Bacteria with a mouth: Discovery and new insights into cell surface structure and macromolecule transport

A bacterium with a "mouth"-like pit structure isolated for the first time in the history of microbiology was a Gram-negative rod, containing glycosphingolipids in the cell envelope, and named Sphingomonas sp. strain A1. The pit was dynamic, with repetitive opening and closing during growth on alginate, and directly included alginate concentrated around the pit, particularly by flagellins, an alginate-binding protein localized on the cell surface. Alginate incorporated into the periplasm was subsequently transferred to the cytoplasm by cooperative interactions of periplasmic solute-binding proteins and an ATP-binding cassette transporter in the cytoplasmic membrane. The mechanisms of assembly, functions, and interactions between the above-mentioned molecules were clarified using structural biology. The pit was transplanted into other strains of sphingomonads, and the pitted recombinant cells were effectively applied to the production of bioethanol, bioremediation for dioxin removal, and other tasks. Studies of the function of the pit shed light on the biological significance of cell surface structures and macromolecule transport in bacteria

Conferring the ability to utilize inorganic polyphosphate on ATP-specific NAD kinase

ATP特異性の獲得メカニズムの解明 : 新薬と新しい物質生産系の開発に期待. 京都大学プレスリリース. 2013-09-11.NAD kinase (NADK) is a crucial enzyme for production of NADP(+). ATP-specific NADK prefers ATP to inorganic polyphosphate [poly(P)] as a phosphoryl donor, whereas poly(P)/ATP-NADK utilizes both ATP and poly(P), and is employed in industrial mass production of NADP(+). Poly(P)/ATP-NADKs are distributed throughout Gram-positive bacteria and Archaea, whereas ATP-specific NADKs are found in Gram-negative α- and γ-proteobacteria and eukaryotes. In this study, we succeeded in conferring the ability to utilize poly(P) on γ-proteobacterial ATP-specific NADKs through a single amino-acid substitution; the substituted amino-acid residue is therefore important in determining the phosphoryl-donor specificity of γ-proteobacterial NADKs. We also demonstrate that a poly(P)/ATP-NADK created through this method is suitable for the poly(P)-dependent mass production of NADP(+). Moreover, based on our results, we provide insight into the evolution of bacterial NADKs, in particular, how NADKs evolved from poly(P)/ATP-NADKs into ATP-specific NADKs

Regulation of pH attenuates toxicity of a byproduct produced by an ethanologenic strain of Sphingomonas sp. A1 during ethanol fermentation from alginate

Marine macroalgae is a promising carbon source that contains alginate and mannitol as major carbohydrates. A bioengineered ethanologenic strain of the bacterium Sphingomonas sp. A1 can produce ethanol from alginate, but not mannitol, whereas the yeast Saccharomyces paradoxus NBRC 0259–3 can produce ethanol from mannitol, but not alginate. Thus, one practical approach for converting both alginate and mannitol into ethanol would involve two-step fermentation, in which the ethanologenic bacterium initially converts alginate into ethanol, and then the yeast produces ethanol from mannitol. In this study, we found that, during fermentation from alginate, the ethanologenic bacterium lost viability and secreted toxic byproducts into the medium. These toxic byproducts inhibited bacterial growth and killed bacterial cells and also inhibited growth of S. paradoxus NBRC 0259–3. We discovered that adjusting the pH of the culture supernatant or the culture medium containing the toxic byproducts to 6.0 attenuated the toxicity toward both bacteria and yeast, and also extended the period of viability of the bacterium. Although continuous adjustment of pH to 6.0 failed to improve the ethanol productivity of this ethanologenic bacterium, this pH adjustment worked very well in the two-step fermentation due to the attenuation of toxicity toward S. paradoxus NBRC 0259–3. These findings provide information critical for establishment of a practical system for ethanol production from brown macroalgae

A novel bleb-dependent polysaccharide export system in nitrogen-fixing Azotobacter vinelandii subjected to low nitrogen gas levels

