假交替单胞菌及其胞外生物活性物质研究进展*

假交替单胞菌是新建立的一个海洋细菌属,它独特的生物学特性引起了越来越多研究者的关注。简介了假交替单胞菌的分类地位、生态分布等特点,重点阐述了假交替单胞菌生物活性物质的种类及其生物活性,最后对假交替单胞菌的应用前景进行了展望。     

海假交替单胞菌fliC-02330基因缺失影响生物被膜形成及厚壳贻贝幼虫附着变态

【背景】假交替单胞菌属是一种广泛分布于海洋环境的革兰氏阴性细菌,存在于海底沉积物中,能分泌大量的胞外产物形成海洋微生物被膜,从而诱导海洋无脊椎动物的附着。【目的】探究海假交替单胞菌鞭毛蛋白fliC基因对生物被膜形成及厚壳贻贝诱导活性的影响。【方法】通过基因敲除构建海假交替单胞菌fliC-02330基因缺失突变菌,研究突变菌和野生菌菌落形态、生物被膜形成能力、胞外物质以及对厚壳贻贝幼虫附着变态的诱导能力等的差异性。【结果】与野生菌相比,突变菌菌落表型出现褶皱,运动能力下降,形成被膜膜厚增加,以及对幼虫附着变态诱导活性下降。共聚焦扫描发现,fliC-02330基因缺失突变菌胞外多糖含量下降,而蛋白含量上升。【结论】海假交替单胞菌鞭毛蛋白fliC-02330基因缺失促进生物被膜形成,但抑制厚壳贻贝幼虫附着变态。本研究为探究细菌鞭毛蛋白基因与厚壳贻贝幼虫的作用机制,以及后续进一步探索微生物参与海洋无脊椎动物附着变态提供一定的理论依据。     

南海孔石藻可培养细菌多样性及代谢产物四溴吡咯研究

钙化藻和微生物在造礁石珊瑚幼虫的附着变态过程中扮演着重要角色,对珊瑚礁生态系统的健康具有重要意义。对南海珊瑚礁区钙化藻的共附生细菌进行分离培养,一方面有利于发掘南海珊瑚礁的微生物资源,另一方面有利于进行微生物与珊瑚、钙化藻等珊瑚礁框架生物的相互作用研究。本研究分离获得孔石藻(Porolithon onkodes)表面共附生细菌,基于16S rRNA基因序列进行系统发育分析,并通过高效液相色谱和质谱对分离得到的假交替单胞菌属菌株的代谢产物进行比较分析。从孔石藻分离获得的369株细菌菌株分别隶属于变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)的5纲12目22科47属的97个种级类群。在属级水平上,假交替单胞菌属(Pseudoalteromonas)的菌株数量最多,分属于7个种。在假交替单胞菌属的代谢产物分析中,发现3株在系统发育树中聚为一簇的假交替单胞菌的代谢产物中存在四溴吡咯,而其余没有。研究结果不仅表明了钙化藻中含有丰富的可培养微生物和潜在新物种资源,还首次发现四溴吡咯在南海珊瑚礁区特定类群菌株的代谢产物中的存在,为珊瑚幼虫附着变态的化学信号研究提供基础。     

深海细菌的分子鉴定分类

从东太平洋海底 5,30 0m深的沉积物中分离到一批嗜冷细菌 ,克隆了其中 1 3株细菌的1 6SrRNA基因 ,通过其序列的测定和比较 ,对这 1 3株细菌进行了分子鉴定 ,结果表明它们分属于副球菌 ,假单胞菌 ,盐单胞菌和假交替单胞菌 4个不同的菌属 ,同时进行了系统发育分析。     

