In the late 1980s, genetic manipulations of lactic acid bacteria (LAB) had begun in earnest with the search for vectors for LAB. The development of chromosomal integration vectors and “food-grade” host/vector expression systems greatly increased the interest in LAB genetics. In the 21st century, scientists are pursuing genome sequencing of industrial LAB and post-genomic research avenues that include the LAB transcriptome, proteome, and metabolome. These omics studies have changed the research objective from a single cell to the meta-analysis of complex systems, including LAB metabolic networks and rapid and systematic responses of LAB to environmental changes. We began researching LAB genetics in the context of yogurt fermentation in 1987 and have actively participated in this constantly evolving field to the present day. Although excellent progress has been made in LAB research, many important LAB have not yet to be genetically manipulated, likely due to a history of selection for tolerance to foreign DNA invasion. Our LAB genetic studies in the past 30 years are introduced here.

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Lactobacillus and Bifidobacterium species are natural inhabitants of the mammalian gastrointestinal (GI) tract. Adhesion to the mucosal surface is an important prerequisite for colonization of the GI tract by non-motile organisms in a constantly changing GI environment. The organisms capable of adhesion to the mucosal surface have a competitive advantage in this ecosystem. In recent years, the development of a repertoire of genetic tools and the advances in analytical techniques have markedly accelerated research aimed at elucidating the molecular mechanisms based on the ability of Lactobacillus and Bifidobacterium to recognize and adhere to mucosal components through diverse cell-surface macromolecules. This review provides molecular insights into the sortase-dependent adhesion factors such as mucus-binding proteins (MucBPs) and pilus, taking into account the findings of our recent study. Moreover, we introduce the competitive exclusion of pathogenic bacteria by Lactobacillus via their adhesion factors.

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Genome finishing still remains a laborious work that includes various validation processes requiring both wet and dry knowledge and consideration, although long-read sequencers such as PacBio RSII have largely contributed to lighten the burden. We here introduce a procedure of post-assemble validation in which draft contigs are circularized into complete chromosome or plasmid sequences. We also describe the DFAST annotation web service specialized for lactic acid bacteria, visualization of BLAST alignments, and correction of contigs by mapping Illumina reads. Supplementary materials are available at our web site, http://www.iu.a.u-tokyo.ac.jp/~kadota/r_seq.html#about_book_JSLAB.

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Using gastric juice tolerant Lactobacillus plantarum FSCM2-12, NBRC 109604 and NBRC 101975, and gastric juice sensitive Lactobacillus brevis FSCT-2, the relationship between the gastric acid tolerance and the morphologic characteristics were examined. Survival ratio of L. plantarum FSCM2-12 cultured in the medium supplemented with glucose, fructose, or sucrose as a carbon source, was more than 70% after treatment with the artificial gastric juice. However, the survival ratio of the strain cultured in the medium supplemented with glycerol or no carbon source, was drastically decreased. L. plantarum FSCM2-12 did not form the biofilm, but clear capsule was observed under the microscope after Hiss staining and by transmission electron microscopic observation. Furthermore, thickness of the capsule showed correlation with gastric juice tolerance. The capsules were thick and the survival ratio after treatment with artificial gastric juice was more than 70% when the strain was cultured in the medium supplemented with glucose, fructose, and sucrose as a carbon sources. Similar phenomena were observed in L. plantarum NBRC 109604 and NBRC 101975, therefore, the high gastric acid tolerance might be provided by thick capsule.

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