南极适冷菌Pseudoaltermonas sp.S-15-13胞外多糖低温保护作用的研究

研究了南极适冷菌Pseudoaltermonas sp.S-15-13胞外多糖(Exopolysaccharides,EPSs)对重复冻融的菌体的保护作用。结果表明,重复冻融循环后,有胞外多糖包被的菌体仍能保持较好的生长能力,无胞外多糖包被的菌体生长缓慢,甚至死亡;不同浓度的胞外多糖均可降低水溶液的冰点,其效果较浓度相同的葡聚糖明显。说明南极适冷菌S-15-13对反复冻融的耐受能力与菌体分泌的胞外多糖直接相关,除了形成机械隔离屏障,胞外多糖还可能通过降低冰点等途径使菌体免受伤害。     

南极菌Pseudoalteromonas sp.S-15-13引导糖基转移酶基因表达对环境因子变化的响应

为了探讨胞外多糖在南极微生物低温适应性中的作用与机制,克隆了南极菌Pseudoalteromonas sp.S-15-13的引导糖基转移酶(GTF)核心片段,并采用Real-Time PCR的方法研究了温度、冻融循环、盐浓度、pH等条件对该糖基转移酶基因(gtf)表达的影响。结果表明,低温有利于gtf的表达,短时的低温刺激(2℃)1 h后,gtf的表达量即上调为对照的1.5倍;而该菌经4和10℃培养24 h后gtf的表达量约为20℃时的8~12倍;经过冻融循环gtf的表达量上调,在第2个冻融循环后gtf 的表达量较对照提高了3.667倍;盐浓度对gtf的影响表现为低促高抑,即NaCl含量为6.0 %时gtf 的表达量是对照(3.0%)的3.59倍,当NaCl含量达9.0 %时gtf 表达量则显著下调;在一定范围内(pH5.0~8.0),pH的改变会促进gtf 的表达,当pH为6.0时gtf 表达量约为pH7.0时的2倍。该结果为探讨胞外多糖在南极微生物环境适应性中的作用与机制提供了科学依据。     

Identification of five strains of antarctic bacteria producing low-temperature lipase

1 IntroductionMicroorganism is an important constituentof the antarctic, arctic and deep sea ecosystemswhich are characterized as permanent lowtemperature. To adapt and survive in this ex-treme environment, these microorganisms pro-duce many distinctive substances such as poly-unsaturated fatty acids (PUFA), low-tempera-ture enzymes and novel pigments, etc. (Thomasand Dieckmann, 2002). Low-temperature en-zymes have a property of higher specific activ-ity (Kcat) at low temperature. Owing to…     

Phylogenetic diversity and phenotypic characterization of cultivable bacterioplankton isolated from polar oceans

A set of 27 marine planktonic bacteria isolated from the polar regions was characterized by 16S rDNA sequencing and physiological and biochemical testing. More than half of these bacteria were positive for caseinase, gelatinase and 13-glucosidase, and could utilize glucose, maltose or malic acid as carbon source for cell growth. Twelve isolates expressed nitrate reduction activities. Except for one antarctic isolate BSwlO175 belonging to Actinobacteria phylum, these isolates were classified as γ-Proteobacteria, suggesting that γ-Proteobacteria dominated in cultivable marine bacterioplankton at both poles. Genus Pseudoalteromonas was the predominant group in the Chukchi Sea and the Bering Sea, and genus ShewaneUa dominated in cultivable bacterioplankton in the Prydz Bay. With sequence similarities above 97%, genus Psychrobacter was found at both poles. These 27 isolates were psychrotolerant, and significant 16S rDNA sequence similarities were found not only between arctic and antarctic marine bacteria （ 〉 99% ）, but also between polar marine bacteria and bacteria from other aquatic environments （ ≥ 98.8% ）, including temperate ocean, deep sea, pond and lake, suggesting that in the polar oceans less temperature-sensitive bacteria may be cosmopolitan and have a bipolar, even global, distribution at the species level.     

