Gut bacterial aromatic amine production: aromatic amino acid decarboxylase and its effects on peripheral serotonin production.

Colonic luminal aromatic amines have been historically considered to be derived from dietary source, especially fermented foods; however, recent studies indicate that the gut microbiota serves as an alternative source of these amines. Herein, we show that five prominent genera of Firmicutes (Blautia, Clostridium, Enterococcus, Ruminococcus, and Tyzzerella) have the ability to abundantly produce aromatic amines through the action of aromatic amino acid decarboxylase (AADC). In vitro cultivation of human fecal samples revealed that a significant positive correlation between aadc copy number of Ruminococcus gnavus and phenylethylamine (PEA) production. Furthermore, using genetically engineered Enterococcus faecalis-colonized BALB/cCrSlc mouse model, we showed that the gut bacterial aadc stimulates the production of colonic serotonin, which is reportedly involved in osteoporosis and irritable bowel syndrome. Finally, we showed that human AADC inhibitors carbidopa and benserazide inhibit PEA production in En. faecalis.

Putrescine Production by Latilactobacillus curvatus KP 3-4 Isolated from Fermented Foods.

Polyamines are aliphatic hydrocarbons with terminal amino groups and are essential for biological activities. It has been reported that polyamines have health-promoting effects in animals, such as the extension of lifespan by polyamine intake. The identification of a high polyamine-producing bacterium from foods could lead to the development of a novel probiotic candidate. We aimed to identify high polyamine-producing bacteria from food, and isolated and collected bacteria from vegetables and fermented foods produced in Japan. We successfully acquired Latilactobacillus curvatus KP 3-4 isolated from Kabura-zushi as a putrescine producing lactic acid bacteria. Comparing the polyamine synthesis capability of L. curvatus KP 3-4 with that of typical probiotic lactic acid bacteria and L. curvatus strains available from the Japan Collection of Microorganisms, it was found that only L. curvatus KP 3-4 was capable of exporting high levels of putrescine into the culture supernatant. The enhancement of putrescine production by the addition of ornithine, and whole-genome analysis of L. curvatus KP 3-4, suggest that putrescine is synthesized via ornithine decarboxylase. The administration of L. curvatus KP 3-4 to germ-free mice increased the concentration of putrescine in the feces.

Next-generation prebiotic promotes selective growth of bifidobacteria, suppressing Clostridioides difficile.

Certain existing prebiotics meant to facilitate the growth of beneficial bacteria in the intestine also promote the growth of other prominent bacteria. Therefore, the growth-promoting effects of ß-galactosides on intestinal bacteria were analyzed. Galactosyl-ß1,4-l-rhamnose (Gal-ß1,4-Rha) selectively promoted the growth of Bifidobacterium. Bifidobacterium longum subsp. longum 105-A (JCM 31944) has multiple solute-binding proteins belonging to ATP-binding cassette transporters for sugars. Each strain in the library of 11 B. longum subsp. longum mutants, in which each gene of the solute-binding protein was disrupted, was cultured in a medium containing Gal-ß1,4-Rha as the sole carbon source, and only the BL105A_0502 gene-disruption mutant showed delayed and reduced growth compared to the wild-type strain. BL105A_0502 homolog is highly conserved in bifidobacteria. In a Gal-ß1,4-Rha-containing medium, Bifidobacterium longum subsp. infantis JCM 1222T, which possesses BLIJ_2090, a homologous protein to BL105A_0502, suppressed the growth of enteric pathogen Clostridioides difficile, whereas the BLIJ_2090 gene-disrupted mutant did not. In vivo, administration of B. infantis and Gal-ß1,4-Rha alleviated C. difficile infection-related weight loss in mice. We have successfully screened Gal-ß1,4-Rha as a next-generation prebiotic candidate that specifically promotes the growth of beneficial bacteria without promoting the growth of prominent bacteria and pathogens.

Analysis of polyamine biosynthetic- and transport ability of human indigenous Bifidobacterium.

