Transformation of Lactiplantibacillus plantarum and Apilactobacillus kunkeei is influenced by recipient cell growth temperature, vector replicon, and DNA methylation

Dennis L Welker; Bailey L Crowley; Justin B Evans; Martin H Welker; Jeff R Broadbent; Robert F Roberts; David A Mills

doi:10.1016/j.mimet.2020.105967

Transformation of Lactiplantibacillus plantarum and Apilactobacillus kunkeei is influenced by recipient cell growth temperature, vector replicon, and DNA methylation

J Microbiol Methods. 2020 Aug:175:105967. doi: 10.1016/j.mimet.2020.105967. Epub 2020 Jun 5.

Authors

Dennis L Welker¹, Bailey L Crowley², Justin B Evans², Martin H Welker³, Jeff R Broadbent⁴, Robert F Roberts⁵, David A Mills⁶

Affiliations

¹ Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322, USA. Electronic address: dennis.welker@usu.edu.
² Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322, USA.
³ Arizona State Museum, University of Arizona, 1013 E. University Blvd., Tucson, AZ 85721, USA.
⁴ Department of Nutrition, Dietetics and Food Sciences, Utah State University, 4700 Old Main Hill, Logan, UT 84322, USA.
⁵ Department of Food Science, Pennsylvania State University, 206 Rodney A. Erickson Food Science Building, University Park, PA 16802, USA.
⁶ Department of Food Science and Technology, Robert Mondavi Institute for Wine and Food Science, University of California, Davis, One Shields Ave., Davis, CA 95616, USA; Foods for Health Institute, University of California, Davis, California, One Shields Ave., Davis, CA 95616, USA; Department of Viticulture and Enology, Robert Mondavi Institute for Wine and Food Science, University of California, Davis, California, One Shields Ave., Davis, CA 95616, USA.

PMID: 32512121
DOI: 10.1016/j.mimet.2020.105967

Abstract

The effects of recipient cell growth temperature, vector choice, and DNA methylation on transformation efficiency were explored for Lactiplantibacillus plantarum strain B38 and Apilactobacillus kunkeei strains YH15 and 3L. All three parameters significantly affected transformation efficiency. L. plantarum B38 and A. kunkeei YH15 transformed at higher efficiencies with the pTW8 vector than with the pTRKH2 vector; conversely, A. kunkeei 3L transformed at higher efficiency with pTRKH2. Mean transformation efficiencies as high as 7.8 × 10⁵ colony forming units (CFU) μg^-1 were obtained with pTW8 for B38, as high as 1.2 × 10⁵ CFU μg^-1 with pTW8 for YH15, and as high as 3.4 × 10⁶ CFU μg^-1 with pTRKH2 for 3L. With respect to methylation, B38 and YH15 transformed at higher efficiencies with DNA that lacked dam methylation, while 3L transformed at higher efficiency with DNA that was dam methylated. Methylation at Escherichia coli dcm sites did not affect the ability of pTRKH2 or pTW8 to transform these strains. Recipient cell growth at 21 °C rather than at 37 °C significantly increased transformation efficiencies when using each strain's preferred vector and methylation state; pTW8 without dam methylation for B38 and YH15 and pTRKH2 with dam methylation for 3L.

Keywords: Apilactobacillus kunkeei; DNA methylation; Electroporation; Lactiplantibacillus plantarum; Lactobacilli; Lactobacillus kunkeei; Lactobacillus plantarum; Recipient cell growth temperature; Transformation; Vector replicon.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Methylation
DNA, Bacterial
Genetic Vectors
Lactobacillus / genetics*
Lactobacillus plantarum / genetics*
Plasmids
Replicon
Temperature
Transformation, Bacterial*

Substances

DNA, Bacterial

Supplementary concepts

Lactobacillus kunkeei