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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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Establishing and optimizing recombineering in L. reuteri. (a) The dsDNA sequence of the targeted rpoB region of L. reuteri is shown aligned with amino acids specified by each codon. On the left the leading and lagging strand are indicated and below the oligonucleotides are listed which result in different mismatches, all resulting in a rifampicin-resistant phenotype. The corresponding amino acid changes are listed on the right. (b) Titrations of the amount of oligonucleotides oJP133 (Ο) and oJP577 (Δ) were performed with 1, 5, 25 and 100 µg of oligonucleotide. Rifampicin resistant colonies derived from the control transformation are represented as a bar graph. Data are expressed as rifampicin-resistant cfu per 109 cells. Data shown are the averages of three independent experiments and error bars represent standard deviation. (c) Assessment of recombineering efficiencies with 100 µg oJP133, oJP311, oJP813 and oJP577 in the wild-type strain (black bars), and in the mismatch repair deficient strains MutS1− (light grey bars) and MutL− (dark grey bars). Data shown are the averages of three independent experiments and error bars represent standard deviation.
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gks147-F2: Establishing and optimizing recombineering in L. reuteri. (a) The dsDNA sequence of the targeted rpoB region of L. reuteri is shown aligned with amino acids specified by each codon. On the left the leading and lagging strand are indicated and below the oligonucleotides are listed which result in different mismatches, all resulting in a rifampicin-resistant phenotype. The corresponding amino acid changes are listed on the right. (b) Titrations of the amount of oligonucleotides oJP133 (Ο) and oJP577 (Δ) were performed with 1, 5, 25 and 100 µg of oligonucleotide. Rifampicin resistant colonies derived from the control transformation are represented as a bar graph. Data are expressed as rifampicin-resistant cfu per 109 cells. Data shown are the averages of three independent experiments and error bars represent standard deviation. (c) Assessment of recombineering efficiencies with 100 µg oJP133, oJP311, oJP813 and oJP577 in the wild-type strain (black bars), and in the mismatch repair deficient strains MutS1− (light grey bars) and MutL− (dark grey bars). Data shown are the averages of three independent experiments and error bars represent standard deviation.

Mentions: To assess the ability of the L. reuteri RecT proteins to support recombineering we placed recT1 under the control of an inducible promoter using the sakacin-based expression vector pSIP411(48) to yield plasmid pJP042. A simple method to quantitatively assess the efficacy of recombineering is to mutate a site in the RNA polymerase gene (rpoB) that results in an amino acid change yielding a rifampicin-resistant phenotype. We designed an 80-mer oligonucleotide (oJP133) targeting the lagging strand of DNA synthesis which has a single centrally located non-complementary base that results in a C•A mismatch (Figure 2a). Incorporation of oJP133 generates the amino acid change H488R in RpoB that confers rifampicin resistance in L. reuteri. After optimization of RecT1 expression conditions (data not shown), transformation of 1 µg oJP133 yielded approximately a 10-fold increase in rifampicin-resistant colonies compared to background levels (Figure 2b). Expression of RecT2 yielded similar recombineering efficiencies (data not shown), and we used RecT1 for all future recombineering experiments in L. reuteri.Figure 2.


High efficiency recombineering in lactic acid bacteria.

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

Establishing and optimizing recombineering in L. reuteri. (a) The dsDNA sequence of the targeted rpoB region of L. reuteri is shown aligned with amino acids specified by each codon. On the left the leading and lagging strand are indicated and below the oligonucleotides are listed which result in different mismatches, all resulting in a rifampicin-resistant phenotype. The corresponding amino acid changes are listed on the right. (b) Titrations of the amount of oligonucleotides oJP133 (Ο) and oJP577 (Δ) were performed with 1, 5, 25 and 100 µg of oligonucleotide. Rifampicin resistant colonies derived from the control transformation are represented as a bar graph. Data are expressed as rifampicin-resistant cfu per 109 cells. Data shown are the averages of three independent experiments and error bars represent standard deviation. (c) Assessment of recombineering efficiencies with 100 µg oJP133, oJP311, oJP813 and oJP577 in the wild-type strain (black bars), and in the mismatch repair deficient strains MutS1− (light grey bars) and MutL− (dark grey bars). Data shown are the averages of three independent experiments and error bars represent standard deviation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3378904&req=5

gks147-F2: Establishing and optimizing recombineering in L. reuteri. (a) The dsDNA sequence of the targeted rpoB region of L. reuteri is shown aligned with amino acids specified by each codon. On the left the leading and lagging strand are indicated and below the oligonucleotides are listed which result in different mismatches, all resulting in a rifampicin-resistant phenotype. The corresponding amino acid changes are listed on the right. (b) Titrations of the amount of oligonucleotides oJP133 (Ο) and oJP577 (Δ) were performed with 1, 5, 25 and 100 µg of oligonucleotide. Rifampicin resistant colonies derived from the control transformation are represented as a bar graph. Data are expressed as rifampicin-resistant cfu per 109 cells. Data shown are the averages of three independent experiments and error bars represent standard deviation. (c) Assessment of recombineering efficiencies with 100 µg oJP133, oJP311, oJP813 and oJP577 in the wild-type strain (black bars), and in the mismatch repair deficient strains MutS1− (light grey bars) and MutL− (dark grey bars). Data shown are the averages of three independent experiments and error bars represent standard deviation.
Mentions: To assess the ability of the L. reuteri RecT proteins to support recombineering we placed recT1 under the control of an inducible promoter using the sakacin-based expression vector pSIP411(48) to yield plasmid pJP042. A simple method to quantitatively assess the efficacy of recombineering is to mutate a site in the RNA polymerase gene (rpoB) that results in an amino acid change yielding a rifampicin-resistant phenotype. We designed an 80-mer oligonucleotide (oJP133) targeting the lagging strand of DNA synthesis which has a single centrally located non-complementary base that results in a C•A mismatch (Figure 2a). Incorporation of oJP133 generates the amino acid change H488R in RpoB that confers rifampicin resistance in L. reuteri. After optimization of RecT1 expression conditions (data not shown), transformation of 1 µg oJP133 yielded approximately a 10-fold increase in rifampicin-resistant colonies compared to background levels (Figure 2b). Expression of RecT2 yielded similar recombineering efficiencies (data not shown), and we used RecT1 for all future recombineering experiments in L. reuteri.Figure 2.

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