Wataru Hashimoto

Azotobacter is a genus of free-living and nitrogen-fixing aerobic bacteria. “ Azotobacter tropicalis” strain KBS was isolated from soil in Japan. The genome sequence of strain KBS comprised a single circular chromosome and two putative plasmids.

グリコサミノグリカン（GAG）は動物の結合組織を中心に各組織の細胞外マトリクスとして存在し、ウロン酸とアミノ糖の二糖の繰り返し配列を基本骨格とする。構成糖、糖結合様式、及び硫酸化レベルの相違により、ヒアルロン酸、コンドロイチン硫酸、ヘパラン硫酸などに分類される。一方、GAGはサプリメントや医療材料としても利用されている。常在細菌叢の中には、GAGを接着や分解の標的とする細菌種が存在する。本稿では、主に、ヒト口腔内と腸内に常在する細菌に焦点をあて、GAG存在下でのヒト常在細菌叢の動態、ならびにGAG資化機構（GAGの二糖への断片化、細胞内への取り込み、単糖への分解、及び単糖の代謝）について概説する。

Glycosaminoglycans (GAGs) occur as extracellular matrix components across animal tissues, including typical connective tissues, and comprise repeating disaccharide units forming a basic scaffold of uronate and amino sugar residues. Based on the sugar component, mode of glycoside bond, and sulfation level, GAGs are classified into hyaluronan, chondroitin sulfate, heparan sulfate, and others. Some GAGs are utilized as supplements and/or medical materials. Among human resident bacterial flora, many bacterial species target GAGs for adherence to host cells and/or degradation for bacterial growth. This minireview focuses on resident bacterial flora primarily inhabiting the oral cavity and intestine and explores the dynamics of human resident bacterial flora targeting GAGs. The review further examines bacterial assimilation mechanisms, including GAG depolymerization into disaccharides, cytoplasmic disaccharide import, degradation into monosaccharide, and subsequent monosaccharide metabolism.

The genus Curtobacterium has been frequently found in soil and plants. In this study, we isolated the bacterial strain designated KT1 from an apple fruit. The 16S rRNA gene sequence of the KT1 was identical to those of Curtobacterium flaccumfaciens and Curtobacterium allii type strains. The average nucleotide identities between the complete genomes of the strain KT1 and the two strains were 89.3% and 89.2%, and the digital DNA–DNA hybridization values were 64.2% and 60.5%, respectively, which were less than the threshold values for species delimitation. Phylogenetic analysis based on orthologs of genus Curtobacterium categorized the strain KT1 into a cluster that contained no representative or type strain genome. The strain KT1 grew at a pH range of 5.0–10.0, while C. flaccumfaciens JCM 1347 (=ATCC 6887) grew at pH 5.0–9.0. KT1 cells had abundant anteiso-heptadecanoic acid. The strain KT1 exclusively possessed a gene island containing genes encoding a CueP family metal-binding protein, heavy metal translocating P-type ATPases, and a metal-sensitive transcriptional regulator. Growth of the strain KT1 was significantly more than that of C. flaccumfaciens JCM 1347 at the concentrations of 2 µg/mL cadmium sulfate and 300 µg/mL copper sulfate, indicating that the genomic island is involved in resistance against heavy metals. IMPORTANCE: Curtobacterium strain KT1 showed low genome sequence identity with known species in the same genus and met the criteria for classification as a novel species. Further phenotypic and physiological characterization revealed notable differences from related species, including carbon source utilization, optimal growth temperature, pH tolerance, and fatty acid profile. Heavy metal resistance of the strain indicated its ability to grow in cadmium/copper-contaminating soils and host plants. Based on the polyphasic data, we propose that the KT1 ^T strain (=DSM 118677 ^T =JCM 37513 ^T ) is a novel species belonging to the genus Curtobacterium and name it as Curtobacterium pomorum sp. nov.