南极深海沉积物中产低温脂肪酶菌株的筛选与基因克隆

在10℃恒温培养条件下,从第29次南极科学考察获得的南极深海沉积物样品中分离筛选到产脂肪酶细菌3株,产脂肪酶真菌1株,对其进行分子生物学鉴定,确定为假交替单胞菌属（Pseudoalteromonas arctica）、芽孢杆菌属（Bacillus pumilus）、Glaciecola sp.以及白冬孢酵母属（Leucosporidium sp.）.通过测定各酶的最适反应温度,获得产低温脂肪酶芽孢杆菌XZ18,该菌所产脂肪酶最适反应温度为30℃,在0℃仍有部分活性.从NaCl浓度和pH值2个条件对产酶条件进行研究,最终确定了该菌最适产酶NaCl浓度5.0%、最适产酶pH值为6,最高产酶量为4.65 U/cm3.通过PCR扩增,获得了脂肪酶全长序列,对该序列进行系统发育分析,发现该脂肪酶基因处于较独立的分支.     

海假交替单胞菌(Pseudoalteromonas marina)pilZ基因缺失抑制厚壳贻贝附着变态

为探究海假交替单胞菌pilZ基因的缺失对生物被膜形成及突变菌生物被膜对厚壳贻贝幼虫附着变态的影响,本文通过同源重组构建pilZ基因缺失菌,分析了基因缺失菌生物被膜的细菌密度、膜厚、c-di-GMP水平和胞外产物含量等特性的变化及其对厚壳贻贝幼虫附着变态的调控作用.结果表明:与野生型菌株相比,pilZ基因缺失菌形成的生物...     

极地产适冷脂肪酶菌株的酶学性质研究

从南北极环境样品中分离到7株产脂肪酶的细菌,经16SrDNA序列分析表明,这些细菌分别属于假交替单胞菌属（Pseudoalteromonas）和嗜冷杆菌属（Psychrobacter）．采用p-NPP法对这7株细菌所产的脂肪酶进行酶学性质研究,结果显示这些酶的最适作用温度在30～40cC、最适作用pH值在7—8之间,在低温下能保持较高的剩余活力,对热敏感,属于适冷脂肪酶．其中假交替单胞菌（Psychrobacter sp．7342）所产脂肪酶具有低温下酶的剩余活力高、有效作用温度和pH范围广、热稳定性较好及对多种金属离子抗性强等特点．该菌株能利用多种单一氮源和碳源产酶,最适产酶温度为25℃．在此基础上进行正交实验分析得到了该菌株的最佳发酵条件为：蛋白胨和淀粉含量各为1．33％,酵母膏含量为0．3％,温度为25℃．     

南极菌Pseudoalteromonas sp. A211-5产α-淀粉酶Amy3809的表达、性质及其降解特性

对具有高淀粉酶活性的南极菌Pseudoalteromonas sp. A211-5的全基因组数据进行了分析和筛选,筛选获得α-淀粉酶疑似序列amy3809,并采用基因工程手段对该基因的功能和性质进行验证和分析。首先,以淀粉酶Amy3809的完整开放阅读框(ORF)为模板设计特异引物,克隆获得amy3809的全序列并对其进行重组表达,获得的重组蛋白采用镍柱进行分离纯化;DNS-还原糖法测定重组酶的酶学性质;薄层层析(TLC)技术对Amy3809的酶解产物进行分析。实验结果:1)克隆获得的amy3809成功地连接到pET-30a载体,并在工程菌E.coli BL21(DE3)中实现了高效表达,纯化的重组酶Amy3809分子量为67 kDa;2)重组酶Amy3809在10~40℃的范围内仍能保持85%以上的酶活,但随着温度的升高酶活迅速降低,70℃时几乎失活,表明该酶具有良好的低温耐受特性及热敏感性;3)最适pH为7.0,在pH 5.0~10.0的范围内仍能保持50%以上的活性;4)金属离子Na+,K+和Ca2+均能提高Amy3809的活性,而Cu2+,Fe2+,Mg2+和EDTA则能显著降低Amy3809的活性;5)Amy3809的酶解产物主要为麦芽四糖、麦芽三糖、麦芽糖和葡萄糖。由此可知,南极菌产的α-淀粉酶Amy3809,具有良好的低温耐受特性,热敏感性和较广的pH耐受范围,并能够有效地将淀粉降解为低聚糖和葡萄糖,因而具有潜在的工业应用前景。     

