<Emphasis Type="Italic">Alkalimonas amylolytica</Emphasis> gen. nov., sp. nov., and<Emphasis Type="Italic"> Alkalimonas delamerensis</Emphasis> gen. nov., sp. nov., novel alkaliphilic bacteria from soda lakes in China and East Africa

Two related novel alkaliphilic and slightly halophilic bacteria are described. They are strain N10 from Lake Chahannor in China and strain 1E1 from Lake Elmenteita in East Africa. Both strains are strictly aerobic, heterotrophic, alkaliphilic, mesophilic, and require NaCl for growth. The optimal conditions for growth were at pH 10–10.5 and 2–3% (w/v) NaCl. Cells of both strains were Gram-negative, rod-shaped, non-spore-forming, and motile with a single polar flagellum. Cellular fatty acids in both strains were predominantly saturated and mono-unsaturated straight-chain fatty acids (16:0, 16:17c and 18:17c). The major isoprenoid quinone of both strains was Q8. The major polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylglycerol phosphate and phosphatidylethanolamine. The guanine plus cytosine (G+C) content of the DNA was 52.5 mol% and 55.4 mol%, respectively. Phylogenetic analysis revealed that the two strains formed a distinct lineage within the gamma-3 subclass of the Proteobacteria. The strains shared a 16S rDNA sequence similarity of 96.1% and showed less than 93.7% of sequence similarity to any other known species. Based on polyphasic data, the two strains were differentiated from currently recognized genera and represent a new genus, Alkalimonas gen. nov., with two species, Alkalimonas amylolytica sp. nov. (type strain is N10^T = AS 1.3430) and Alkalimonas delamerensis sp. nov. ( type strain is 1E1^{P, T} = CBS 391.94). The GenBank accession numbers for the 16S rRNA gene sequence of strains N10 and 1E1 are AF250323 and X92130, respectively.Communicated by K. Horikoshi     

Characterization and high-quality draft genome sequence of Herbivorax saccincola A7, an anaerobic,alkaliphilic, thermophilic,cellulolytic, and xylanolytic bacterium

An anaerobic, cellulolytic-xylanolytic bacterium, designated strain A7, was isolated from a cellulose-degrading bacterial community inhabiting bovine manure compost on Ishigaki Island, Japan, by enrichment culture using unpretreated corn stover as the sole carbon source. The strain was Gram-positive, non-endospore forming, non-motile, and formed orange colonies on solid medium. Strain A7 was identified as Herbivorax saccincola by DNA-DNA hybridization, and phylogenetic analysis based on 16S rRNA gene sequences showed that it was closely related to H. saccincola GGR1 (= DSM 101079^T). H. saccincola A7 (= JCM 31827 = DSM 104321) had quite similar phenotypic characteristics to those of strain GGR1. However, the optimum growth of A7 was at alkaline pH (9.0) and 55 °C, compared to pH 7.0 at 60 °C for GGR1, and the fatty acid profile of A7 contained 1.7-times more C_17:0 iso than GGR1. The draft genome sequence revealed that H. saccincola A7 possessed a cellulosome-like extracellular macromolecular complex, which has also been found for Clostridium thermocellum and C. clariflavum. H. saccincola A7 contained more glycoside hydrolases (GHs) belonging to GH families-11 and -2, and more diversity of xylanolytic enzymes, than C. thermocellum and C. clariflavum. H. saccincola A7 could grow on xylan because it encoded essential genes for xylose metabolism, such as a xylose transporter, xylose isomerase, xylulokinase, and ribulose-phosphate 3-epimerase, which are absent from C. thermocellum. These results indicated that H. saccincola A7 has great potential as a microorganism that can effectively degrade lignocellulosic biomass.     

