Antibacterial and antilarval activity of deep-sea bacteria from sediments of the West Pacific Ocean

Abstract

Deep-sea microorganisms are a new source of bioactive compounds. In this study, crude ethyl acetate extracts of 176 strains of deep-sea bacteria, isolated from sediments of the West Pacific Ocean, were screened for their antibacterial activity against four test bacterial strains isolated from marine biofilms. Of these, 28 deep-sea bacterial strains exhibited antibacterial activity against one or more of the bacteria tested. Active deep-sea bacterial strains belonged mainly to the genera of Pseudomonas, Psychrobacter and Halomonas. Additionally, antilarval activity of 56 deep-sea bacterial strains was screened using Balanus amphitrite larvae. Seven bacterial strains produced metabolites that had strong inhibitive effects on larval settlement. None of these metabolites showed significant toxicity. The crude extract of one deep-sea Streptomyces strain could completely inhibit larval settlement at a concentration of 25 μg ml^?1.     

寡营养细菌及其生态作用和应用的研究进展

寡营养细菌是自然环境中特别是在寡营养环境中广泛分布的一种细菌,对寡营养生态环境起着至关重要的作用。近年来,随着分子生物学技术的飞速发展,寡营养细菌开始被广泛深入的研究。其分离培养技术,细菌形态特征,在生态中的作用和应用是目前微生物领域和环境科学领域的前沿课题,本文对这几个方面近年来的研究状况进行了综述,并展望了未来的发展趋势。     

极区低温海洋细菌及其产酶情况的初步研究

通过对大量极区低温海洋菌株的分离、筛选及进一步的生理生化特性研究,获得1株最适生长温度为15℃、生长温度上限为35℃、产蛋白酶及多种多糖水解酶的耐冷细菌。该菌过氧化氢酶为阳性,具有弱嗜盐性;蔗糖、可溶性淀粉是有利于菌株生长的碳源物质,而酵母膏则是效果最佳的氮源物质。该菌株所产蛋白酶的最适作用温度为55℃,而淀粉酶、琼脂酶及纤维素酶的最适作用温度皆为35℃。     

Depth and movement behaviour of the Pacific sleeper shark in the north-east Pacific Ocean

Satellite-linked radio telemetry was used to study the geographic movements and vertical movement behaviour of the Pacific sleeper shark Somniosus pacificus . The fish were tagged near Steller sea lion Eumetopias jubatus rookeries in the Gulf of Alaska during periods when Steller sea lions pups were most vulnerable to predation; when Steller sea lion pups first enter the water (July to August) and when Steller sea lion pups are weaned (April to May). Final locations recovered from most Pacific sleeper sharks (76%) were within 100 km of release locations, 16% were within 100–250 km and 8% were within 250–500 km. The most striking behavioural feature was their extensive, nearly continuous vertical movements. Median daily depth range was 184 m; the most time (61%) was spent between 150 and 450 m, but ascents above 100 m were common (58% of days). Median vertical movement rate was 6 km day⁻¹ and steady. The longest period of continuous vertical movement (> 60 m h⁻¹) was 330 h. Systematic vertical oscillations were most common (60%), followed by diel vertical migrations (25%) and irregular vertical movements (15%). The Pacific sleeper sharks travelled below the photic zone during the day and approached the surface at night. Pacific sleeper sharks appear to employ a stealth and ambush hunting strategy that incorporates slow vertical oscillations to search for prey, and cryptic colouration and cover of darkness to avoid detection by potential prey. The depth and geographic range of Pacific sleeper shark and Steller sea lions overlap near four important Steller sea lion rookeries in the northern Gulf of Alaska, so the potential exists for predation to occur. None of the tissues in the stomachs of the 198 Pacific sleeper sharks collected during a companion diet study, however, were identified as Steller sea lion.     