Bifidobacteria are members of the human intestinal microbiota, being numerically dominant in the colon of infants, and also being prevalent in the large intestine of adults. In this study, we measured the concentrations of major polyamines (putrescine, spermidine, and spermine) in cells and culture supernatant of 13 species of human indigenous Bifidobacterium at growing and stationary phase. Except for Bifidobacterium bifidum and Bifidobacterium gallicum, 11 species contained spermidine and/or spermine when grown in Gifu-anaerobic medium (GAM). However, Bifidobacterium scardovii and Bifidobacterium longum subsp. infantis, which contain spermidine when grown in GAM, did not contain spermidine when grown in polyamine-free 199 medium. Of the tested 13 Bifidobacterium species, 10 species showed polyamine transport ability. Combining polyamine concentration analysis in culture supernatant and in cells, with basic local alignment search tool analysis suggested that novel polyamine transporters are present in human indigenous Bifidobacterium. ABBREVIATIONS: Put: putrescine; Spd: spermidine; Spm: spermine; GAM: Gifu anaerobic medium; BHI: brain-heart infusion.

Functional analysis of arginine decarboxylase gene speA of Bacteroides dorei by markerless gene deletion.

Polyamine concentrations in the intestine are regulated by their biosynthesis by hundreds of gut microbial species and these polyamines are involved in host health and disease. However, polyamine biosynthesis has not been sufficiently analyzed in major members of the human gut microbiota, possibly owing to a lack of gene manipulation systems. In this study, we successfully performed markerless gene deletion in Bacteroides dorei, one of the major members of the human gut microbiota. The combination of a thymidine kinase gene (tdk) deletion mutant and a counter-selection marker tdk, which has been applied in other Bacteroides species, was used for the markerless gene deletion. Deletion of tdk in B. dorei caused 5-fluoro-2΄-deoxyuridine resistance, suggesting the utility of B. dorei Δtdk as the host for future markerless gene deletions. Compared to parental strains, an arginine decarboxylase gene (speA) deletion mutant generated in this system showed a severe growth defect and decreased concentration of spermidine in the cells and culture supernatant. Collectively, our results indicate the accessibility of gene deletion and the important role of speA in polyamine biosynthesis in B. dorei.

Comprehensive analysis of polyamine transport and biosynthesis in the dominant human gut bacteria: Potential presence of novel polyamine metabolism and transport genes.

Recent studies have reported that polyamines in the colonic lumen might affect animal health and these polyamines are thought to be produced by gut bacteria. In the present study, we measured the concentrations of three polyamines (putrescine, spermidine, and spermine) in cells and culture supernatants of 32 dominant human gut bacterial species in their growing and stationary phases. Combining polyamine concentration analysis in culture supernatant and cells with available genomic information showed that novel polyamine biosynthetic proteins and transporters were present in dominant human gut bacteria. Based on these findings, we suggested strategies for optimizing polyamine concentrations in the human colonic lumen via regulation of genes responsible for polyamine biosynthesis and transport in the dominant human gut bacteria.

Use of Gifu Anaerobic Medium for culturing 32 dominant species of human gut microbes and its evaluation based on short-chain fatty acids fermentation profiles.

Recently, a "human gut microbial gene catalogue," which ranks the dominance of microbe genus/species in human fecal samples, was published. Most of the bacteria ranked in the catalog are currently publicly available; however, the growth media recommended by the distributors vary among species, hampering physiological comparisons among the bacteria. To address this problem, we evaluated Gifu anaerobic medium (GAM) as a standard medium. Forty-four publicly available species of the top 56 species listed in the "human gut microbial gene catalogue" were cultured in GAM, and out of these, 32 (72%) were successfully cultured. Short-chain fatty acids from the bacterial culture supernatants were then quantified, and bacterial metabolic pathways were predicted based on in silico genomic sequence analysis. Our system provides a useful platform for assessing growth properties and analyzing metabolites of dominant human gut bacteria grown in GAM and supplemented with compounds of interest.