海洋细菌LP621 右旋糖苷酶分批发酵动力学研究

对海洋细菌Pseudoalteromonas tetraodonis LP621右旋糖苷酶进行了50L发酵罐分批发酵的代谢特性的研究.结果表明LP621右旋糖苷酶合成和菌体生长呈部分生长偶联型.据分批发酵试验结果采用Logistic方程、Luedeking-Piret方程建立了描述菌株生长、产酶以及葡萄糖底物消耗分批发酵动力学模型.动力学模型计算值结果与实验值拟合良好,较好反映了菌株右旋糖苷酶分批发酵过程的动力学特征.     

Binding of thorium(IV) to carboxylate, phosphate and sulfate functional groups from marine exopolymeric substances (EPS)

Th(IV) isotopes are important proxies in oceanographic investigations, and are used as tracers of particle dynamics and particulate organic matter (POC) fluxes out of the euphotic zone through the use of ²³⁴Th/POC ratios. These approaches rely on empirically determined and variable POC to ²³⁴Th ratios, which might be controlled, in parts, by the abundance of exopolymeric substances (EPS). EPS contain acidic polysaccharides (APS) and are excreted by both phytoplankton and bacteria. To this end, radiotracer experiments with EPS from microbial cultures were conducted to determine the binding environment of ²³⁴Th(IV)-binding ligands in colloids and suspended particles in marine systems. In these experiments, the ²³⁴Th distribution during isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE) was related to the functional group composition of EPS and of colloidal organic matter (COM) isolated from the Gulf of Mexico (GOM) using cross-flow ultrafiltration. EPS was extracted from phytoplankton (Emiliania huxleyi and Synechococcus elongatus) and bacteria (Sagittula stellata and Roseobacter gallaeciensis) cultures by repeated alcohol precipitation. Phosphate and sulfate concentrations were determined using ion chromatography (IC). IEF profiles indicated that 49% to 65% of the ²³⁴Th-labeled EPS from plankton and bacteria as well as COM samples from the GOM was found concentrated below pH of 4, near an isoelectric point, pH_IEP, of about 2. The carboxylic acid maxima for extracted EPS and COM samples appeared close to the pH_IEF of ²³⁴Th(IV). The phosphate maximum appeared at the same pH_IEF as ²³⁴Th(IV) for EPS from R. gallaeciensis and S. elongatus. The sulfate maximum was found at the same pH_IEF as ²³⁴Th(IV) for EPS from S. elongatus and COM. The molecular weight (MW) of the strongly Th(IV)-binding ligand varied from 1 to 14 kDa, depending on the species, but was about 10 kDa in COM. Thus, depending on the species of plankton or bacteria, the MW and specific functional group composition of the strongly ²³⁴Th(IV)-binding amphiphilic biomolecule can vary. Therefore, different acidic functional groups can, at times, contribute to the binding of Th(IV) to the EPS chelating ligand, which can also have different MWs. This implies that the binding environment for Th(IV), which is present at total concentrations at least a million times lower than the acid functional groups, consists of strong polydentate chelate complexes in clustered structures of carboxylate, sulfates and/or phosphates. The combination of strongly chelating groups and amphiphilicity gives this biomolecule the unique properties of a “sticky” ligand.     

海洋细菌QY202产κ-卡拉胶酶的分离纯化和性质研究

为获得高效降解卡拉胶菌株,从青岛太平角海域采集的角叉菜表面分离到1株高产κ-卡拉胶酶的海洋交替假单胞菌(Pseudoalteromonas sp.)QY202,经硫酸铵沉淀、脱盐、DEAE阴离子交换层析等步骤从该菌株发酵液上清中分离纯化得到1种专一性降解κ-卡拉胶的κ-卡拉胶酶,并研究了该酶的基本酶学性质.结果表明该酶被纯化了23.1倍,回收率为43.9%,分子量大小为33.2 kDa.酶的最适反应温度为40 ℃,最适反应pH为8.0,在0～40 ℃,pH=7.0～8.0之间酶活力较稳定.酶对底物κ-卡拉胶的米氏常数Km值为1.6 mg/mL.Na~+、K~+对酶活有促进作用,而Hg~(2+)、Cu~(2+)强烈抑制酶的活性.酶解κ-卡拉胶的主产物为硫酸新κ-卡拉二糖和硫酸新κ-卡拉四糖.     