西太平洋多金属结核区12个站位沉积物中可培养潜在腐殖质转化菌的多样性

【背景】海洋环境作为地球上最大的有机碳库贮藏着大量腐殖质,其中可能蕴藏着丰富的腐殖质转化菌。【目的】从深海沉积物环境中分离具有潜在腐殖质转化能力的细菌,为难降解天然有机多聚物的生物转化提供菌种资源。【方法】利用以腐殖质为唯一碳源的培养基,对西太平洋多金属结核区12个站位沉积物样品中的腐殖质转化菌进行富集培养和纯化,通过16S rRNA基因测序分析比对初步确定分离菌株的分类地位,并利用含苯胺蓝的培养基筛选潜在的腐殖质转化菌。【结果】从12个沉积物样品中共分离获得菌株276株,隶属于放线菌纲(Actinobacteria)、纤维粘网菌纲(Cytophagia)、黄杆菌纲(Flavobacteria)、 α变形菌纲(Alphaproteobacteria)和γ变形菌纲(Gammaproteobacteria)中的14个目37个属56个种(含1个潜在新属和2个新种),其中49个种呈现木质素修饰酶阳性。【结论】利用以腐殖质为唯一碳源和能源的培养基可以分离获得具有较高多样性的潜在腐殖质转化菌。     

Polar lipid biomarkers of free-living bacteria from oligotrophic marine waters

Free-living marine bacteria isolated from oligotrophic Mediterranean waters were enriched in culture to characterize their phospholipid fatty acids (PLFAs). Odd chain iso- and anteiso-FAMEs and n–16:0 were the predominant structural PLFAs, together with a homologous series identified as mid-chain methoxy FAMEs. The dominant methoxy fatty acids identified were 9-CH₃O-15:0, 9-CH₃-16:0 and 11-CH₃O-17:0, occuring as pairs of stereoimers. Methoxy fatty acids accounted for up to 37% of PLFAs of free-living bacteria, which sets them as promising new biomarkers for bacteria of oligotrophic waters. Although similar homologues have already been characterized in a variety of eukaryotes and prokaryotes, methoxy fatty acids are identified here for the first time in marine bacteria. Analytical difficulties that may hinder the characterization of these biomarkers are presented, and structural elucidation keys by gas chromotography coupled to mass spectrometry are discussed. Whilst bacterial branched fatty acids were transferred to storage lipids of bacterivorous flagellates methoxy acids were not transferred to higher trophic levels in the studied conditions.     

印度洋表层海水石油降解菌的多样性分析

【目的】为了研究印度洋石油降解菌多样性,并获得新的石油降解菌。【方法】本研究通过印度洋表层海水样品采集、以柴油与原油1:1混合物作为碳源,从中富集、分离筛选石油降解菌,并通过PCR-DGGE对13个站点富集菌群的菌群结构进行分析。【结果】通过形态观察、生理生化反应和16SrRNA分析,共得到共29个属的51株不同的细菌,它们主要是属于α亚群和γ亚群。其中,Alcanivorax属(占18%),Novosphingobium属(占10%)和Marinobacter(占6%)Thalassospira(占6%)为主要的优势菌。通过生态多样性分析表明,Shannon-Winner指数(H)为4.57968,说明其具有较高的多样性;均匀度指数(E)为0.8664771,表示其分布比较均匀。单菌实验表明,49株具有石油降解能力其中,Sinomonas,Knoellia,Mesoflavibacter等属的细菌为首次发现有降解能力。DGGE分析表明Alcanivorax属的细菌是印度洋表层海水中的重要石油降解菌。【结论】本研究首次揭示了印度洋表层海水中石油降解菌的多样性,并获取了若干在海洋石油污染生物修复中具有应用前景的降解菌。     

