The Chromosomal Gene Manipulation Method for Lactobacillus delbrueckii subsp. bulgaricus Using a Conjugative Shuttle Vector pGMβ1

Lactobacillus bulgaricus is an industrial strain that has been used in the dairy products since ancient times. Because of the difficulty of chromosomal gene manipulation, there have been few reports of gene deletion, insertion, or replacement. We have developed a system that enables chromosomal gene manipulation of L. bulgaricus using a conjugal transfer vector and easily vector construction in E. coli. As an example, we have deleted a regulatory gene for the extracellular polysaccharide synthesis of L. bulgaricus to elucidate the function of the gene in question. Methods for constructing vectors for chromosomal integration, conjugation experiment, and obtaining deletion strains by double recombination were presented in detail. This conjugative shuttle vector, pGMβ1, has been deposited at Addgene ( https://www.addgene.org )and can be used by anyone for academic purposes.