<Emphasis Type="Italic">Halobacillus locisalis</Emphasis> sp. nov., a halophilic bacterium isolated from a marine solar saltern of the Yellow Sea in Korea

A Gram-positive, motile, endospore-forming and rod-shaped halophilic bacterial strain MSS-155 (KCTC 3788 and KCCM 41687) was isolated from a marine solar saltern of the Yellow Sea in Korea and was subjected to a polyphasic taxonomic study. This organism grew at temperature of 10.0–42.0°C with an optimum of 35°C. Strain MSS-155 grew optimally in the presence of 10% NaCl and did not grow in the absence of NaCl. The cell wall peptidoglycan type of strain MSS-155 was A4 based on l-Orn-d-Asp. Strain MSS-155 was also characterized chemotaxonomically by having menaquinone-7 (MK-7) as the predominant isoprenoid quinone and anteiso-C_15:0 as the major fatty acid. The DNA G+C content was 44.0 mol%. Phylogenetic analysis based on 16S rDNA sequences showed that strain MSS-155 falls within the radiation of the cluster comprising Halobacillus species. Levels of 16S rDNA sequence similarity between strain MSS-155 and the type strains of four Halobacillus species were in the range 97.6–98.8%. Strain MSS-155 exhibited levels of DNA-DNA relatedness of 6.2–11.2% to the type strains of Halobacillus species described previously. On the basis of phenotypic properties, phylogeny, and genomic data, strain MSS-155 should be placed in the genus Halobacillus as a member of a novel species, for which we propose the name Halobacillus locisalis sp. nov.Communicated by W.D. Grant     

A Na+/H+ antiporter gene of the moderately halophilic bacterium Halobacillus dabanensis D-8T: cloning and molecular characterization

A gene encoding a Na(+)/H(+) antiporter was cloned from a chromosomal DNA of Halobacillus dabanensis strain D-8(T) by functional complementation. Its presence enabled the antiporter-deficient Escherichia coli strain KNabc to survive in the presence of 0.2 M NaCl or 5 mM LiCl. The gene was sequenced and designated as nhaH. The deduced amino-acid sequence of NhaH consists of 403 residues with a calculated molecular mass of 43,481 Da, which was 54% identical and 76% similar to the NhaG Na(+)/H(+) antiporter of Bacillus subtilis. The hydropathy profile was characteristic of a membrane protein with 12 putative transmembrane domains. Everted membrane vesicles prepared from E. coli cells carrying nhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with highest activities at pH 8.5-9.0 and at pH 8.5, respectively. Moreover, nhaH confers upon E. coli KNabc cells the ability to grow under alkaline conditions.     

Production,optimization and purification of a novel extracellular protease from the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus karajensis</Emphasis>

The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion occurred at 34°C, pH 8.0–8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with 68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography. The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic activity of the enzyme was observed at 50°C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still remained. The maximum enzyme activity was obtained at a broad pH range of 8.0–10.0, with 55 and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view.     

一株中度嗜盐细菌whb45的鉴定及其抗菌与抗肿瘤活性筛选

从盐场中分离鉴定中度嗜盐细菌并对其潜在的抗菌和抗肿瘤活性进行评价。从山东威海的鹿道口盐场分离嗜盐细菌,对菌株whb45进行形态学和生理生化特性研究,测定其16SrRNA序列并通过同源性比对进行系统发育分析,采用抗菌和细胞毒模型进行活性筛选。试验结果表明,菌株whb45为中度嗜盐细菌,whb45与Halobacillus trueperi在形态和生理生化特征方面最接近,16SrRNA序列相似性为99%。whb45的粗提物对多种细菌、真菌和肿瘤细胞的生长都具有较强的抑制作用,可以作为发现生物活性物质的潜在的新来源。     

新型YdjM超家族成员的钠/氢逆向转运蛋白功能鉴定

在原核生物中,钠/氢逆向转运蛋白具有催化细胞内的Na~+、Li~+或K~+等碱基阳离子的排出,换取外部质子,以降低有毒碱性金属阳离子的细胞质浓度和维持细胞内pH稳态起到了至关重要的作用。为了进一步挖掘中度嗜盐菌Halobacillus Y5中具有盐碱耐受性的钠/氢逆向转运蛋白基因并对其功能进行鉴定,我们首先提取该菌的基因组DNA,然后采用Sau3AI随机酶切及功能互补的方法获得了一个新型的钠/氢逆向转运蛋白基因Ha_ydjM。生物信息学分析表明,该基因属于YdjM超家族成员,是一个未知功能的膜蛋白,系统发育分析证实,其与来自Halobacillus sp. Marseille-P 3789的YdjM(蛋白登录号WP_101846656. 1)家族成员聚在一起但形成独立分支。研究发现,该基因能够恢复大肠杆菌突变株KNabc对0. 2mol/L NaCl和5mmol/L Li Cl的耐受特性,并且耐受碱性pH 8. 0。功能分析显示,该蛋白呈现pH依赖的钠/氢逆向转运蛋白活性,转运动力学分析表明,Na~+、K~+、Li~+在KNabc中K_m值分别是0. 43±0. 05mmol/L、0. 49±0. 06mmol/L、0. 64±0. 06mmol/L,即对Na~+、K~+、Li~+的亲和力分别是Na~+ K~+ Li~+。综上所述,Ha_ydjM代表了一种新型的钠/氢逆向转运蛋白,这丰富了YdjM超家族成员,并为其他未知膜蛋白功能分析提供依据。     

<Emphasis Type="Italic">Halobacillus naozhouensis</Emphasis> sp. nov., a moderately halophilic bacterium isolated from a sea anemone

