Bifidobacteria are a major component of the intestinal microbiota in humans, particularly breast-fed infants. Therefore, elucidation of the mechanisms by which these bacteria colonize the intestine is desired. One approach is transposon mutagenesis, a technique currently attracting much attention because, in combination with next-generation sequencing, it enables exhaustive identification of genes that contribute to microbial fitness. We now describe a transposon mutagenesis system for subsp. 105-A (JCM 31944) based on IS, a native IS family insertion sequence. To build this system, xylose-inducible or constitutive bifidobacterial promoters were tested to drive the expression of full-length or a truncated form at the N terminus of the IS transposase. An artificial transposon plasmid, pBFS12, in which IS terminal inverted repeats are separated by a 3-bp spacer, was also constructed to mimic the transposition intermediate of IS elements. The introduction of this plasmid into a strain expressing transposase resulted in the insertion of the plasmid with an efficiency of >10 CFU/μg DNA. The plasmid targets random 3- to 4-bp sequences, but with a preference for noncoding regions. This mutagenesis system also worked at least in NCC2705. Characterization of a transposon insertion mutant revealed that a putative α-glucosidase mediates palatinose and trehalose assimilation, demonstrating the suitability of transposon mutagenesis for loss-of-function analysis. We anticipate that this approach will accelerate functional genomic studies of subsp. Several hundred species of bacteria colonize the mammalian intestine. However, the genes that enable such bacteria to colonize and thrive in the intestine remain largely unexplored. Transposon mutagenesis, combined with next-generation sequencing, is a promising tool to comprehensively identify these genes but has so far been applied only to a small number of intestinal bacterial species. In this study, a transposon mutagenesis system was established for subsp. , a representative health-promoting species. The system enables the identification of genes that promote colonization and survival in the intestine and should help illuminate the physiology of this species.

Recently, there has been an increasing interest in site-specific modifications of antibodies used in immunoassays for disease diagnosis and as antibody therapeutics, such as antibody-drug conjugates. Previously, we established a site-specific chemical conjugation system using an IgG-Fc binding chemical conjugation affinity peptide (CCAP). CCAP could be used only for the modification of human IgG owing to the lack of affinity of CCAP to rodent IgG molecules. In this study, novel CCAP reagents are proposed, which can be used for both human and mouse IgG, based on the Staphylococcus aureus protein A domain-derived affinity peptides Z34C and Z33. Compared to the activity of a conventional randomly modified antibody, mouse IgG modified using this method had favorable features in two immunoassays, demonstrating the advantages of the proposed CCAP method in preserving antibody functionality during conjugation.

Transposon mutagenesis systems are still under development in bifidobacteria, partly because intrinsic active insertion sequences are not well characterized in bifidobacteria. Here, we isolated an active insertion sequence, ISBlo11, from Bifidobacterium longum 105-A using a sacB-based counterselection system, which is generally used to screen for active insertion sequences from bacterial genomes. ISBlo11 is 1432 bp long and belongs to the IS3 family. It has a single ORF encoding a transposase and 25-bp inverted repeats at its termini. Full-length copies of ISBlo11 are specifically conserved among certain B. longum genomes and exist in different sites. Transposition analysis of an artificial ISBlo11 transposon using an Escherichia coli conjugation system revealed that ISBlo11 has adequate transposition activity, comparable to the reported activity of IS629, another IS3 family element initially isolated from Shigella sonnei. ISBlo11 also showed low transposition selectivity for non-conserved 3- or 4-bp target sequences. These characteristics of ISBlo11 seem suitable for the development of a new transposon mutagenesis system in bifidobacteria.

No abstract

A BSTRACT Introduction: The Japanese government has promoted policies ensuring standardized medical care across the secondary medical care areas (SMCAs); however, these efforts have not been evaluated, making the current conditions unclear. Multidimensional indicators could identify these differences; thus, this study examined the regional characteristics of the medical care provision system for 21 SMCAs in Hokkaido, Japan, and the changes from 1998 to 2018. Materials and Methods: This study evaluated the characteristics of SMCAs by principal component analysis using multidimensional data related to the medical care provision system. Factor loadings and principal component scores were calculated, with the characteristics of each SMCA visually expressed using scatter plots. Additionally, data from 1998 to 2018 were analyzed to clarify the changes in SMCAs’ characteristics. Results: The primary and secondary principal components were Medical Resources and Geographical Factors , respectively. The Medical Resources components included the number of hospitals, clinics, and doctors, and an area’s population of older adults, accounting for 65.28% of the total variance. The Geographical Factors components included the number of districts without doctors and the population and a land area of these districts, accounting for 23.20% of the variance. The accumulated proportion of variance was 88.47%. From 1998 to 2018, the area with the highest increase in Medical Resources was Sapporo, with numerous initial medical resources (−9.283 to −10.919). Discussion: Principal component analysis summarized multidimensional indicators and evaluated SMCAs in this regional assessment. This study categorized SMCAs into four quadrants based on Medical Resources and Geographical Factors . Additionally, the difference in principal component scores between 1998 and 2018 emphasized the expanding gap in the medical care provision system among the 21 SMCAs.