Glycosaminoglycans (GAGs), comprising uronic acids and amino sugars, are widely distributed in human tissues such as the intestine and oral cavity. Various bacteria colonize these tissues by assimilating GAGs. During GAG degradation, 4-deoxy- l - threo -5-hexosulose uronate (DHU) is produced. Pectin, an abundant plant component, is also degraded into DHU. DHU is metabolized in a stepwise manner by the isomerase KduI or its nonhomologous isofunctional enzyme DhuI, followed by the reductase KduD or DhuD, belonging to the same reductase–dehydrogenase family. Previous studies have found that the genes encoding isomerase and reductase ( kduI–kduD and dhuD–dhuI , respectively) are usually organized in clusters. Therefore, it was believed that the kduI–kduD and dhuD–dhuI clusters evolved independently. However, the discovery of a hybrid kduI–dhuD cluster raised questions regarding the evolution of these clusters. This study investigated the diversity of clusters through a pan-genomic phylogenetic analysis across 3,550 bacterial strains. Among 16 possible cluster structures, 10 types were involved in DHU metabolism. Bacteroidota possessed a hybrid-type kduI–dhuD cluster, while Bacillota, but not Pseudomonadota or Bacteroidota, possessed the cluster dhuD–dhuI . Using public data sets from the human fecal microbiome and environmental habitats, we detected the prevalence of kduI–dhuD and dhuD–dhuI clusters in gut microbes. Although DHU is generated from oligomerized GAG degradation by unsaturated glucuronyl hydrolase (UGL), the UGL gene was frequently found in pathogenic strains containing kduD–kduI , dhuD–dhuI , kduI–dhuD , or dhuD–kduI , indicating that the acquisition of these clusters is advantageous for human colonization. IMPORTANCE: Glycosaminoglycans (GAGs), crucial components of the extracellular matrix, play vital roles in host infection by pathogenic bacteria and host colonization by commensal bacteria. The dhuD–dhuI cluster is well conserved within certain phyla, and it appears to have a strong association with GAG metabolism. In contrast, kduI -containing clusters are more widely distributed across bacterial species. Based on the possession ratios of genes encoding the enzymes involved in the production of 4-deoxy- l - threo -5-hexosulose uronate, this study indicates that the substrates differ depending on the specific cluster type.

Brown algae are promising sources of blue carbon for biofuel production. The nonmotile Sphingomonas sp. strain A1 imports alginate, a major component of brown algae, by alginate-binding proteins AlgQ1/Q2 and an ATP-binding cassette transporter. Previously, we showed that metabolically engineered ethanol-fermentative strains A1 (A1-EPv14 and A1-EPv104) produced bioethanol from alginate and that repeated subculturing of strain A1 on soft agar plates inducibly activated motility, resulting in expression of chemotaxis toward alginate. This study examined the possibility of bioethanol production directly from brown algae, and what triggers chemotaxis and how chemotaxis affects ethanol yield. Strain A1-EPv104 produced ethanol from brown algae as well as alginate. Gene disruption and complementation revealed that AlgQ2, not AlgQ1, was involved in alginate chemotaxis. Compared with nonmotile strain A1-EPv14, strain A1-EPv14c exhibiting alginate chemotaxis produced ethanol from alginate more rapidly. This is the first report on chemotaxis-associated biotechnology for the rapid production of bioethanol from blue carbon alginate.

Yeast obtained from the water containing raisins has been conventionally used for breadmaking. It is assumed that people consumed naturally fermented raisin water as wine in ancient times. In this study, we demonstrated spontaneous fermentation by yeast that colonizes raisins. Raisin water samples containing Saccharomyces and non-Saccharomyces species underwent alcohol fermentation. As the fermentation progressed, the diversity and abundance of Aspergillus decreased. A significant difference in β diversity was detected between the microbiomes of raw grapes and raisins, with raisins containing a higher ratio of Saccharomycetaceae. The yeast colonizing raisin fermented alcohol more efficiently than those on raw grapes. Outdoor-dried raisins harbored a distinct eukaryotic microbiome that successfully fermented alcohol, indicating that these yeasts migrate from the environment. Therefore, our data suggest that sun-drying grapes can be utilized for food preservation and making alcoholic beverages, validating the ancient practice of wine-making from raisins due to spontaneous fermentation.