印度洋可培养解有机磷细菌的多样性及解磷特性

【目的】筛选获得印度洋海水解有机磷细菌,研究其解有机磷作用机理,初步了解该地区可培养有机磷细菌的系统发育多样性。【方法】采用卵磷脂培养基从分离自印度洋的细菌中筛选解有机磷细菌,根据16S rRNA基因序列确定获得的有机磷细菌的分类地位;同时挑选解磷效果显著的3株细菌利用液体发酵进行产酶和解磷特性分析。【结果】自916株细菌中得到99株具有解有机磷活性的细菌,分属于16个属。在以卵磷脂为其有机磷来源时,菌株India-BSP-1 (Cobetia sp.)、India-BSP-21 (Pelagibaca sp.)、India-BSP-23 (Pelagibacterium sp.)的培养液中磷酸盐(DIP)浓度曲线为N型,并且碱性磷酸酶的检测活性滞后于DIP的生成。【结论】印度洋海水中解有机磷细菌种类多样性丰富,疑似有新种;解有机磷细菌的解磷特性受DIP和碱性磷酸酶相互作用的影响。     

Biodiversity and function of bacteria in the Southern Ocean

A short overview of the biodiversity of Antarctic marine bacteria is given with respect to morphology and metabolic activity. The importance of spatial and temporal variability is described. The physiological adaptation and ecological function of Antarctic marine bacterioplankton are discussed.     

南海南沙海域沉积物中可培养微生物及其多样性分析

【目的】为了从南沙海域中分离获得微生物菌种资源,【方法】本文通过沉积物采样、可培养菌分离及16S rRNA鉴定,【结果】从22个站点的沉积物样品中获得349株细菌,分属于87个种。发现产芽孢细菌分布最广,并在10个站点的分离株中占多数;它们是Bacillus,Halobacillus,Brevibacillus,Paenibacillus,Pontibacillus和Thalassobacillus。其中芽孢杆菌（Bacillus）无论在数量上还是种类上都最多,分别属于34种,其中有8个可能的新种。此外,g-Proteobacteria是分离率较高的另一亚群;其中,假单胞菌（Pseudomonas）,海杆菌（Marinobacter）,食烷菌（Alcanivorax）属的细菌最多。统计还发现,在深度750~2000 m之间,低GC含量的细菌最丰富,而深度2000 m以下,分离株则全部为g-Proteobacteria。【结论】南沙沉积物可培养微生物中产芽孢细菌及 g-Proteobacteria比较丰富;其中,产芽孢细菌的多样性最高,具有进一步研究开发价值。     

一株寡营养细菌胞外多糖的摇瓶发酵研究

从新疆的寡营养环境——古尔班通古特沙漠中分离到一株寡营养细菌Azotobacter sp．(1～15mg碳/L培养基),通过进行Azotobacter sp．菌的单因子优化培养基的试验、摇瓶培养工艺条件的优化试验(培养温度、培养时间、初始pH值、溶氧量),确定了菌种生长与营养需求等主要因子与胞外多糖产量、粘度的关系,结果表明,摇瓶发酵的最适宜条件为:以蔗糖为碳源,碳酸钙含量为2g/L,初始pH值为7左右,种龄72～84h,磷酸二氢钾、硫酸镁的含量分别为0．3g/L、0．1g/L,接种体积分数15%,于37℃摇瓶培养72h,250mL摇瓶装液量为50mL,在适宜条件下粘多糖的产量最大可达到1145．94μg/mL,粘性可达9200 mPa·s。     

Isolation and identification of bacteria from spent nuclear fuel pools

The aim of the present research was to isolate and identify bacteria from spent nuclear fuel pools of a Spanish nuclear power plant. Water samples were collected and inoculated onto different culture media to isolate the highest number of species. 16S rDNA fragments from colonies growing on solid media were amplified and analysed by denaturing gradient gel electrophoresis. Sequencing revealed the presence of 21 different bacteria belonging to several phylogenetic groups (, , and -Proteobacteria, Actinomycetales, Flavobacterium, and the Bacillus/Staphylococcus group). The isolation of these microorganisms in this particular environment (oligotrophic and radioactive) is highly interesting because of the possibility of their being used for the bioremediation of radionuclide-contaminated waters.     