Biological characteristics of marine bacterium S-9801 strain and its culture conditions of pigment production

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光棘球海胆(Strongylocentrotus nudus)酚氧化酶氧化产物的抗菌特性研究

本文通过线性连续梯度非变性电泳结合邻苯二酚发色法分离纯化了光棘球海胆(Strongylocentrotus nudus)体腔液上清中的酚氧化酶(PO),然后通过生长曲线测定法进行了光棘球海胆PO氧化产物的抗菌特性分析。结果显示:(1)光棘球海胆体腔液上清中存在3种PO(Sn PO1,Sn PO2,Sn PO3),在聚丙烯酰胺凝胶中与邻苯二酚反应分别呈现褐色、黄色和紫色;(2)以多巴胺为底物时,Sn PO1的氧化产物对灿烂弧菌Vibrio splendidus、哈维氏弧菌Vibrio harveyi和金黄色葡萄球菌Staphylococcus aureus有抗菌作用,Sn PO2氧化产物对哈维氏弧菌、金黄色葡萄球菌和拟诺卡式菌Nocardiopsis sp.有抗菌作用,Sn PO3氧化产物仅对哈维氏弧菌和拟诺卡式菌有抗菌作用;(3)以左旋多巴为底物时,仅Sn PO2氧化产物对拟诺卡式菌有抗菌作用;(4)光棘球海胆PO氧化产物对假交替单胞菌Pseudoalteromonas nigrifaciens、希瓦氏菌Shewanella baltica和溶壁微球菌Micrococcus lysodeikticus的生长无明显影响。上述结果表明,在光棘球海胆中,PO氧化产物具有窄谱抗菌活性,多巴胺氧化产物的抗菌活力高于左旋多巴氧化产物,并且不同PO亚型的氧化产物具有不同的抗菌谱和抗菌特性。     

盐度和营养限制对盐田底栖硅藻披针舟形藻生长及胞外多糖产率的影响

从台南盐场藻垫中分离得到底栖硅藻披针舟形藻(Navicula lanceolata),该藻生长的最适盐度是1.6% NaCl.在最适盐度之上,随着盐度增大,藻细胞生长受到轻微抑制.氮磷营养限制条件下披针舟形藻生长受到严重抑制,氮营养限制比磷营养限制对藻生长的抑制作用更显著.高盐度和氮磷营养限制条件下,披针舟形藻胞外多糖(EPS)产率都明显增加,氮营养限制比磷营养限制对EPS产率的促进作用更显著.这表明在盐田高盐和氮磷营养限制条件下披针舟形藻产生更多的EPS,有利于藻垫的形成和稳定.     

Gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp.SM9913

Pseudoalteromonas sp.SM9913 is a phychrotrophic bacterium isolated from the deep-sea sediment.The genes encoding chaperones DnaJ and DnaK of P.sp.SM9913 were cloned by normal PCR and TAIL-PCR(GenBank accession Nos DQ640312,DQ504163).The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria,and show some cold-adapted characteristics in their structures when compared with those from psychro-,meso-and themophilic bacteria.It is indicated that chaperones DnaJ and DnaK of P.sp.SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding,assembling and translocation of proteins at low temperature.This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.     

Exopolysaccharides produced by bacteria isolated from the pelagic Southern Ocean — Role in Fe binding,chemical reactivity,and bioavailability

