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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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Related in: MedlinePlus

Converting vancomycin-resistant L. reuteri to vancomycin-sensitive by a single amino acid change. (a) The determined 3D structure of the d-ala-d-ala ligase (Ddl) protein of E.coli (PDB ID 1IOV; blue) and the modeled protein structure of Ddl of L. reuteri (green) were superimposed to locate the putative active site residues in Ddl of L. reuteri which, in E. coli, form a hydrogen-bonded network (Y216–E15–S150; boxed region). (b) Superimposing the determined structures of the Ddl proteins of the E. coli wild-type (blue) and a mutant (PDB ID 1IOW; grey) show that replacing a tyrosine at position 216 with a phenylalanine (Y216F) does not form a hydrogen-bond between F216–E15, and changes the enzymatic activity from a dipeptide ligase to a depsipeptide ligase. (c) We predicted the active site residues of Ddl-L. reuteri by superimposing the wild-type E. coli Ddl protein (blue) with the predicted protein structure of L. reuteri (green), and residues F258–E17–S186 in Ddl-L. reuteri correspond to the E. coli triad Y216–E15–S15, respectively. According to the E. coli model changing the phenylalanine at position 258 to a tyrosine (F258Y) in Ddl-L. reuteri would establish a hydrogen-bond between F258–E17 and may therefore yield dipeptide ligase activity which subsequently would result in a vancomycin-sensitive phenotype. (d) The dsDNA sequence of the ddl region of L. reuteri is shown aligned with the encoded amino acids for each triplet. Italic sequence represents the ApoI restriction site that is mutated by incorporation of oJP810. On the left are the leading and lagging strand indicated, and below is oJP810 showing the different mismatches with the resulting amino acid change (F258Y) on the right. (e) Susceptibility of L. reuteri wild-type (left) and the mutant RPRB3003 (right) to vancomycin using an Etest assay. The MIC is determined by identifying where bacterial growth intersects the Etest strip.
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gks147-F5: Converting vancomycin-resistant L. reuteri to vancomycin-sensitive by a single amino acid change. (a) The determined 3D structure of the d-ala-d-ala ligase (Ddl) protein of E.coli (PDB ID 1IOV; blue) and the modeled protein structure of Ddl of L. reuteri (green) were superimposed to locate the putative active site residues in Ddl of L. reuteri which, in E. coli, form a hydrogen-bonded network (Y216–E15–S150; boxed region). (b) Superimposing the determined structures of the Ddl proteins of the E. coli wild-type (blue) and a mutant (PDB ID 1IOW; grey) show that replacing a tyrosine at position 216 with a phenylalanine (Y216F) does not form a hydrogen-bond between F216–E15, and changes the enzymatic activity from a dipeptide ligase to a depsipeptide ligase. (c) We predicted the active site residues of Ddl-L. reuteri by superimposing the wild-type E. coli Ddl protein (blue) with the predicted protein structure of L. reuteri (green), and residues F258–E17–S186 in Ddl-L. reuteri correspond to the E. coli triad Y216–E15–S15, respectively. According to the E. coli model changing the phenylalanine at position 258 to a tyrosine (F258Y) in Ddl-L. reuteri would establish a hydrogen-bond between F258–E17 and may therefore yield dipeptide ligase activity which subsequently would result in a vancomycin-sensitive phenotype. (d) The dsDNA sequence of the ddl region of L. reuteri is shown aligned with the encoded amino acids for each triplet. Italic sequence represents the ApoI restriction site that is mutated by incorporation of oJP810. On the left are the leading and lagging strand indicated, and below is oJP810 showing the different mismatches with the resulting amino acid change (F258Y) on the right. (e) Susceptibility of L. reuteri wild-type (left) and the mutant RPRB3003 (right) to vancomycin using an Etest assay. The MIC is determined by identifying where bacterial growth intersects the Etest strip.