Characterization of Psychrotrophic Bacteria in the Surface and Deep-Sea Waters from the Northwestern Pacific Ocean Based on 16S Ribosomal DNA Analysis

Seventy-eight 4°C-culturable bacteria were isolated using ZoBell 2216E medium from surface (0–200 m) and deep-sea (1000–9671 m) waters in the northwestern Pacific Ocean. Growth studies indicated that all 4°C-culturable bacteria were psychrotrophs. Six phylotypes were observed in the surface water samples and 8 phylotypes in the deep-sea waters. Phylogenetic characterization based on 16S ribosomal DNA sequence analysis of the representative phylotypes revealed that some bacterial genera, Pseudoalteromonas, Photobacterium, and Vibrio, were common to surface and deep-sea waters, and others, Pseudomonas and Halomonas, specifically occurred in surface water. Overall, the members of Vibrionaceae appear to be dominant in both habitats. Received June 13, 2000; accepted March 8, 2001.     

Bacterial diversity in surface sediments from the Pacific Arctic Ocean

In order to assess bacterial diversity within four surface sediment samples (0–5 cm) collected from the Pacific Arctic Ocean, 16S ribosomal DNA clone library analysis was performed. Near full length 16S rDNA sequences were obtained for 463 clones from four libraries and 13 distinct major lineages of Bacteria were identified (α, β, γ, δ and ε-Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Actinobacteria, Firmicutes, Planctomycetes, Spirochetes, and Verrucomicrobia). α, γ, and δ-Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria were common phylogenetic groups from all the sediments. The γ-Proteobacteria were the dominant bacterial lineage, representing near or over 50% of the clones. Over 35% of γ-Proteobacteria clones of four clone library were closely related to cultured bacterial isolates with similarity values ranging from 94 to 100%. The community composition was different among sampling sites, which potentially was related to geochemical differences.     

中西太平洋鲣鱼的年龄鉴定和生长特性

鱼类的年龄和生长等生物学参数对于准确评估渔业资源非常重要.本文于2007年10月至2008年1月,利用围网渔船在中西太平洋海域采集了262尾鲣鱼样本,现场测定其叉长（278～746 mm）和体质量（345～9905 g）,并搜集第一背鳍鳍棘用于鉴定其年龄和生长状况.结果表明：鲣鱼叉长（L, mm）与体质量(M,g)的关系式为M=3.612×10^-6L^{3. 278} （R²=0.9782）,性别对其影响不显著（F=2.002,P>0.05）;经赤池信息量标准（AIC）评估,在幂函数、线性及指数3种回归关系中,线性回归模型最好地拟合了鲣鱼叉长与鳍棘截面半径之间的关系(AIC=2257.4);采用Fraser-Lee法求得的鲣鱼1～5龄平均逆算叉长分别为398.4、494.2、555.4、636.8和728.8 mm;经残差平方和分析,雌雄鲣鱼的生长状况间不存在显著性差异(F=0.670;df=182;P>0.05),推得鲣鱼von Bertalanffy生长方程为L_t=706.51(1-e^{-0.64(t+0.037)}).     

Occurrence and diversity of mesophilic Shewanella strains isolated from the North-West Pacific Ocean

Although bacteria of the genus Shewanella belong to one of the readily cultivable groups of "Gammaproteobacteria", little is known about the occurrence and abundance of these microorganisms in the marine ecosystem. Studies revealed that of 654 isolates obtained from marine invertebrates (ophiuroid Amphiopholis kochii, sipuncula Phascolosoma japonicum, and holothurian Apostichopus japonicus, Cucumaria japonica), seawater and sediments of the North-West Pacific Ocean (i.e. the Sea of Japan and Iturup Is, Kurile Islands), 10.7% belonged to the genus Shewanella. The proportion of viable Shewanella species varied from 4% to 20% depending on the source of isolation. From the isolation study, representative strains of different phenotypes (from seventy presumptive Shewanella strains) were selected for detailed characterization using phenotypic, chemotaxonomic, and phylogenetic testing. 16S rDNA sequence-based phylogenetic analysis confirmed the results of tentative identification and placed the majority of these strains within only a few species of the genus Shewanella with 98-99% of 16S rDNA sequences identity mainly with S. japonica and S. colwelliana, suggesting that the strains studied might belong to these species. Numerically dominant strains of S. japonica were metabolically active and produced proteinases (gelatinases, caseinases), lipases, amylases, agarases, and alginases. Shewanella strains studied demonstrated weak antimicrobial and antifungal activities that might be an indication of their passive role in the colonization on living and non-living surfaces.     