北欧海海水可培养细菌多样性

【目的】为了解北欧海表层海水可培养细菌多样性与所在水文环境的关系。【方法】利用2216E、R2A和海水培养基对该海域暖流区、寒流区、海盆区及交汇区等多个区域不同站位的表层海水样品中的可培养细菌进行分离培养,通过16S r RNA基因测序对分离的菌株进行分类鉴定,并构建系统发育树进行系统发育分析。【结果】从北欧海表层海水中共分离到407株细菌,通过RFLP分析选取其中154株进行测序,结果表明此154株细菌分属于3个门,18个属,27个种。3个门包括变形菌门、厚壁菌门和放线菌门,优势属为假交替单胞菌属、嗜冷杆菌属等,优势种为食琼脂假交替单胞菌、海雪嗜冷杆菌等,并分离到闪烁交替单胞菌等多株嗜冷菌。比较不同区域的微生物多样性可以看出,γ-变形菌纲的细菌在各个区域均占较高比例。交汇区的细菌多样性最高,分离到了10个不同属的细菌,而海盆区细菌多样性最低,只分离到了4种。除了海盆区外,其他3个区域的样品中都分离到了特有的类群。【结论】从以上结果可以看出,北欧海域有较为丰富的微生物资源,且交汇区微生物多样性较其他区域高。     

海洋细菌A78的鉴定及褐藻胶裂解酶特性的研究

对分离自日本海域海带的产褐藻胶裂解酶的细菌A78菌株进行鉴定,通过生理生化特征及16S rDNA基因序列分析,鉴定为假交替单胞菌(Pseudoalteromonas nigrifaciens).不同浓度褐藻酸钠诱导实验表明,菌株A78产酶最佳诱导物浓度为1％褐藻酸钠.不同的温度梯度(20～55℃)及pH值梯度(4～10...     

南极交替单胞菌R11-5产卡拉胶酶的发酵条件优化

【背景】极地寒冷环境中发现了大量具有潜在应用前景的冷适应酶,同时也存在种类繁多的海藻多糖降解菌,因此极端环境微生物是筛选获得新颖、高效多糖降解酶的重要新源泉。由于筛选培养基通常并非野生菌发酵产酶的最优条件,为了使野生菌的产酶效率达到最高,需要对其培养条件进行优化,从而为其深入研究及开发利用提供依据。【目的】对一株产卡拉胶酶的南极菌株进行种属鉴定,并采用响应面法对该菌的发酵产酶条件进行优化。【方法】通过16SrRNA基因对产卡拉胶酶的南极菌株进行种属鉴定,采用响应面法优化南极菌株产酶发酵条件。【结果】该南极菌属于交替单胞菌属(Alteromonas),命名为交替单胞菌R11-5。发酵条件优化结果显示,7个环境因子影响交替单胞菌R11-5的产酶量。利用Design-Expert软件中的Plackett-Burman设计实验,筛选出影响交替单胞菌R11-5产酶量的4个主要因素分别为培养温度、牛肉膏浓度、卡拉胶浓度和Ca~(2+)浓度。通过Box-Behnken设计和响应面分析得到交替单胞菌R11-5最佳产酶发酵条件为:温度15.0°C,牛肉膏浓度11.0 g/L,卡拉胶浓度3.0 g/L,Ca~(2+)浓度5.0 mmol/L。优化后发酵上清液酶产量达到87.193 U/mL,与优化前相比提高了1.8倍。【结论】响应面法提高了南极交替单胞菌R11-5卡拉胶酶的产量,为其开发应用提供了科学依据。     

普通卷甲虫肠道可培养细菌的分离、鉴定及产消化酶活性

目的分离培养普通卷甲虫肠道中的可培养细菌,筛选有产消化酶活性的细菌,推测其在协助普通卷甲虫消化食物中的作用。方法通过传统分离培养法分离普通卷甲虫肠道中的可培养细菌,利用平板透明圈法筛选产淀粉酶、蛋白酶、纤维素酶和脂肪酶活性的细菌,利用水解圈与菌落直径的比值,比较不同细菌的产消化酶活性。利用SPSS 20.0软件进行统计学分析,组间比较采用单因素方差分析。结果在普通卷甲虫肠道中分离出4个属9种细菌,其中气单胞菌属3种,假芽胞杆菌属和柠檬酸杆菌属各2种,芽胞杆菌属和假单胞菌属各1种。9种细菌中弗氏柠檬酸杆菌、豚鼠气单胞菌、南海假芽胞杆菌等3种细菌可产蛋白酶,嗜水气单胞菌、波特卡伦柠檬酸杆菌、水生气单胞菌、豚鼠气单胞菌和南海假芽胞杆菌等5种细菌可产纤维素酶,嗜水气单胞菌、波特卡伦柠檬酸杆菌、印度芽胞杆菌、水生气单胞菌、嗜盐假芽胞杆菌、豚鼠气单胞菌和南海假芽胞杆菌等7种细菌可产淀粉酶,未筛选到产脂肪酶细菌。统计学分析表明,3种产蛋白酶细菌和5种产纤维素酶细菌的产酶活性差异无统计学意义。而7种产淀粉酶细菌产酶的活力间差异有统计学意义,水生气单胞菌的产淀粉酶活性能力最强。结论普通卷甲虫肠道可培养细菌结构简单,但有消化酶活性的细菌种类多,5种细菌有产2种以上消化酶功能,说明肠道细菌可能在普通卷甲虫食物消化中起着重要作用。     

