The Chromosomal Gene Manipulation Method for Lactobacillus delbrueckii subsp. bulgaricus Using a Conjugative Shuttle Vector pGMß1.

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, pGMß1, has been deposited at Addgene ( https://www.addgene.org )and can be used by anyone for academic purposes.

Immunomodulatory effects of extracellular glyceraldehyde 3-phosphate dehydrogenase of exopolysaccharide-producing Lactiplantibacillus plantarum JCM 1149.

Probiotic lactic acid bacteria evoke immunomodulatory effects in the host; however, the reasons for the different effects of various species and strains remain to be elucidated. To clarify the critical immunomodulatory components and impact of exopolysaccharide (EPS) in Lactiplantibacillus plantarum, 11 types of L. plantarum strains were compared for the production of EPS, inflammatory cytokines, interleukin-6 and -12, and the anti-inflammatory cytokine, interleukin-10, from THP-1 differentiated dendritic cells. EPS in the fermented medium correlated with cytokine-inducing activities. L. plantarum JCM 1149, with the highest production of EPS, also induced interleukin-6, -10, and -12 among the 11 tested strains. Notably, the cytokine-producing activities overlapped with the protein fraction in gel filtration chromatography but not with EPS, which has been reported to exert immunomodulatory effects. The 41 kDa protein that coexisted with EPS was purified as a major active component and identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a known moonlighting protein. GAPDH secretion was reduced when EPS synthesis inhibitors were added to the culture medium. RNA sequencing of GAPDH-treated THP-1 cells revealed an up-regulation in the expression of genes involved in transcriptional regulation, cell surface receptor signalling, immune response, and matrix components. Here, we report, to our knowledge for the first time, that the cell surface-associated L. plantarum GAPDH plays a crucial role in cytokine production in THP-1 cells, but EPS with less activity may help GAPDH secretion.

Intracellular pH Determination for the Study of Acid Tolerance of Lactic Acid Bacteria.

It is important to assess acid tolerance in lactic acid bacteria, particularly for probiotics, although it involves multiple mechanisms. Measuring the difference between intracellular and extracellular pH (ΔpH) using the fluorescent probe CFDA-SE is particularly effective for such assessments because it gives direct information on the level of tolerance in the extracellular acidic pH range from 7 to 2.5. It also enables acid adaptation to be induced and observed by slowly introducing HCl into the medium and decreasing the extracellular pH. The difference of acid tolerance between anaerobic and aerobic conditions in lactic acid bacteria can also be evaluated by measuring ΔpH.