Mentions: To emphasize the utility of recombineering we converted L. reuteri from vancomycin resistant to vancomycin sensitive by mutating the active site of Ddl directly in the chromosome. Vancomycin inhibits the peptidoglycan biosynthesis by binding to muramylpentapeptides that terminate in d-alanyl-d-alanine (d-Ala-d-Ala) and blocks the addition of these precursors into the peptidoglycan backbone (54,55). Many lactobacilli incorporate d-alanyl-d-lactate (d-Ala-d-Lac) into their muramylpentapeptides, and this is the basis for their vancomycin resistance as peptides that terminate in d-Ala-d-Lac are bound 1000-fold less efficiently by vancomycin compared to peptides terminating in d-Ala-d-Ala (56,57). The enzyme responsible for the addition of d-Ala or d-lac is d-Ala-d-Ala ligase (Ddl). In E. coli Ddl is a dipeptide ligase as it catalyzes the formation of d-Ala-d-Ala whereas in L. mesenteorides, and probably in many lactobacilli, Ddl is a depsipeptide ligase as it catalyzes the formation of d-Ala-d-Lac. Superimposing the structures of Ddl-E. coli and Ddl-L. reuteri show that both proteins are structurally related (Figure 5a). In vitro analyses in E. coli and L. mesenteroides have previously identified active site residues in Ddl that upon mutation could switch from a dipeptide ligase to a depsipeptide ligase and vice versa(34,58). In Ddl-E. coli the residues Y216–E15–S150 form a hydrogen-bonding triad (Figure 5b) that is linked with the specificity of the enzyme although the exact mechanism for the shift in specificity is unknown (34). By replacing the tyrosine at position 216 with a phenylalanine (Y216F) in Ddl-E. coli there is no hydrogen bond formed between F216–E15, and as a consequence Ddl-Y216F has gained depsipeptide activity (Figure 5b) (34). To translate this to L. reuteri, we identified the predicted active site residues in Ddl- L. reuteri by superimposing Ddl-L. reuteri with Ddl-E. coli (Fig 5c). The L. reuteri triad is predicted to be F258–E17–S186, and mutating phenylalanine at position 258 to a tyrosine (F258Y) would, based on the E. coli model, yield a hydrogen-bond between Y258-E17 and change subsequently the enzyme activity from a depsipeptide ligase to a dipeptide ligase. As a result the cells render sensitivity to vancomycin.Figure 5.


High efficiency recombineering in lactic acid bacteria.