阿拉伯海假交替单胞菌N1230-9两个甲基受体趋化蛋白的功能鉴定

【目的】作为海洋中的特有及优势种群,假交替单胞菌(Pseudoalteromonas)普遍拥有多个甲基受体趋化蛋白(methyl-accepting chemotaxis protein, MCP),探究这些趋化受体的功能。【方法】以太平洋表层海水来源的一株阿拉伯海假交替单胞菌(Pseudoalteromonas arabiensis) N1230-9为研究对象,利用软琼脂平板法测试该菌株对23种碳源的趋化能力,继而利用同源重组策略构建2个含sCache结构域MCP编码基因(woc28264和woc27036)缺失突变体,并分析突变体对10种碳源的趋化能力。【结果】菌株N1230-9对海藻糖、麦芽糖、蔗糖、N-乙酰氨基葡萄糖、l-苹果酸、乙酸钠、丙酸钠、丙酮酸钠、柠檬酸和琥珀酸10种碳源具有趋化能力。WOC28264是l-苹果酸和蔗糖的特异性趋化受体,WOC27036则是柠檬酸和琥珀酸的特异性趋化受体。此外,WOC28264和WOC27036还均是N-乙酰氨基葡萄糖和海藻糖的趋化受体。【结论】WOC28264和WOC27036存在重叠的碳源效应物。     

北极海洋沉积物中锰细菌的分离与系统发育

对中国第二次北极科学考察采集的北极海洋沉积物中的锰细菌进行了筛选、分离和系统发育分析。根据其在筛选平板上菌落的形态学特征,分别从站位P11和S11采集的沉积物中分离到了21株和19株锰细菌。系统发育分析表明,两个站位的锰细菌群落组成有着明显的差别。站位P11分离的可培养锰细菌主要由细菌域（Bacteria）中变形杆菌门的γ-变形杆菌纲（γ-Proteobacteria）和放线菌纲（Actinobacteria）组成,二者分别占86％和14％;γ-变形杆菌纲主要包括嗜冷杆菌属（Psychrobacter）、希瓦氏菌属（Shewanella）、假交替单胞菌属（Pseudoaheromonas）、不动杆菌属（Acinetobacter）、海杆菌属（Marinobacter）,其中以嗜冷杆菌属为主,其比例可达67％。从站位S11分离到的可培养锰细菌主要包括细菌域中变形杆菌门的α-变形杆菌纲（α-Proteobacteria）和γ-变形杆菌纲以及拟杆菌门（Bacteroides）中的黄杆菌纲（Flavobaeteria）;γ-变形杆菌纲主要包括希瓦氏菌属、海单胞菌属（Marinomonas）和交替单胞菌属（Aheromonas）,α-变形杆菌纲主要为鞘氨醇单胞菌属（Sphingomonas）。实验菌株均对Mn^2＋有着较强的抗性,其中以菌株Marinomonas sp．S11-S-4耐受性最高。     

Molecular investigation of the distribution, abundance and diversity of the genus Pseudoalteromonas in marine samples