Alginate, a heteropolysaccharide composed of α-L-guluronic acid (G) and β-D-mannuronic acid (M), comprises poly-G, poly-M, and mixed poly-MG regions. Alginate lyases, classified within the polysaccharide lyase (PL) family, degrade alginate into unsaturated saccharides via β-elimination. Due to the abundance of alginate in brown algae, various marine bacteria produce alginate lyases for its assimilation. Recently, alginate lyases have also been identified in gut bacteria such as those of the genus Bacteroides. In this study, we purified an alginate lyase from enrichment culture supernatants containing alginate, using a human fecal sample, and isolated B. xylanisolvens strain MK6803, which can grow on alginate as a sole carbon source-unlike the type strain B. xylanisolvens XB1A. Draft genome sequencing of strain MK6803 revealed an alginate-metabolizing gene cluster encoding three alginate lyases belonging to PL6_1, PL17_2, and PL38, along with a putative oxidoreductase. This gene cluster was shared with B. ovatus CP926 and B. xylanisolvens CL11T00C41, but not with the type strain XB1A. Bacteroides species lacking this gene cluster exhibited no alginate assimilation, even if they possessed genes encoding one or more of the three alginate lyases. This suggests that the presence of the putative oxidoreductase, alongside the lyases, is essential for alginate assimilation in Bacteroides species. Phylogenetic analysis indicated horizontal gene transfer within the genus Bacteroides. These findings highlight the role of alginate metabolism in the adaptation of human gut microbiota.

Iron is essential for bacterial survival; however, the relationship of Pseudomonas species between iron import systems and environmental adaptation remains poorly understood. Multi‐locus sequence and pan‐genome analyses using 320 Pseudomonas genomes classified the species into four groups: P. aeruginosa , P. putida , P. syringae , and P. fluorescens , each exhibiting distinct patterns of iron importer and siderophore synthetic gene cluster. The P. aeruginosa group contained fewer iron importers, whereas the P. putida group exhibited a higher prevalence of xenosiderophore importers. The Fe ²⁺ ‐importing Efe system was predominantly detected in the P. syringae and P. fluorescens groups, both of which include plant pathogens and plant growth‐promoting rhizobacteria. Reanalysis of publicly available transcriptome data revealed efeU , foxA , and fpvA were significantly upregulated in plant roots but not in insect hosts, and these genes were specifically suppressed during plant immune responses. Growth test and in planta competitive assay using P. fluorescens wild‐type and Efe system‐deficient mutant suggested that the Efe system gives an advantage under plant‐related environments. Phylogenetic analysis indicated the Pseudomonas Efe system was inherited through vertical gene transfer from a common ancestor. Our comprehensive analysis revealed the distribution of iron importers across Pseudomonas groups, highlighting the importance of these systems in environmental adaptation.

Glycosaminoglycans (GAGs), comprising uronic acids and amino sugars, are widely distributed in human tissues such as the intestine and oral cavity. Various bacteria colonize these tissues by assimilating GAGs. 4-Deoxy-L- threo -5-hexosulose uronate (DHU) is produced during the degradation of GAGs. Pectin, abundant in plants, is also degraded into DHU. DHU is metabolized in a stepwise manner by the isomerase KduI or DhuI, followed by the reductase KduD or DhuD. Previous studies have shown that the two genes encoding isomerase and reductase ( kduI-kduD and dhuD-dhuI , respectively) are designated in an operon. Therefore, it was believed that dhuD - dhuI and kduI - kduD operons evolved independently. Nevertheless, the discovery of a hybrid kduI - dhuD operon raised questions regarding the evolution of these operons. This study was conducted to investigate the factors driving the diversity of operons through a pan-genomic phylogenetic analysis across 3550 bacterial strains. Seven types of DHU metabolism-related operons were identified. The phylum Bacteroidota possesses a hybrid-type kduI - dhuD operon rather than kduI - kduD or kduD - kduI operon. The phylum Bacillota, but not Pseudomonadota or Bacteroidota, possesses the dhuD - dhuI operon; however, dhuI - dhuD operon was not detected in any bacterial strain. Although DHU is generated from the degradation of oligomerized GAG by an unsaturated glucuronyl hydrolase (UGL), the UGL gene was found in strains positive for kduD - kduI , dhuD - dhuI , kduI - dhuD , and dhuD-kduI operons at high ratios, indicating that the acquisition of these operons is advantageous for colonization on human hosts. (230 words) IMPORTANCE: Glycosaminoglycans (GAGs), crucial components of the extracellular matrix, play a vital role in host infection by pathogenic bacteria as well as in the colonization of the host by commensal bacteria. The isomerases KduI and DhuI are nonhomologous isofunctional enzymes. The dhuD - dhuI operon is well conserved within certain phyla and appears to have a strong association with GAG metabolism. In contrast, the kduI system is more widely distributed across various species. Based on the possession ratios of genes encoding each enzyme that produces 4-deoxy-L- threo -5-hexosulose uronate, this study indicated that the substrates targeted by each metabolic system vary depending on the specific operon type.