Anaerobic,alkaliphilic, saccharolytic bacterium <Emphasis Type="Italic">Alkalibacter saccharofermentans</Emphasis> gen. nov., sp. nov. from a soda lake in the Transbaikal region of Russia

Three strains of new obligately anaerobic alkaliphilic bacteria have been isolated as a saccharolytic component from the cellulolytic community of alkaline Lake Nizhnee Beloe (Transbaikal region, Russia), a lake with low salt concentration. DNA analysis of these strains showed an interspecies level of DNA similarity of 96–100%. Strain Z-79820 was selected for further investigations. Cells were Gram-positive, asporogenous, nonmotile short rods with pointed ends. The strain was a true alkaliphile: growth occurred from pH 7.2 to 10.2 with the optimum at pH 9.0. Strain Z-79820 was halotolerant and could grow in medium with up to 10% (w/v) NaCl, with the optimum between 0 and 4% NaCl. The new isolate obligately depended on Na⁺ ions in the form of carbonates or chlorides. Total Na⁺ content needed for optimal growth was 0.46 M Na⁺, with a wide range from 0.023–0.9 M Na⁺ at which growth also occurred. The isolate was a mesophile and grew at temperatures from 6 to 50°C (slow growth at 6 and 15°C) with an optimum at 35°C. The organotrophic organism fermented ribose, xylose, glucose, mannose, fructose, sucrose, mannitol, and peptone. The products of glucose fermentation were acetate, ethanol, formate, H₂, and CO₂. Yeast extract was required for some anabolic needs. The DNA G+C content of the type strain Z-79820 was 42.1 mol%. The new bacterium fell into the 16S rRNA gene cluster XV of the Gram-positive bacteria with low G+C content, where it formed an individual branch. Based on its growth characteristics and genotype traits, we propose the new genus and species named Alkalibacter saccharofermentans with the type strain Z-79820 (=DSM14828), Uniqem-218 (Institute Microbiology, RAS; ).     

Characterization and gene cloning of a novel β-mannanase from alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. N16-5

An alkaline -mannanase was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. N16-5. The enzyme had optimum activity at pH 9.5 and 70°C. It was composed of a single polypeptide chain with a molecular weight of 55 kDa deduced from SDS-PAGE, and its isoelectric point was around pH 4.3. The enzyme efficiently hydrolyzed galactomannan and glucomannan, producing a series of oligosaccharides and monosaccharides. The -mannanase gene (manA) contained an open reading frame (ORF) of 1,479 bp, encoding a 32-amino acids signal peptide, and a mature protein of 461 amino acids, with a calculated molecular mass of 50,743 Da. Strain N16-5 ManA, deduced from the manA ORF, exhibited relatively high amino acid similarity to the members of the glycosyl hydrolase family 5. The eight conserved active-site amino acids in family 5 glycosyl hydrolase were found in the deduced amino acid sequence of strain N16-5 ManA.     

An improved physical and genetic map of the genome of alkaliphilic Bacillus sp. C-125

Among alkaliphilic bacteria reported so far, Bacillus sp. C-125 is the strain most thoroughly characterized physiologically, biochemically, and genetically. A physical map of the chromosome of this strain was constructed to facilitate further genome analysis, and the genome size was revised from 3.7 to 4.25 Mb. Complete digestion of the chromosomal DNA with two rare cut restriction endonucleases, AscI and Sse8387I, each yielded 20 fragments ranging in size from 20 to 600 kb. Seventeen linking clones were isolated in each instance to join the adjacent AscI or Sse8387I fragments in the chromosomal map. All AscI linking clones isolated were sequenced and analyzed by comparison with the BSORF database to map the genes in the chromosome of strain C-125. Several ORFs showing significant similarities to those of B. subtilis in the AscI linking clones were positioned on the physical map. The oriC region of the C-125 chromosome was identified by southern blot analysis with a DNA probe containing the gyrB region. Received: May 6, 1998 / Accepted: May 26, 1998     

Sucrose transport by the alkaliphilic, thermophilic Bacillus sp. strain TA2.A1 is dependent on a sodium gradient

An alkaliphilic Bacillus designated strain TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70°C. Sodium chloride (>5 mM) was an obligate requirement for the growth of strain TA2.A1 on sucrose, and growth on sucrose was inhibited by monensin, an ionophore that collapses the sodium gradient (ΔpNa⁺) across the cell membrane. Sucrose transport by strain TA2.A1 was sodium dependent and was inhibited by monensin. The K_t for sucrose tran-sport was 33 μM and the Eadie–Hofstee plot was linear, suggesting one high-affinity uptake system for sucrose. The affinity for sodium was low (0.5 mM), and the Hill plot had a slope of 1.6, suggesting that sodium binding was noncooperative and that the sucrose transporter had more than one binding site for sodium. Based on these results, Bacillus strain TA2.A1 uses a sodium gradient for sucrose uptake, in addition to the sodium-dependent glutamate uptake system reported previously. Received: March 15, 2000 / Accepted: July 17, 2000     