The genus Pseudoalteromonas has attracted interest because it has frequently been found in association with eukaryotic hosts, and because many Pseudoalteromonas species produce biologically active compounds. One distinct group of Pseudoalteromonas species is the antifouling subgroup containing Pseudoalteromonas tunicata and Ps. ulvae, which both produce extracellular compounds that inhibit growth and colonization by different marine organisms. PCR primers targeting the 16S rRNA gene of the genus Pseudoalteromonas and the antifouling subgroup were developed and applied in this study. Real-time quantitative PCR (qPCR) was applied to determine the relative bacterial abundance of the genus and the antifouling subgroup, and denaturing gradient gel electrophoresis (DGGE) was applied to study the diversity of the genus in 11 different types of marine samples from Danish coastal waters. The detection of Ps. tunicata that contain the antifouling subgroup was achieved through specific PCR amplification of the antibacterial protein gene (alpP). The Pseudoalteromonas species accounted for 1.6% of the total bacterial abundance across all samples. The Pseudoalteromonas diversity on the three unfouled marine organisms Ciona intestinalis, Ulva lactuca and Ulvaria fusca was found to be low, and Ps. tunicata was only detected on these three hosts, which all contain accessible cellulose polymers in their cell walls.     

A new family of Alteromonadaceae fam. nov., including the marine proteobacteria species Alteromonas, Pseudoalteromonas, Idiomarina i Colwellia

The taxonomic position of the marine genera Alteromonas, Pseudoalteromonas, Idiomarina, and Colwellia within the gamma subclass of the class Proteobacteria were specified on the basis of their phenotypic, genotypic, and phylogenetic characteristics. Gram-negative aerobic bacteria of the genera Alteromonas, Pseudoalteromonas, and Idiomarina and facultatively anaerobic bacteria of the genus Colwellia were found to form a phylogenetic cluster with a 16S rRNA sequence homology of 90% or higher. The characteristics of these genera presented in this paper allow their reliable taxonomic identification. Based on the analysis of our experimental data and analyses available in the literature, we propose to combine the genera Alteromonas, Pseudoalteromonas, Idiomarina, and Colwellia into a new family, Alteromonadaceae fam. nov., with the type genus Alteromonas.     

Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents

The newly established genus Pseudoalteromonas contains numerous marine species which synthesize biologically active molecules. The production of a range of compounds which are active against a variety of target organisms appears to be a unique characteristic for this genus and may greatly benefit Pseudoalteromonas cells in their competition for nutrients and colonization of surfaces. Species of Pseudoalteromonas are generally found in association with marine eukaryotes and display anti-bacterial, bacteriolytic, agarolytic and algicidal activities. Moreover, several Pseudoalteromonas isolates specifically prevent the settlement of common fouling organisms. While a wide range of inhibitory extracellular agents are produced, compounds promoting the survival of other marine organisms living in the vicinity of Pseudoalteromonas species have also been found.     

Marine bacteria from Danish coastal waters show antifouling activity against the marine fouling bacterium Pseudoalteromonas sp. strain S91 and zoospores of the green alga Ulva australis independent of bacteriocidal activity

The aims of this study were to determine if marine bacteria from Danish coastal waters produce antifouling compounds and if antifouling bacteria could be ascribed to specific niches or seasons. We further assess if antibacterial effect is a good proxy for antifouling activity. We isolated 110 bacteria with anti-Vibrio activity from different sample types and locations during a 1-year sampling from Danish coastal waters. The strains were identified as Pseudoalteromonas, Phaeobacter, and Vibrionaceae based on phenotypic tests and partial 16S rRNA gene sequence similarity. The numbers of bioactive bacteria were significantly higher in warmer than in colder months. While some species were isolated at all sampling locations, others were niche specific. We repeatedly isolated Phaeobacter gallaeciensis at surfaces from one site and Pseudoalteromonas tunicata at two others. Twenty-two strains, representing the major taxonomic groups, different seasons, and isolation strategies, were tested for antiadhesive effect against the marine biofilm-forming bacterium Pseudoalteromonas sp. strain S91 and zoospores of the green alga Ulva australis. The antiadhesive effects were assessed by quantifying the number of strain S91 or Ulva spores attaching to a preformed biofilm of each of the 22 strains. The strongest antifouling activity was found in Pseudoalteromonas strains. Biofilms of Pseudoalteromonas piscicida, Pseudoalteromonas tunicata, and Pseudoalteromonas ulvae prevented Pseudoalteromonas S91 from attaching to steel surfaces. P. piscicida killed S91 bacteria in the suspension cultures, whereas P. tunicata and P. ulvae did not; however, they did prevent adhesion by nonbactericidal mechanism(s). Seven Pseudoalteromonas species, including P. piscicida and P. tunicata, reduced the number of settling Ulva zoospores to less than 10% of the number settling on control surfaces. The antifouling alpP gene was detected only in P. tunicata strains (with purple and yellow pigmentation), so other compounds/mechanisms must be present in the other Pseudoalteromonas strains with antifouling activity.     