The alginate biofilm-producing bacterium Azotobacter vinelandii aerobically fixes nitrogen by oxygensensitivenitrogenases. Here we investigated the bacterial response to nitrogen/oxygen gas mixtures. A. vinelandii cellswere cultured in nitrogen-free minimal media containing gas mixtures differing in their ratios of nitrogen and oxygen.The bacteria did not grow at oxygen concentrations >75 % but grew well in the presence of 5 % nitrogen/25 % oxygen.Growth of wild-type and alginate-deficient strains when cultured with 50 % oxygen did not differ substantially, indicatingthat alginate is not required for the protection of nitrogenases from oxygen damage. In response to decreasing nitrogenlevels, A. vinelandii produced greater amounts of alginate, accompanied by the formation of blebs on the cell surface. Theencystment of vegetative cells occurred in tandem with the release of blebs and the development of a multilayered exine.Immunoelectron microscopy using anti alginate-antibody revealed that the blebs contained alginate molecules. Bycontrast, alginate-deficient mutants could not form blebs. Taken together, our data provide evidence for a novel blebdependentpolysaccharide export system in A. vinelandii that is activated in response to low nitrogen gas levels. [IntMicrobiol 2013; 16(1):35-44

Hydration of vinyl ether groups by unsaturated glycoside hydrolases and their role in bacterial pathogenesis

Many pathogenic microorganisms invade mammalian and/or plant cells by producing polysaccharide-degrading enzymes (lyases and hydrolases). Mammalian glycosaminoglycans and plant pectins that form part of the cell surface matrix are typical targets for these microbial enzymes. Unsaturated glycoside hydrolase catalyzes the hydrolytic release of an unsaturated uronic acid from oligosaccharides, which are produced through the reaction of matrix-degrading polysaccharide lyase. This enzymatic ability suggests that unsaturated glycoside hydrolases function as virulence factors in microbial infection. This review focuses on the molecular identification, bacterial distribution, and structure/function relationships of these enzymes. In contrast to general glycoside hydrolases, in which the catalytic mechanism involves the retention or inversion of an anomeric configuration, unsaturated glycoside hydrolases uniquely trigger the hydrolysis of vinyl ether groups in unsaturated saccharides but not of their glycosidic bonds. [Int Microbiol 2007; 10(4):233-243

マイクロサテライト ブンセキ ニ モトヅク ニホン ノ エミュー シヨウ シュウダン ニ オケル イデンテキ タヨウド

エミュー（Dromaius novaehollandiae）は食肉，卵およびオイルを生産する新規動物資源となることが期待されている。しかしながら，エミュー産業の歴史は浅く，その生産形質の遺伝的改良はほとんど進んでいない。我々は，日本で最大規模となる北海道網走市のエミュー牧場の個体群を対象としてマイクロサテライト解析に基づく遺伝的多様度を経年的に調査した。検出されたアレルの数（NA）は2013，2014，2015および2016年でそれぞれ4.83，4.17，4.17および7.17であり，ヘテロ接合率（HE/HO）はそれぞれ0.466/0.339，0.426/0.325，0.433/0.384および0.550/0.347であった。近交係数（FIS）は調査したすべての世代において正の値を示し，2016年に孵化した個体では0.369と最も高い値が観察された。Structureプログラムを用いた解析では，本集団は3つのクラスターに分かれ，2016年に孵化した個体群は明らかに他の世代とは異なる遺伝的構成を示した。またアレル共有率に基づく系統樹は5つのクレードを示し，2016年に孵化した個体の約半数は一つのクレードに属した。本研究は，網走市のエミュー集団は遺伝的多様度が低いこと，遺伝的に3－5の異なる系統から構成されること，ならびに2016年に孵化した個体の遺伝的構成が他の世代とは異なることを確認した。The emu (Dromaius novaehollandiae) is predicted to be a new livestock animal for oil, meat and egg production. However, the genetic structure of emu populations in Japanese farms is scarcely known. The aim of this study was to determine the genetic diversity and population structure in the largest emu farm in Japan. We collected feather pulps of emu chicks (N＝131) from 40, 20, 23, and 48 individuals hatched at 2013, 2014, 2015, and 2016, respectively, in the Okhotsk Emu farm in Abashiri, Hokkaido, Japan. Using six microsatellite markers, we investigated the genetic diversity and structure of this farmed emu population. The number of alleles (NA) were 4.83, 4.17, 4.17, and 7.17, in individuals hatched in 2013, 2014, 2015, and 2016, respectively. Expected and observed heterozygosity (HE ; HO, respectively) was 0.466/0.339, 0.426/0.325, 0.433/0.384, and 0.550/0.347, in each year, respectively. A high inbreeding coefficient (FIS) was observed in all tested generations (0.113-0.369). The Structure program and unrooted phylogenetic tree analysis showed that the Abashiri emu population is largely divided into three to five different clades. Our results suggested that the genetic diversity in the Abashiri emu population is low, and that it contains three to five genetic lineages. These data may help guide a more sustainable breeding of emus in Japan

