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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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Non-selected recombineering in L. reuteri. (a) One hundred colonies derived from transformation with 100 µg oJP577, which upon incorporation yields a rifampicin-resistant phenotype (RpoB H488R), were patched onto MRS agar without antibiotic selection (left—MRS), and onto MRS agar containing rifampicin (right—MRS + RIF). (b) Overview of a selection of genes mutated by non-selected recombineering in the L. reuteri ATCC PTA 6475 chromosome. The locations of the mutated genes on the L. reuteri ATCC PTA 6475 chromosome are indicated based on the closed genome of the closely related strain L. reuteri F275 (NCBI accession number NC_010609). The numbers displayed represent the last four digits of the locus tag (HMPREF0535_XXXX) for each of the genes targeted and the recombineering frequency shown in parentheses. The rpoB gene (DNA directed RNA polymerase; locus tag HMPREF0536_0828) and the ddl gene (d-Ala-d-Ala ligase, locus tag HMPREF0536_1572) are listed using their gene names based on experimental evidence. ND: not determined; ori: origin of replication.
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gks147-F3: Non-selected recombineering in L. reuteri. (a) One hundred colonies derived from transformation with 100 µg oJP577, which upon incorporation yields a rifampicin-resistant phenotype (RpoB H488R), were patched onto MRS agar without antibiotic selection (left—MRS), and onto MRS agar containing rifampicin (right—MRS + RIF). (b) Overview of a selection of genes mutated by non-selected recombineering in the L. reuteri ATCC PTA 6475 chromosome. The locations of the mutated genes on the L. reuteri ATCC PTA 6475 chromosome are indicated based on the closed genome of the closely related strain L. reuteri F275 (NCBI accession number NC_010609). The numbers displayed represent the last four digits of the locus tag (HMPREF0535_XXXX) for each of the genes targeted and the recombineering frequency shown in parentheses. The rpoB gene (DNA directed RNA polymerase; locus tag HMPREF0536_0828) and the ddl gene (d-Ala-d-Ala ligase, locus tag HMPREF0536_1572) are listed using their gene names based on experimental evidence. ND: not determined; ori: origin of replication.

Mentions: For genes other than rpoB, a selection of genes was chosen that targeted different regions of the chromosome of L. reuteri. For each gene a 90-mer oligonucleotide was designed with multiple consecutive mismatches. With exception of oJP475 and oJP810, recombineering oligonucleotides were designed such that an in-frame stop codon and a restriction endonuclease recognition site (either EcoRI, BamHI or HindIII) were created upon incorporation. For all genes 100 µg recombineering oligonucleotide was transformed and after recovery serial dilutions were spread on MRS plates without antibiotic selection. From the pool of transformants approximately 300 colonies were subjected to screening. We applied two screening strategies. The first strategy involves identification of mutations by restriction digest when the mutation being incorporated generated a new restriction site. PCR amplification of a 1-kb fragment in which the target site is located centrally was performed. Amplicons were subsequently subjected to restriction digest analysis with the appropriate enzyme. A partial or completely digested amplicon was indicative of incorporation of the recombineering oligonucleotide. The rationale for the partial digest is that one strand is targeted by oligonucleotide recombineering, i.e. the PCR product can be derived from mixed genotypes yielding a mix of amplicons. Alternatively, a fully digested amplicon can be expected if the mixed genotypes separated during recovery. The second method is a mismatch amplification mutation analysis–PCR (MAMA–PCR) (32,33), and relies on the use of an oligonucleotide of which the 3′-end is complementary to the recombinant sequence. Upon identification of a recombinant genotype by either method, cells were streaked on plate to separate the genotypes followed by screening to identify a pure genotype. The oligonucleotide sequences related to the targets listed in Figure 3b are available upon request.


High efficiency recombineering in lactic acid bacteria.

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

Non-selected recombineering in L. reuteri. (a) One hundred colonies derived from transformation with 100 µg oJP577, which upon incorporation yields a rifampicin-resistant phenotype (RpoB H488R), were patched onto MRS agar without antibiotic selection (left—MRS), and onto MRS agar containing rifampicin (right—MRS + RIF). (b) Overview of a selection of genes mutated by non-selected recombineering in the L. reuteri ATCC PTA 6475 chromosome. The locations of the mutated genes on the L. reuteri ATCC PTA 6475 chromosome are indicated based on the closed genome of the closely related strain L. reuteri F275 (NCBI accession number NC_010609). The numbers displayed represent the last four digits of the locus tag (HMPREF0535_XXXX) for each of the genes targeted and the recombineering frequency shown in parentheses. The rpoB gene (DNA directed RNA polymerase; locus tag HMPREF0536_0828) and the ddl gene (d-Ala-d-Ala ligase, locus tag HMPREF0536_1572) are listed using their gene names based on experimental evidence. ND: not determined; ori: origin of replication.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks147-F3: Non-selected recombineering in L. reuteri. (a) One hundred colonies derived from transformation with 100 µg oJP577, which upon incorporation yields a rifampicin-resistant phenotype (RpoB H488R), were patched onto MRS agar without antibiotic selection (left—MRS), and onto MRS agar containing rifampicin (right—MRS + RIF). (b) Overview of a selection of genes mutated by non-selected recombineering in the L. reuteri ATCC PTA 6475 chromosome. The locations of the mutated genes on the L. reuteri ATCC PTA 6475 chromosome are indicated based on the closed genome of the closely related strain L. reuteri F275 (NCBI accession number NC_010609). The numbers displayed represent the last four digits of the locus tag (HMPREF0535_XXXX) for each of the genes targeted and the recombineering frequency shown in parentheses. The rpoB gene (DNA directed RNA polymerase; locus tag HMPREF0536_0828) and the ddl gene (d-Ala-d-Ala ligase, locus tag HMPREF0536_1572) are listed using their gene names based on experimental evidence. ND: not determined; ori: origin of replication.
Mentions: For genes other than rpoB, a selection of genes was chosen that targeted different regions of the chromosome of L. reuteri. For each gene a 90-mer oligonucleotide was designed with multiple consecutive mismatches. With exception of oJP475 and oJP810, recombineering oligonucleotides were designed such that an in-frame stop codon and a restriction endonuclease recognition site (either EcoRI, BamHI or HindIII) were created upon incorporation. For all genes 100 µg recombineering oligonucleotide was transformed and after recovery serial dilutions were spread on MRS plates without antibiotic selection. From the pool of transformants approximately 300 colonies were subjected to screening. We applied two screening strategies. The first strategy involves identification of mutations by restriction digest when the mutation being incorporated generated a new restriction site. PCR amplification of a 1-kb fragment in which the target site is located centrally was performed. Amplicons were subsequently subjected to restriction digest analysis with the appropriate enzyme. A partial or completely digested amplicon was indicative of incorporation of the recombineering oligonucleotide. The rationale for the partial digest is that one strand is targeted by oligonucleotide recombineering, i.e. the PCR product can be derived from mixed genotypes yielding a mix of amplicons. Alternatively, a fully digested amplicon can be expected if the mixed genotypes separated during recovery. The second method is a mismatch amplification mutation analysis–PCR (MAMA–PCR) (32,33), and relies on the use of an oligonucleotide of which the 3′-end is complementary to the recombinant sequence. Upon identification of a recombinant genotype by either method, cells were streaked on plate to separate the genotypes followed by screening to identify a pure genotype. The oligonucleotide sequences related to the targets listed in Figure 3b are available upon request.

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