As a result of ubiquitous excretion by micro-organisms, extracellular polymeric substances are reported in high concentrations in marine systems. The majority of this material is exopolysaccharide (EPS). Despite previous studies showing that EPS can affect carbon as well as trace metal cycling, little is known about the effect on Fe – a critical nutrient limiting primary productivity in up to 40% of the ocean. Here, we have characterised an EPS purified from bacteria isolated from the pelagic Southern Ocean (Pseudoalteromonas sp.) and investigated its role in Fe chemical speciation, solubility, as well as bioavailability for two keystone Southern Ocean phytoplankton strains. This EPS has an average molecular weight of 4.6 MDa, exhibiting mainly –OH, COO– and –NH₂ functional groups. An asymmetrical flow field-flow fractionation coupled online with UV-spectrophotometer, differential refractive index, and multiangle laser light scattering (aFlFFF-UV-DRI-MALS) demonstrates that this EPS is polydisperse with three, not well resolved, size populations having molar masses in the range from 0.57 to 15.8 MDa. Fe was exclusively associated with the medium size fraction of this EPS and was the most abundant trace metal with 2.2 nM Fe per nM EPS. Only a third of this Fe was chemically labile, and the strength of Fe-EPS complexes increased with equilibration time. 1 nM EPS is efficient to retain Fe in solution, mainly in the colloidal phase (0.02–0.2 μm). Fe bound to the EPS was highly bioavailable (25% as much as for inorganic Fe). Due to combined effect of EPS on Fe solubility and bioavailability, it can increase the residence time of bioavailable Fe in the euphotic zone, therefore possibly sustaining and controlling primary productivity in sensitive oceanic regions, such as the Southern Ocean.     

鸭毛藻内生真菌Hypocreales sp.的化学成分研究

从采自大连近海的红藻鸭毛藻（Symphyocladia latiuscula）中分离到一株肉座菌目真菌（Hypocreales sp．）,对其发酵代谢产物的化学成分进行了研究。利用正相硅胶柱层析、葡聚糖凝胶Sephadex LH-20柱层析、制备薄层层析（PTLC）以及重结晶等分离手段,并通过一维、二维核磁共振技术、质谱技术等从该菌发酵液中分离鉴定了10个化舍物：双酚A（1）;邻羟基苯甲酸（2）;吲哚甲酸（3）;吲哚乙酸（4）;N-乙酰色胺（5）;（22E,24R）-麦角甾-7,9,22三烯-3β-醇（6）;过氧化麦角甾醇（7）;（22E,24R）-5α,6α-环氧麦角甾-8,22-二烯-3β,7α-二醇（8）;（22E,24R）-麦角甾-7,22-二烯-6β-甲氧基-3β,5α-二醇（9）;啤酒甾醇（10）。这些化合物均为首次从该菌中分离得到,其中化合物1为首次作为天然产物分离得到。     

斑节对虾弧菌的病原生物学研究

对患弧茵病的养殖斑节对虾作作了细菌学分析，取典型症状的淑死对虾的血淋巴和肝胰腺组织进行细菌分离，纯化，保种，并将分离菌进行回接感染，发现有很强的致病作用，且产生与自然发病相拟的症状，从感染弧菌病的病虾体内进行细菌的分离，纯化和保种，经栓验发现，分离菌和回接感染后的再分离菌均为革兰氏阴性短杆菌，极生单鞭毛，有动力，对O／129弧菌抑制剂敏感，发酵葡萄糖产酸不产气，氧化酶和过氧化氢酶反应呈阳性，不利用乳糖，蔗糖，蜜二糖，能利用柠檬酸盐，甘露醇，果糖和半乳糖，在无盐的蛋白胨水和10％NaCl的蛋白砾水中不生长，在3％，6％，8％NaCl的蛋白砾水中生长良好，吲哚反应呈阳性，V－P反应呈阴性，赖氨酸脱酸酶，鸟氨酸脱羧酶，明胶酶和淀粉酶试验呈阳性，β－半乳糖苷酶，色氨酸脱氨酶，苯丙氨酸脱氨酶和精氨酸双水解酶反应呈阴性，可还原硝酸盐，经鉴定，该病原菌为副溶血弧菌Vibria parahaemolyticus.     

深海芽孢杆菌E401B03 次生代谢产物分离和结构鉴定

利用硅胶柱、凝胶柱及高效液相(HPLC)等色谱方法,对采自中国南海的深海芽孢杆菌 E401B03发酵液的乙酸乙酯相浸膏进行了分离,通过1H-NMR、13C-NMR、EIMS 等方法鉴定了7个化合物: N-苯乙基乙酰胺、3-(羟基乙酰基)-吲哚、3-(2-羟基乙基)-吲哚、环(缬-缬)二肽、尿嘧啶、胸腺嘧啶、对羟基苯甲醛