Biofuel production based on carbohydrates from both brown and red macroalgae: Recent developments in key biotechnologies.

Marine macroalgae (green, red and brown macroalgae) have attracted attention as an alternative source of renewable biomass for producing both fuels and chemicals due to their high content of suitable carbohydrates and to their advantages over terrestrial biomass. However, except for green macroalgae, which contain relatively easily-fermentable glucans as their major carbohydrates, practical utilization of red and brown macroalgae has been regarded as difficult due to the major carbohydrates (alginate and mannitol of brown macroalgae and 3, 6-anhydro-L-galactose of red macroalgae) not being easily fermentable. Recently, several key biotechnologies using microbes have been developed enabling utilization of these brown and red macroalgal carbohydrates as carbon sources for the production of fuels (ethanol). In this review, we focus on these recent developments with emphasis on microbiological biotechnologies

ビセイブツ エネルギー オ モチイル グルタチオン ノ セイサン

京都大学0048新制・論文博士農学博士乙第4635号論農博第967号新制||農||346(附属図書館)学位論文||S57||N1313(農学部図書室)UT51-57-E581(主査)教授 木村 光, 教授 栃倉 辰六郎, 教授 上久保 正学位規則第5条第2項該当Kyoto UniversityDFA

Structural and mutational analysis of amino acid residues involved in ATP specificity of Escherichia coli acetate kinase.

Acetate kinase (AK) generally utilizes ATP as a phosphoryl donor, but AK from Entamoeba histolytica (PPi-ehiAK) uses pyrophosphate (PPi), not ATP, and is PPi-specific. The determinants of the phosphoryl donor specificity are unknown. Here, we inferred 5 candidate amino acid residues associated with this specificity, based on structural information. Each candidate residue in Escherichia coli ATP-specific AK (ATP-ecoAK), which is unable to use PPi, was substituted with the respective PPi-ehiAK amino acid residue. Each variant ATP-ecoAK had an increased Km for ATP, indicating that the 5 residues are the determinants for the specificity to ATP in ATP-ecoAK. Moreover, Asn-337 of ATP-ecoAK was shown to be particularly significant for the specificity to ATP. The 5 residues are highly conserved in 2625 PPi-ehiAK homologs, implying that almost all organisms have ATP-dependent, rather than PPi-dependent, AK

Structural insights into alginate binding by bacterial cell-surface protein.

A gram-negative Sphingomonas sp. strain A1 inducibly forms a mouth-like pit on the cell surface in the presence of alginate and directly incorporates polymers into the cytoplasm via the pit and ABC transporter. Among the bacterial proteins involved in import of alginate, a cell-surface EfeO-like Algp7 shows an ability to bind alginate, suggesting its contribution to accumulate alginate in the pit. Here, we show identification of its positively charged cluster involved in alginate binding using X-ray crystallography, docking simulation, and site-directed mutagenesis. The tertiary structure of Algp7 was determined at a high resolution (1.99Å) by molecular replacement, although no alginates were included in the structure. Thus, an in silico model of Algp7/oligoalginate was constructed by docking simulation using atomic coordinates of Algp7 and alginate oligosaccharides, where some charged residues were found to be potential candidates for alginate binding. Site-directed mutagenesis was conducted and five purified mutants K68A, K69A, E194A, N221A, and K68A/K69A were subjected to a binding assay. UV absorption difference spectroscopy along with differential scanning fluorimetry analysis indicated that K68A/K69A exhibited a significant reduction in binding affinity with alginate than wild-type Algp7. Based on these data, Lys68/Lys69 residues of Algp7 probably play an important role in binding alginate