A moderately halophilic, Gram-positive, catalase- and oxidase-positive, rod-shaped, aerobic bacterium, designated strain JSM 071068^T, was isolated from a sea anemone (Anthopleura xanthogrammica) collected from the Naozhou Island on the Leizhou Bay in the South China Sea. Cells were motile by means of peritrichous flagella and formed ellipsoidal endospores lying in subterminal swollen sporangia. Strain JSM 071068^T was able to grow with 1–20% (w/v) total salts (optimum, 6–9%), at pH values of 6.0–10.0 (optimum, pH 7.5) and a temperature range of 10–35°C (optimum, 25°C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The predominant menaquinone was MK-7 and the major cellular fatty acids were anteiso-C_15:0, anteiso-C_17:0 and iso-C_15:0. The genomic DNA G + C content was 42.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 071068^T belonged to the genus Halobacillus. The 16S rRNA gene sequence similarities between strain JSM 071068^T and the type strains of the recognized Halobacillus species ranged from 97.9% (with Halobacillus alkaliphilus) to 95.3% (with Halobacillus kuroshimensis). The levels of DNA–DNA relatedness between the new isolate and the type strains of H. alkaliphilus, Halobacillus campisalis, Halobacillus halophilus and Halobacillus seohaensis were 25.6, 22.1, 10.8 and 13.2%, respectively. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 071068^T represents a new species of the genus Halobacillus, for which the name Halobacillus naozhouensis sp. nov. is proposed, with JSM 071068^T (=DSM 21183^T =KCTC 13234^T) as the type strain. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 071068^T is EU925615.     

A primary sodium pump gene of the moderate halophile Halobacillus dabanensis exhibits secondary antiporter properties

The primary sodium pump has been proved to be involved in Na(+) extrusion of bacteria. In our present study, a novel gene encoding a putative primary sodium pump was cloned from chromosomal DNA of moderate halophile Halobacillus dabanensis D-8 by functional complementation, which expression resulted in the growth of antiporter-deficient Escherichia coli strain KNabc in the presence of 0.2 M NaCl. The gene was sequenced and designated nap. The deduced amino acid sequence of Nap has 56% identity to NADH dehydrogenase of Bacillus cereus and 55% to NADH oxidase of Bacillus halodurans C-125. E. coli KNabc carrying nap exhibited resistance to uncoupler CCCP (carbonyl-cyanide m-chlorophenylhydrazone). Everted membrane vesicles prepared from E. coli KNabc carrying nap exhibited secondary Na(+)/H(+) antiporter activity, and nap also supported the growth of respiratory-deficient E. coli ANN0222 lacking NADH dehydrogenase. Based on these results, we proposed that Nap possessed both characteristics of secondary Na(+)/H(+) antiporter and primary sodium pump.     

Incorporation of [14C]Methanol and [14C]Bicarbonate by Hyphomicrobium sp. 53-49 under Various Growth Conditions: Aerobic,Denitrifying and Autotrophic

A study was made of the incorporation of methanol and bicarbonate into the cell constituents of denitrifying or aerobic methanol grown and autotrophic H₂–O₂–CO₂ grown Hyphomicrobium sp. 53-49. Cells were incubated with [¹⁴C]methanol or [¹⁴C]bicarbonate, and the distribution of the radioactivity in the nonvolatile constituents of ethanol extracts of cells was examined. When denitrifying grown cells were incubated with [¹⁴C]methanol, the major part of the radioactivity was fixed to serine as the first stable compound. Aerobic methanol grown cells also fixed [¹⁴C]methanol mainly to serine. These results suggest that methanol grown cells assimilate methanol by the serine pathway. When denitrifying or aerobic methanol grown cells were incubated with [¹⁴C]bicarbonate, malate was mainly observed as a nonvolatile compound in the initial period of the incubation. Autotrophic grown cells also fixed the major part of [¹⁴C]bicarbonate to malate. In this case, phosphoglyceric acid was found in the phosphorylated compounds area.     

中度嗜盐菌LY9的分离鉴定及其淀粉酶特性研究

目的:从运城盐湖中分离获得中度嗜盐菌,并对其进行分离鉴定及酶学特性研究.方法:采用淀粉底物平板法获得高产胞外淀粉酶的嗜盐菌株LY9.16S rRNA序列分析,结合形态学和生理生化特征分析对其进行分类鉴定.研究不同物理化学因素对淀粉酶活性的影响.结果:系统发育分析表明该菌为Halobacillus属成员,鉴定并命名为Halobacillus sp.LY9,为中度嗜盐菌.该淀粉酶最适作用温度和pH值分别为60℃和8.0,同时在较宽pH范围内(4.0～12.0)保持高活力,表现出了较强的抗酸碱能力.Cu2+可明显抑制该酶活性,其它金属离子则基本无影响;该酶不受EDTA的抑制,表明该酶不属于金属蛋白酶,但SDS却能使酶活明显降低.结论:LY9的分离鉴定及其淀粉酶特性研究对促进运城盐湖生物资源的开发利用具有重要意义.     

中度嗜盐菌Halobacillus sp. LY5的分离、鉴定及其胞外脂肪酶特性研究

利用吐温平板筛选法,从山西运城盐湖中分离获得一株高产胞外脂肪酶的中度嗜盐菌。通过形态学观察,生理生化特征及16S rRNA序列分析,初步鉴定并命名该菌为Halobacillus sp.LY5。酶学性质研究表明,该脂肪酶可在较宽温度范围内(30℃~90℃)保持高活力;在NaCl浓度为10%的反应缓冲体系(pH值8.0)中,温度为50℃时,酶活性最佳。金属离子除Fe3+外,对酶活性均具有明显的抑制作用;而EDTA和SDS亦可不同程度的抑制酶活性。结果表明LY5所产脂肪酶可能存在某些特殊性质。