16S rDNA sequence analysis of culturable marine biofilm forming bacteria from a ship's hull

Marine bacteria from the hull of a ship in the form of biofilms or microfouling were isolated, cultured, and identified by phylogenetic analysis using 16S rDNA sequences. With an average length of 946 bp, all the 16 sequences were classified using the Ribosomal database project (RDP) and were submitted to the National Center for Biotechnology Information. Phylogenetic analysis using 16S rDNA sequences indicated that the 16 strains belonged to the Firmicutes (IK-MB6 Exiguobacterium aurantiacum, IK-MB7 Exiguobacterium arabatum, IK-MB8 Exiguobacterium arabatum, IK-MB9 Jeotgalibacillus alimentarius, IK-MB10 Bacillus megaterium, IK-MB11 Bacillus pumilus, IK-MB12 Bacillus pumilus, IK-MB13 Bacillus pumilus, IK-MB14 Bacillus megaterium), High GC, Gram-positive bacteria (IK-MB2 Micrococcus luteus, IK-MB5 Micrococcus luteus, IK-MB16 Arthrobacter mysorens), G-Proteobacteria (IK-MB3 Halomonas aquamarina, IK-MB15 Halotalea alkalilenta), CFB group bacteria (IK-MB1 Myroides odoratimimus), and Enterobacteria (IK-MB4 Proteus mirabilis). Among the 16 strains, representatives of the Firmicutes were dominant (56.25%) compared to the high GC, Gram-positive bacteria (18.75%), G-Proteobacteria (12.5%), CFB group bacteria (6.25%), and Enterobacteria (6.25%). Analysis revealed that majority of marine species found in marine biofilm are of anthropogenic origin.     

海洋细菌Agarivorans albus NBRC102603琼胶酶的分离纯化

利用盐析、分子筛和离子交换等方法对海洋细菌Agarivorans albus NBRC102603分泌的琼胶酶粗酶液进行分离纯化,得到琼胶酶A和琼胶酶B.琼胶酶A纯化倍数为17.51倍,酶比活力为881.82 U/mg;琼胶酶B纯化倍数为16.64倍,酶比活力为838.32 U/mg.纯化的琼胶酶经SDS-PAGE检测,显示为单一条带,其相对分子质量分别为酶A 8.36×104和酶B 3.68×104.     

Antibacterial and antilarval activity of deep-sea bacteria from sediments of the West Pacific Ocean

Deep-sea microorganisms are a new source of bioactive compounds. In this study, crude ethyl acetate extracts of 176 strains of deep-sea bacteria, isolated from sediments of the West Pacific Ocean, were screened for their antibacterial activity against four test bacterial strains isolated from marine biofilms. Of these, 28 deep-sea bacterial strains exhibited antibacterial activity against one or more of the bacteria tested. Active deep-sea bacterial strains belonged mainly to the genera of Pseudomonas, Psychrobacter and Halomonas. Additionally, antilarval activity of 56 deep-sea bacterial strains was screened using Balanus amphitrite larvae. Seven bacterial strains produced metabolites that had strong inhibitive effects on larval settlement. None of these metabolites showed significant toxicity. The crude extract of one deep-sea Streptomyces strain could completely inhibit larval settlement at a concentration of 25 microg ml(-1).