia coli for over a decade, the successful adaptation of ssDNA recombineering to gram-positive bacteria has not been reported. Here we describe the development and application of ssDNA recombineering in lactic acid bacteria. Mutations were incorporated in the chromosome of Lactobacillus reuteri and Lactococcus lactis without selection at frequencies ranging between 0.4% and 19%. Whole genome sequence analysis showed that ssDNA recombineering is specific and not hypermutagenic. To highlight the utility of ssDNA recombineering we reduced the intrinsic vancomymycin resistance of L. reuteri >100-fold. By creating a single amino acid change in the D-Ala-D-Ala ligase enzyme we reduced the minimum inhibitory concentration for vancomycin from >256 to 1.5 µg/ml, well below the clinically relevant minimum inhibitory concentration. Recombineering thus allows high efficiency mutagenesis in lactobacilli and lactococci, and may be used to further enhance beneficial properties and safety of strains used in medicine and industry. We expect that this work will serve as a blueprint for the adaptation of ssDNA recombineering to other gram-positive bacteria.

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Overview of recombineering in L. reuteri. (i) Electrocompetent cells in which RecT is expressed are transformed with a recombineering oligonucleotide. Transformation efficiencies of ∼ ≥105cfu/µg DNA are required for high recombineering efficiencies. Wavy black lines with a red dot represent recombineering oligonucleotides with multiple non-complementary bases; the green circle represents a bacterial cell; pJP042 is the sakacin-based expression vector that contains recT; the black double helix represents chromosomal DNA; expressed RecT proteins are denoted in the cell. (ii) An oligonucleotide identical to the lagging strand contains multiple non-complementary bases that avoid the mismatch repair system resulting in increased recombineering efficiency. (iii) Viable cells are recovered on antibiotic-free plates and recombinants are detected by a mismatch amplification mutation assay-PCR (MAMA-PCR). Two oligonucleotides (blue) will yield a 1-kb fragment, whereas a third oligonucleotide (red) will only be extended by the polymerase when the mutations are incorporated in the chromosome yielding a second amplicon of 500 bp. As the recombineering oligonucleotide only targets one strand during DNA replication the colonies will be of mixed genotype. The red dot on the chromosome indicates that the mutations are incorporated. (iv) Single colony purification is performed to separate the wild-type genotype from the mutant genotype. (v) MAMA-PCR is repeated as described in section iii to identify a pure genotype mutant. A 1:1 ratio of wild-type and mutant genotypes is suggestive that during replication a single chromosome is being replicated per cell. (vi) The recombineering plasmid pJP042 can now be cured from the mutant strain by passaging bacteria without antibiotic selection to yield a plasmid-free derivative (vii).
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gks147-F1: Overview of recombineering in L. reuteri. (i) Electrocompetent cells in which RecT is expressed are transformed with a recombineering oligonucleotide. Transformation efficiencies of ∼ ≥105cfu/µg DNA are required for high recombineering efficiencies. Wavy black lines with a red dot represent recombineering oligonucleotides with multiple non-complementary bases; the green circle represents a bacterial cell; pJP042 is the sakacin-based expression vector that contains recT; the black double helix represents chromosomal DNA; expressed RecT proteins are denoted in the cell. (ii) An oligonucleotide identical to the lagging strand contains multiple non-complementary bases that avoid the mismatch repair system resulting in increased recombineering efficiency. (iii) Viable cells are recovered on antibiotic-free plates and recombinants are detected by a mismatch amplification mutation assay-PCR (MAMA-PCR). Two oligonucleotides (blue) will yield a 1-kb fragment, whereas a third oligonucleotide (red) will only be extended by the polymerase when the mutations are incorporated in the chromosome yielding a second amplicon of 500 bp. As the recombineering oligonucleotide only targets one strand during DNA replication the colonies will be of mixed genotype. The red dot on the chromosome indicates that the mutations are incorporated. (iv) Single colony purification is performed to separate the wild-type genotype from the mutant genotype. (v) MAMA-PCR is repeated as described in section iii to identify a pure genotype mutant. A 1:1 ratio of wild-type and mutant genotypes is suggestive that during replication a single chromosome is being replicated per cell. (vi) The recombineering plasmid pJP042 can now be cured from the mutant strain by passaging bacteria without antibiotic selection to yield a plasmid-free derivative (vii).

Mentions: In this study we describe the development of targeted ssDNA recombineering in Lactobacillus reuteri ATCC PTA 6475, a human isolate which is a candidate probiotic with anti-inflammatory properties (25,26) without the need for antibiotic selection (Figure 1). Also, we extended this technology to Lactococcus lactis which is widely used in the dairy industry for fermentation processes and has been applied as a biotherapeutic delivery vehicle (3,27). RecT-mediated recombineering is not hypermutagenic, and may serve as a powerful and novel tool for strain improvement through directed evolution by genetic engineering in LAB. The use of recombineering in the development of LAB as biotherapeutics is discussed.Figure 1.