Bioactivity,Chemical Profiling,and 16S rRNA-Based Phylogeny of <Emphasis Type="BoldItalic">Pseudoalteromonas</Emphasis> Strains Collected on a Global Research Cruise

One hundred one antibacterial Pseudoalteromonas strains that inhibited growth of a Vibrio anguillarum test strain were collected on a global research cruise (Galathea 3), and 51 of the strains repeatedly demonstrated antibacterial activity. Here, we profile secondary metabolites of these strains to determine if particular compounds serve as strain or species markers and to determine if the secondary metabolite profile of one strain represents the bioactivity of the entire species. 16S rRNA gene similarity divided the strains into two primary groups: One group (51 strains) consisted of bacteria which retained antibacterial activity, 48 of which were pigmented, and another group (50 strains) of bacteria which lost antibacterial activity upon sub-culturing, two of which were pigmented. The group that retained antibacterial activity consisted of six clusters in which strains were identified as Pseudoalteromonas luteoviolacea, Pseudoalteromonas aurantia, Pseudoalteromonas phenolica, Pseudoalteromonas ruthenica, Pseudoalteromonas rubra, and Pseudoalteromonas piscicida. HPLC-UV/VIS analyses identified key peaks, such as violacein in P. luteoviolacea. Some compounds, such as a novel bromoalterochromide, were detected in several species. HPLC-UV/VIS detected systematic intra-species differences for some groups, and testing several strains of a species was required to determine these differences. The majority of non-antibacterial, non-pigmented strains were identified as Pseudoalteromonas agarivorans, and HPLC-UV/VIS did not further differentiate this group. Pseudoalteromonas retaining antibacterial were more likely to originate from biotic or abiotic surfaces in contrast to planktonic strains. Hence, the pigmented, antibacterial Pseudoalteromonas have a niche specificity, and sampling from marine biofilm environments is a strategy for isolating novel marine bacteria that produce antibacterial compounds.     

Growth patterns of two marine isolates: adaptations to substrate patchiness?

During bottle incubations of heterotrophic marine picoplankton, some bacterial groups are conspicuously favored. In an earlier investigation bacteria of the genus Pseudoalteromonas rapidly multiplied in substrate-amended North Sea water, whereas the densities of Oceanospirillum changed little (H. Eilers, J. Pernthaler, and R. Amann, Appl. Environ. Microbiol. 66:4634-4640, 2000). We therefore studied the growth patterns of two isolates affiliating with Pseudoalteromonas and Oceanospirillum in batch culture. Upon substrate resupply, Oceanospirillum lagged threefold longer than Pseudoalteromonas but reached more than fivefold-higher final cell density and biomass. A second, mobile morphotype was present in the starved Oceanospirillum populations with distinctly greater cell size, DNA and protein content, and 16S rRNA concentration. Contrasting cellular ribosome concentrations during stationary phase suggested basic differences in the growth responses of the two strains to a patchy environment. Therefore, we exposed the strains to different modes of substrate addition. During cocultivation on a single batch of substrates, the final cell densities of Oceanospirillum were reduced three times as much as those Pseudoalteromonas, compared to growth yields in pure cultures. In contrast, the gradual addition of substrates to stationary-phase cocultures was clearly disadvantageous for the Pseudoalteromonas population. Different growth responses to substrate gradients could thus be another facet affecting the competition between marine bacteria and may help to explain community shifts observed during enrichments.     

Genome sequences of six Pseudoalteromonas strains isolated from Arctic sea ice

Yu et al. (Polar Biol. 32:1539-1547, 2009) isolated 199 Pseudoalteromonas strains from Arctic sea ice. We sequenced the genomes of six of these strains, which are affiliated to different Pseudoalteromonas species based on 16S rRNA gene sequences, facilitating the study of physiology and adaptation of Arctic sea ice Pseudoalteromonas strains.