Flavobacterium longum sp. nov. and Flavobacterium urocaniciphilum sp. nov., isolated from a wastewater treatment plant, and emended descriptions of Flavobacterium caeni and Flavobacterium terrigena.

Two Gram-staining-negative, strictly aerobic, non-endospore-forming, non-motile, rod-shaped bacteria, designated strains YIT 12745T and YIT 12746T, were isolated from sludge from a wastewater treatment plant. 16S rRNA gene sequence analyses indicated that these strains belonged to the genus Flavobacterium. In these analyses, strains YIT 12745T and YIT 12746T were most closely related to the type strains of Flavobacterium caeni and Flavobacterium terrigena, with 16S rRNA gene sequence similarity values of 94.9% and 96.2%, respectively. For both novel strains, menaquinone (MK-6) was the only respiratory quinone. The major fatty acids of strain YIT 12745T were iso-C15:1 G (14.4%), iso-C16:0 (13.2%), C15:0 (12.9%), iso-C15:0 (12.9%) and iso-C17:0 3-OH (11.5%). Those of strain YIT 12746T were iso-C15:0 (21.5%), iso-C16:0 (13.3%), C15:0 (12.0%) and iso-C15:1 G (11.9%). The genomic DNA G+C contents of strains YIT 12745T and YIT 12746T were 48.7 and 30.9 mol%, respectively. From their differential phenotypic and phylogenetic characteristics, these strains are considered to represent two novel species of the genus Flavobacterium, for which the names Flavobacterium longum sp. nov. (type strain YIT 12745T=JCM 19141T=DSM 27077T) and Flavobacterium urocaniciphilum sp. nov. (type strain YIT 12746T=JCM 19142T=DSM 27078T) are proposed. Emended descriptions of Flavobacterium caeni and Flavobacterium terrigena are also proposed.

Michael addition-aromatization reaction of dienylimines bearing a leaving group and its application to the preparation of thiol-selective labeling reagents capable of forming strong carbon-sulfur bonds.

The reaction of a dienylimine with thiols was found to proceed smoothly to afford the corresponding indolines bearing aromatic carbon-sulfur bonds as a result of a Michael addition-aromatization sequence. Furthermore, this reaction was applied to the development of fluorogenic dienylimines that could be used as thiol-selective fluorescent labeling reagents.

Selective induction of human gut-associated acetogenic/butyrogenic microbiota based on specific microbial colonization of indigestible starch granules.

Prediction of individualized responses is one of biggest challenges in dietary intervention to modulate human gut microbiota. Bacterial interspecies competition for dietary factors should underlie the inter-subject heterogeneity of microbial responses. Microscale localization of bacterial species around intestinal food structures could provide direct evidence for understanding this, however, little information is currently available. Here we analyzed human fecal sections and found multiple types of bacterial colonization of food structures. The most eminent one was dense and frequent colonization of starch granules by Bifidobacterium adolescentis. After intake of raw potato starch (pSt), B. adolescentis dramatically increased in every carrier of the species, accompanied by an increase in bifidobacterial metabolite acetate. In the other subjects, Eubacterium rectale and its metabolite butyrate increased, but it was suppressed in B. adolescentis carriers. A correlation analysis indicated the contribution of these species to respective metabolites. In vitro analyses of isolates of major gut bacterial species confirmed that these species are major colonizers of pSt and that B. adolescentis can colonize pSt even in the presence of the known starch granule-degrading bacterium Ruminococcus bromii. Collectively, we propose that specific binding of B. adolescentis or E. rectale to pSt selectively induces acetogenic or butyrogenic response of gut microbiota, where the former determines the response of the latter.

Spatial distribution of live gut microbiota and bile acid metabolism in various parts of human large intestine.

Gut microbiomics is based on analysis of both live and dead cells in the stool. However, to understand the ecology of gut microbiota and their symbiotic relationships with hosts, spatial distribution of live bacteria must be examined. Here, we analyzed the live composition of luminal microbiota (LM) and mucosa-associated microbiota (MAM) in the ascending and descending colons and the rectums of 10 healthy adults and compared it with the total composition. The abundance of Lachnospiraceae in live LM decreased along the gut length and was significantly lower than that in total LM. Contrastingly, the abundance of Bacteroidaceae and Bifidobacteriaceae in live LM was higher than that in total LM, suggesting differences in death rate during gut migration. Live Enterobacteriaceae levels in MAM were significantly higher in rectum than in the ascending and descending colons and in LM. High-performance liquid chromatographic analysis of luminal bile acids revealed that 7α-dehydroxylation occurred towards the rectum. In live LM where a bile acid-inducible gene could be detected, 7α-dehydroxylation rates were higher than those in the group without the gene. Overall, we showed differences in live bacteria composition among three gut sites and between LM and MAM, highlighting the importance of understanding their spatial distribution.

Critical roles of a housekeeping sortase of probiotic Bifidobacterium bifidum in bacterium-host cell crosstalk.

Bifidobacterium bifidum YIT 10347 (BF-1) is adhesive in vitro. Here we studied the molecular aspects of the BF-1 adhesion process. We identified and characterized non-adhesive mutants and found that a class E housekeeping sortase was critical for the adhesion to mucin. These mutants were significantly less adhesive to GCIY cells than was the wild type (WT), which protected GCIY cells against acid treatment more than did a non-adhesive mutant. The non-adhesive mutants aberrantly accumulated precursors of putative sortase-dependent proteins (SDPs). Recombinant SDPs bound to mucin. Disruption of the housekeeping sortase influenced expression of SDPs and pilus components. Mutants defective in a pilin or in an SDP showed the same adhesion properties as WT. Therefore, multiple SDPs and pili seem to work cooperatively to achieve adhesion, and the housekeeping sortase is responsible for cell wall anchoring of its substrates to ensure their proper biological function.

Identification of active denitrifiers in rice paddy soil by DNA- and RNA-based analyses.

Denitrification occurs markedly in rice paddy fields; however, few microbes that are actively involved in denitrification in these environments have been identified. In this study, we used a laboratory soil microcosm system in which denitrification activity was enhanced. DNA and RNA were extracted from soil at six time points after enhancing denitrification activity, and quantitative PCR and clone library analyses were performed targeting the 16S rRNA gene and denitrification functional genes (nirS, nirK and nosZ) to clarify which microbes are actively involved in denitrification in rice paddy soil. Based on the quantitative PCR results, transcription levels of the functional genes agreed with the denitrification activity, although gene abundance did not change at the DNA level. Diverse denitrifiers were detected in clone library analysis, but comparative analysis suggested that only some of the putative denitrifiers, especially those belonging to the orders Neisseriales, Rhodocyclales and Burkholderiales, were actively involved in denitrification in rice paddy soil.