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

Converting vancomycin-resistant L. reuteri to vancomycin-sensitive by a single amino acid change. (a) The determined 3D structure of the d-ala-d-ala ligase (Ddl) protein of E.coli (PDB ID 1IOV; blue) and the modeled protein structure of Ddl of L. reuteri (green) were superimposed to locate the putative active site residues in Ddl of L. reuteri which, in E. coli, form a hydrogen-bonded network (Y216–E15–S150; boxed region). (b) Superimposing the determined structures of the Ddl proteins of the E. coli wild-type (blue) and a mutant (PDB ID 1IOW; grey) show that replacing a tyrosine at position 216 with a phenylalanine (Y216F) does not form a hydrogen-bond between F216–E15, and changes the enzymatic activity from a dipeptide ligase to a depsipeptide ligase. (c) We predicted the active site residues of Ddl-L. reuteri by superimposing the wild-type E. coli Ddl protein (blue) with the predicted protein structure of L. reuteri (green), and residues F258–E17–S186 in Ddl-L. reuteri correspond to the E. coli triad Y216–E15–S15, respectively. According to the E. coli model changing the phenylalanine at position 258 to a tyrosine (F258Y) in Ddl-L. reuteri would establish a hydrogen-bond between F258–E17 and may therefore yield dipeptide ligase activity which subsequently would result in a vancomycin-sensitive phenotype. (d) The dsDNA sequence of the ddl region of L. reuteri is shown aligned with the encoded amino acids for each triplet. Italic sequence represents the ApoI restriction site that is mutated by incorporation of oJP810. On the left are the leading and lagging strand indicated, and below is oJP810 showing the different mismatches with the resulting amino acid change (F258Y) on the right. (e) Susceptibility of L. reuteri wild-type (left) and the mutant RPRB3003 (right) to vancomycin using an Etest assay. The MIC is determined by identifying where bacterial growth intersects the Etest strip.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks147-F5: Converting vancomycin-resistant L. reuteri to vancomycin-sensitive by a single amino acid change. (a) The determined 3D structure of the d-ala-d-ala ligase (Ddl) protein of E.coli (PDB ID 1IOV; blue) and the modeled protein structure of Ddl of L. reuteri (green) were superimposed to locate the putative active site residues in Ddl of L. reuteri which, in E. coli, form a hydrogen-bonded network (Y216–E15–S150; boxed region). (b) Superimposing the determined structures of the Ddl proteins of the E. coli wild-type (blue) and a mutant (PDB ID 1IOW; grey) show that replacing a tyrosine at position 216 with a phenylalanine (Y216F) does not form a hydrogen-bond between F216–E15, and changes the enzymatic activity from a dipeptide ligase to a depsipeptide ligase. (c) We predicted the active site residues of Ddl-L. reuteri by superimposing the wild-type E. coli Ddl protein (blue) with the predicted protein structure of L. reuteri (green), and residues F258–E17–S186 in Ddl-L. reuteri correspond to the E. coli triad Y216–E15–S15, respectively. According to the E. coli model changing the phenylalanine at position 258 to a tyrosine (F258Y) in Ddl-L. reuteri would establish a hydrogen-bond between F258–E17 and may therefore yield dipeptide ligase activity which subsequently would result in a vancomycin-sensitive phenotype. (d) The dsDNA sequence of the ddl region of L. reuteri is shown aligned with the encoded amino acids for each triplet. Italic sequence represents the ApoI restriction site that is mutated by incorporation of oJP810. On the left are the leading and lagging strand indicated, and below is oJP810 showing the different mismatches with the resulting amino acid change (F258Y) on the right. (e) Susceptibility of L. reuteri wild-type (left) and the mutant RPRB3003 (right) to vancomycin using an Etest assay. The MIC is determined by identifying where bacterial growth intersects the Etest strip.
Mentions: To emphasize the utility of recombineering we converted L. reuteri from vancomycin resistant to vancomycin sensitive by mutating the active site of Ddl directly in the chromosome. Vancomycin inhibits the peptidoglycan biosynthesis by binding to muramylpentapeptides that terminate in d-alanyl-d-alanine (d-Ala-d-Ala) and blocks the addition of these precursors into the peptidoglycan backbone (54,55). Many lactobacilli incorporate d-alanyl-d-lactate (d-Ala-d-Lac) into their muramylpentapeptides, and this is the basis for their vancomycin resistance as peptides that terminate in d-Ala-d-Lac are bound 1000-fold less efficiently by vancomycin compared to peptides terminating in d-Ala-d-Ala (56,57). The enzyme responsible for the addition of d-Ala or d-lac is d-Ala-d-Ala ligase (Ddl). In E. coli Ddl is a dipeptide ligase as it catalyzes the formation of d-Ala-d-Ala whereas in L. mesenteorides, and probably in many lactobacilli, Ddl is a depsipeptide ligase as it catalyzes the formation of d-Ala-d-Lac. Superimposing the structures of Ddl-E. coli and Ddl-L. reuteri show that both proteins are structurally related (Figure 5a). In vitro analyses in E. coli and L. mesenteroides have previously identified active site residues in Ddl that upon mutation could switch from a dipeptide ligase to a depsipeptide ligase and vice versa(34,58). In Ddl-E. coli the residues Y216–E15–S150 form a hydrogen-bonding triad (Figure 5b) that is linked with the specificity of the enzyme although the exact mechanism for the shift in specificity is unknown (34). By replacing the tyrosine at position 216 with a phenylalanine (Y216F) in Ddl-E. coli there is no hydrogen bond formed between F216–E15, and as a consequence Ddl-Y216F has gained depsipeptide activity (Figure 5b) (34). To translate this to L. reuteri, we identified the predicted active site residues in Ddl- L. reuteri by superimposing Ddl-L. reuteri with Ddl-E. coli (Fig 5c). The L. reuteri triad is predicted to be F258–E17–S186, and mutating phenylalanine at position 258 to a tyrosine (F258Y) would, based on the E. coli model, yield a hydrogen-bond between Y258-E17 and change subsequently the enzyme activity from a depsipeptide ligase to a dipeptide ligase. As a result the cells render sensitivity to vancomycin.Figure 5.

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