Purification and characterisation of a serine peptidase from the marine psychrophile strain PA-43

An extracellular serine peptidase, purified from the culture supernatant of the sub-Arctic psychrophilic bacterium strain PA-43, is monomeric, with a relative molecular mass of 76000, and an unusually low pI of 3.8. The peptidase is active towards N-succinyl AAPF p-nitroanilide and N-succinyl AAPL p-nitroanilide, indicating a chymotrypsin-like substrate specificity. It is inhibited by the serine peptidase inactivator phenylmethylsulfonyl fluoride, but not by EDTA or EGTA, suggesting that added metal ions are not necessary for activity. The enzyme is most active at pH 8.3 and at 55-60 degrees C, although it is unstable at 60 degrees C. It is nevertheless remarkably stable for an enzyme from a psychrophilic microorganism, remaining active after 1 week at 20 degrees C and after five freeze-thaw cycles. Comparison of the N-terminal 40 amino acid residues with other archived sequences revealed highest similarity to the alkaline serine protease (aprx) from Bacillus subtilis.     

Expression of chromate resistance genes from Shewanella sp. strain ANA-3 in Escherichia coli

The plasmidic chromate resistance genes chrBAC from Shewanella sp. strain ANA-3 were transferred to Escherichia coli . Expression of chrA alone, on a high- or low-copy number plasmid, conferred increased chromate resistance. In contrast, expression of the complete operon chrBAC on a high-copy number plasmid did not result in a significant increase in resistance, although expression on a low-copy number plasmid made the cells up to 10-fold more resistant to chromate. The chrA gene also conferred increased chromate resistance when expressed in Pseudomonas aeruginosa . The chrR gene from the P. aeruginosa chromosome was necessary for full chromate resistance conferred by chrA . A diminished chromate uptake in cells expressing the chrA gene suggests that chromate resistance is due to chromate efflux.     

Studies of iron-uptake mechanisms in two bacterial species of the shewanella genus adapted to middle-range (Shewanella putrefaciens) or antarctic (Shewanella gelidimarina) temperatures

Iron(III)-uptake mechanisms in bacteria indigenous to the Antarctic, which is the most Fe-deficient continent on Earth, have not been extensively studied. The cold-adapted, Antarctic bacterium, Shewanella gelidimarina, does not produce detectable levels of the siderophore, putrebactin, in the supernatant of Fe(III)-deprived cultures. This is distinct from the putrebactin-producing bacterium from the same genus, Shewanella putrefaciens, which is adapted to middle-range temperatures. The production of putrebactin by S. putrefaciens is optimal, when the pH value of the medium is 7.0. According to the strong positive response from whole cells in the Chrome Azurol S (CAS) agar diffusion assay, Shewanella gelidimarina appears to produce cell-associated siderophores. In the RP-HPLC trace of an Fe(III)-loaded extract from the cell-associated components of S. gelidimarina cultured in media with [Fe(III)] ca. 0 microM, a peak appears at [MeCN] ca. 77%, which decreases in intensity in a parallel experiment in which [Fe(III)] ca. 5 microM, and is barely detectable in Fe(III)-replete media ([Fe(III)] ca. 20 microM). The Fe(III)-dependence of this peak suggests that the attendant species, which is significantly more hydrophobic than putrebactin (RP-HPLC elution: [MeCN] ca. 14%), is associated with Fe(III)-management in S. gelidimarina. This study highlights the diversity in Fe(III)-uptake mechanisms in Shewanella species adapted to different environmental and thermal niches.     

Chitin oligosaccharide deacetylase from Shewanella baltica ATCC BAA-1091

Chitin oligosaccharide deacetylase (COD) from bacteria that have been examined so far typically comprise two carbohydrate-binding domains (CBDs) and one polysaccharide deacetylase domain. In contrast, Shewanella baltica ATCC BAA-1091 COD (Sb-COD) has only one CBD, yet exhibits chitin-binding properties and substrate specificities similar to those of other CODs.     

