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The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective
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Published on: June 16, 2022

High efficiency recombineering in lactic acid bacteria.

Jan-Peter van Pijkeren1, Robert A Britton

  • 1Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.

Nucleic Acids Research
|February 14, 2012
PubMed
Summary

Scientists developed a new genome engineering method, single-stranded DNA (ssDNA) recombineering, for gram-positive bacteria like Lactobacillus and Lactococcus. This technique enables precise mutations without antibiotic selection, enhancing bacterial strain development for industry and medicine.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genome Engineering

Background:

  • Oligonucleotide-mediated recombineering using single-stranded DNA (ssDNA) is established in E. coli for targeted chromosomal mutations.
  • Adaptation of ssDNA recombineering to gram-positive bacteria, particularly lactic acid bacteria, has been lacking.

Purpose of the Study:

  • To develop and apply ssDNA recombineering in gram-positive lactic acid bacteria.
  • To demonstrate the efficiency and specificity of ssDNA recombineering in these organisms.
  • To showcase the utility of ssDNA recombineering for strain improvement.

Main Methods:

  • Development of ssDNA recombineering protocols for Lactobacillus reuteri and Lactococcus lactis.
  • Introduction of targeted point mutations into the bacterial chromosome without antibiotic selection.
  • Whole genome sequencing to assess mutation specificity and potential off-target effects.
  • Phenotypic analysis of vancomycin resistance in engineered L. reuteri strains.

Main Results:

  • Successful implementation of ssDNA recombineering in L. reuteri and L. lactis, achieving mutation frequencies from 0.4% to 19%.
  • Whole genome sequencing confirmed the specificity of ssDNA recombineering, showing no evidence of hypermutagenicity.
  • A single amino acid change in D-Ala-D-Ala ligase drastically reduced vancomycin resistance in L. reuteri (>100-fold).

Conclusions:

  • ssDNA recombineering is an efficient and specific mutagenesis tool for Lactobacillus and Lactococcus species.
  • This method facilitates targeted genetic modification in gram-positive bacteria, enabling strain improvement for industrial and medical applications.
  • The established protocols serve as a foundation for adapting ssDNA recombineering to other gram-positive bacterial species.