一株产蛋白酶嗜碱菌株的分离、鉴定及酶学特性

【目的】筛选产蛋白酶嗜碱菌并对其进行鉴定和特性分析。【方法】利用碱性脱脂牛奶培养基分离纯化产蛋白酶嗜碱菌,通过形态特征、生理生化、16S rRNA基因序列分析以及DNA-DNA杂交实验确定菌株的分类地位,利用酪蛋白水解法分析所产蛋白酶的pH和温度作用范围、稳定性和耐氧化剂能力。【结果】从我国西藏盐碱湖样品中分离到一株产碱性蛋白酶的菌株ZL223,该菌株为革兰氏阳性菌,最适生长温度为37℃,最适生长pH9.0,16S rRNA基因序列分析显示,菌株ZL223与假强芽孢杆菌Bacillus pseudo firmus OF4亲缘关系最近,16S rRNA基因序列相似性为98.6%,DNA-DNA杂交结果显示与B.pseudofirmus OF4同源性为86%。菌株ZL223产生的蛋白酶作用的最适pH为12.0,最适温度为40℃。【结论】结合生理生化指标测定的结果,鉴定菌株为假强芽孢杆菌ZL223(B.pseudofirmus ZL223)。该菌株产生的碱性蛋白酶具有较高的pH适应性,值得进一步研究。     

Isolation and characterization of bacteriophage BCJA1, a novel temperate bacteriophage active against the alkaliphilic bacterium, Bacillus clarkii

The isolation and characterization of a novel bacteriophage active against the obligately alkaliphilic bacterium Bacillus clarkii is described. The bacteriophage, designated BCJA1, is a member of the Siphoviridae family with a B1 morphology. It possesses an isometric head, which measures 65 nm between opposite apices, and a noncontractile tail of 195 nm length. It had a buoyant density of 1.518 g/ml and an estimated particle mass of 37 × 10⁷ daltons. BCJA1 was stable over the pH range of 6–11. A one-step growth experiment conducted at pH 10 demonstrated a latent period of about 40 min and a burst size of approximately 40. The purified bacteriophage appeared to consist of 10 proteins with the major head and tail proteins likely to be of molecular weight 36 500 and 28 000, respectively. The genome size was estimated to be between 32.1 and 34.8 kb. The percent G + C content of purified bacteriophage DNA was 45.6. The wildtype bacteriophage is temperate but a clear plaque mutant was isolated. Received: May 25, 1997 / Accepted: August 5, 1997     

Evidence for a Na+ / H+ electrogenic antiporter in an alkaliphilic cyanobacterium Synechocystis

Abstract An alkaliphilic cyanobacterium characterized as a Synechocystis species was purified from a soil sample taken from a village in Java, Indonesia, by its preferential growth at elevated pH; it grew optimally at pH 9.5. Phosphorus nuclear magnetic resonance studies showed that the organism can maintain a ΔpH of over 2 pH units at an external pH of 10. It was observed that the viability of the organism in the dark was dependent on sodium ions. Evidence from experiments in which the extrusion of Na⁺ was measured from cells subjected to an alkali shock suggests that the organism possesses a Na⁺ / H⁺ electrogenic antiporter which is used for the maintenance of pH homeostasis.     

Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens

An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from␣seawater and identified here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence identity to Cel5A of B. agaradhaerens DSM8721^T. The enzyme activity increased around 4-fold by the addition of 0.2–2.0 M NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). KCl, Na₂SO₄, NaBr, NaNO₃, CH₃COONa, LiCl, NH₄NO₃, and NH₄Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7–9.4 and 60 °C with 0.2 M NaCl, but pH 6.5–7 and 50 °C without NaCl; enzyme activity increased approximately 6-fold at 60 °C with 0.2 M NaCl compared to that at 50 °C without NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe²⁺ and Hg²⁺ ions), regardless whether NaCl was present or not. * The nucleotide sequence of 16S rRNA of this strain has been submitted to DDBJ, EMBL, and GenBank databases under accession no. AB211544.