一个降解染料的希瓦氏菌新种——中国希瓦氏菌

从细胞形态、生理生化特性和16S rRNA基因和gyrB基因序列同源性分析等方面对一株广谱高效染料降解菌D14进行了鉴定.菌株D14的细胞壁革兰氏染色为阴性,细胞为杆状,大小为0.6μm～1.0μm×1.0μm～4.0μm,周身纤毛,极生单鞭毛,其生长pH范围为pH 7.0～10.0,最适生长pH为8.0,生长温度范围为4℃～40℃,最适生长温度为20℃～30℃.该菌株具有还原三价铁,液化明胶、Tween40和Tween 80,产生H2S的能力.在乳酸钠存在的条件下,能还原硝酸盐、亚硝酸盐、铁氧化物和硫代硫酸钠.可利用D-半乳糖、D-葡萄糖、蔗糖、丙酸钠、L-亮氨酸等多种有机物为碳源.BIOLOG细菌自动鉴定系统鉴定结果表明该菌株应归属于希瓦菌属.16S rDNA和gyrB基因序列分析结果表明,菌株D14与其亲缘关系最近的菌株Shewanella putrefacien ATCC8071T的16S rDNA序列相似值为97%(小于97.7%),gyrB基因序列相似值为87%(小于90%).菌体所含有的主要脂肪酸为iso-15:0,16:1ω7c,15:0和16:0,DNA中(G C)mol%含量为49.3.结合菌株D14的生理生化特征和分子生物学特性,将菌株定为希瓦氏菌属的一个新种,命名为中国希瓦氏菌(Shewanella cinica)D14T.     

脱色希瓦氏菌(Shewanella decolorationis) S12还原不同电子受体的厌氧发酵罐培养方法

脱色希瓦氏菌Shewanella decolorationisS12在厌氧环境下能够使用多种电子受体进行厌氧呼吸。为了取得足够的细胞量用于膜蛋白质组学等科学研究的需要,本研究选取无机小分子(硝酸钠)、金属离子(柠檬酸铁)和有机大分子(偶氮染料苋菜红)作为电子受体,在使用确定成分的无机盐培养基条件下,使用不同浓度的电子供体和碳源对S12进行厌氧条件下静置和发酵罐的优化培养,采用连续补充电子受体的培养方式,确认了电子供体和碳源的合适浓度,建立了S12厌氧发酵罐培养方法。相比传统的静置厌氧培养,厌氧发酵罐培养方法在保证了严格厌氧条件下高效率还原电子受体的同时,还极大的提高了细胞生长密度。连续补充电子受体的厌氧发酵罐培养的S12最大细胞密度最大分别可达到静置厌氧培养细胞密度的325,304,369倍,而生长时间也比静置厌氧培养分别缩短了26.5%,17.6%,7.5%。这为需要大量细胞和蛋白的细菌厌氧呼吸生长实验建立了可行方法,对于进行兼性厌氧呼吸的微生物的大规模厌氧培养具有借鉴意义。     

Mcc在脱色希瓦氏菌S12电极呼吸中的作用

【目的】研究脱色希瓦氏菌S12周质空间c型细胞色素Mcc的功能,进一步探索和补充微生物胞外电子传递过程的机制。【方法】借助自杀质粒敲除mcc基因,通过细胞浓度测定和激光共聚焦显微镜比较分析突变株和野生株之间的浮游细胞和生物膜的生长情况,并比较分析二者在微生物燃料电池电极还原、铁还原和胞外偶氮染料还原过程中的功能。【结果】Mcc缺失对铁还原和偶氮还原没有影响,但却造成电极呼吸活性下降34.1%;与野生株相比,mcc突变株的好氧生长和厌氧浮游细胞生长无明显影响,但却显著抑制了电极表面生物膜的形成。【结论】Mcc是希瓦氏菌S12电极呼吸过程中周质空间电子传递的重要组分之一,缺失会显著抑制其电极呼吸效率以及生物膜的形成。     

Purification of a ccb-type quinol oxidase specifically induced in a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F

We investigated for the first time the respiratory chain system of a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F. A membrane-bound ccb-type quinol oxidase, from cells grown at 60 MPa pressure, was purified to an electrophoretically homogeneous state. The purified enzyme complex consisted of four kinds of subunits with molecular masses of 98, 66, 18.5, and 15 kDa, and it contained 0.96 mol of protoheme and 1.95 mol of covalently bound heme c per mol of enzyme. Only protoheme in the enzyme reacted with CO and CN⁻, and the catalytic activity of the enzyme was 50% inhibited by 4 μM CN⁻. The isoelectric point of the native enzyme complex was determined to be 5.0. This enzyme was specifically induced only under conditions of elevated hydrostatic pressure, and high levels were expressed in cells grown at 60 MPa. The membranes isolated from cells grown at atmospheric pressure (0.1 MPa) exhibited high levels of both cytochrome c oxidase and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPDH2)-oxidase activity. These results suggest the presence of two kinds of respiratory chains regulated in response to pressure in the deep-sea bacterium DB-172F. Received: November 25, 1997 / Accepted: December 25, 1997