High efficiency recombineering in lactic acid bacteria.

van Pijkeren JP, Britton RA - Nucleic Acids Res. (2012)

Overview of recombineering in L. reuteri. (i) Electrocompetent cells in which RecT is expressed are transformed with a recombineering oligonucleotide. Transformation efficiencies of ∼ ≥105cfu/µg DNA are required for high recombineering efficiencies. Wavy black lines with a red dot represent recombineering oligonucleotides with multiple non-complementary bases; the green circle represents a bacterial cell; pJP042 is the sakacin-based expression vector that contains recT; the black double helix represents chromosomal DNA; expressed RecT proteins are denoted in the cell. (ii) An oligonucleotide identical to the lagging strand contains multiple non-complementary bases that avoid the mismatch repair system resulting in increased recombineering efficiency. (iii) Viable cells are recovered on antibiotic-free plates and recombinants are detected by a mismatch amplification mutation assay-PCR (MAMA-PCR). Two oligonucleotides (blue) will yield a 1-kb fragment, whereas a third oligonucleotide (red) will only be extended by the polymerase when the mutations are incorporated in the chromosome yielding a second amplicon of 500 bp. As the recombineering oligonucleotide only targets one strand during DNA replication the colonies will be of mixed genotype. The red dot on the chromosome indicates that the mutations are incorporated. (iv) Single colony purification is performed to separate the wild-type genotype from the mutant genotype. (v) MAMA-PCR is repeated as described in section iii to identify a pure genotype mutant. A 1:1 ratio of wild-type and mutant genotypes is suggestive that during replication a single chromosome is being replicated per cell. (vi) The recombineering plasmid pJP042 can now be cured from the mutant strain by passaging bacteria without antibiotic selection to yield a plasmid-free derivative (vii).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3378904&req=5

gks147-F1: Overview of recombineering in L. reuteri. (i) Electrocompetent cells in which RecT is expressed are transformed with a recombineering oligonucleotide. Transformation efficiencies of ∼ ≥105cfu/µg DNA are required for high recombineering efficiencies. Wavy black lines with a red dot represent recombineering oligonucleotides with multiple non-complementary bases; the green circle represents a bacterial cell; pJP042 is the sakacin-based expression vector that contains recT; the black double helix represents chromosomal DNA; expressed RecT proteins are denoted in the cell. (ii) An oligonucleotide identical to the lagging strand contains multiple non-complementary bases that avoid the mismatch repair system resulting in increased recombineering efficiency. (iii) Viable cells are recovered on antibiotic-free plates and recombinants are detected by a mismatch amplification mutation assay-PCR (MAMA-PCR). Two oligonucleotides (blue) will yield a 1-kb fragment, whereas a third oligonucleotide (red) will only be extended by the polymerase when the mutations are incorporated in the chromosome yielding a second amplicon of 500 bp. As the recombineering oligonucleotide only targets one strand during DNA replication the colonies will be of mixed genotype. The red dot on the chromosome indicates that the mutations are incorporated. (iv) Single colony purification is performed to separate the wild-type genotype from the mutant genotype. (v) MAMA-PCR is repeated as described in section iii to identify a pure genotype mutant. A 1:1 ratio of wild-type and mutant genotypes is suggestive that during replication a single chromosome is being replicated per cell. (vi) The recombineering plasmid pJP042 can now be cured from the mutant strain by passaging bacteria without antibiotic selection to yield a plasmid-free derivative (vii).
Mentions: In this study we describe the development of targeted ssDNA recombineering in Lactobacillus reuteri ATCC PTA 6475, a human isolate which is a candidate probiotic with anti-inflammatory properties (25,26) without the need for antibiotic selection (Figure 1). Also, we extended this technology to Lactococcus lactis which is widely used in the dairy industry for fermentation processes and has been applied as a biotherapeutic delivery vehicle (3,27). RecT-mediated recombineering is not hypermutagenic, and may serve as a powerful and novel tool for strain improvement through directed evolution by genetic engineering in LAB. The use of recombineering in the development of LAB as biotherapeutics is discussed.Figure 1.

Bottom Line: To highlight the utility of ssDNA recombineering we reduced the intrinsic vancomymycin resistance of L. reuteri >100-fold.By creating a single amino acid change in the D-Ala-D-Ala ligase enzyme we reduced the minimum inhibitory concentration for vancomycin from >256 to 1.5 µg/ml, well below the clinically relevant minimum inhibitory concentration.Recombineering thus allows high efficiency mutagenesis in lactobacilli and lactococci, and may be used to further enhance beneficial properties and safety of strains used in medicine and industry.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.

ABSTRACT
The ability to efficiently generate targeted point mutations in the chromosome without the need for antibiotics, or other means of selection, is a powerful strategy for genome engineering. Although oligonucleotide-mediated recombineering (ssDNA recombineering) has been utilized in Escherichia coli for over a decade, the successful adaptation of ssDNA recombineering to gram-positive bacteria has not been reported. Here we describe the development and application of ssDNA recombineering in lactic acid bacteria. Mutations were incorporated in the chromosome of Lactobacillus reuteri and Lactococcus lactis without selection at frequencies ranging between 0.4% and 19%. Whole genome sequence analysis showed that ssDNA recombineering is specific and not hypermutagenic. To highlight the utility of ssDNA recombineering we reduced the intrinsic vancomymycin resistance of L. reuteri >100-fold. By creating a single amino acid change in the D-Ala-D-Ala ligase enzyme we reduced the minimum inhibitory concentration for vancomycin from >256 to 1.5 µg/ml, well below the clinically relevant minimum inhibitory concentration. Recombineering thus allows high efficiency mutagenesis in lactobacilli and lactococci, and may be used to further enhance beneficial properties and safety of strains used in medicine and industry. We expect that this work will serve as a blueprint for the adaptation of ssDNA recombineering to other gram-positive bacteria.

Show MeSH
Related in: MedlinePlus