Estimating and comparing the diversity of marine actinobacteria

This paper reviews the application of species richness estimators to microbial diversity data and describes phylogenetic approaches to comparing microbial communities. The techniques are demonstrated using a community of marine actinobacteria. Results demonstrate that marine environments harbour massive actinobacterial diversity. Furthermore, these predictions are likely to be severe underestimates due to the use of arbitrary OTU definitions.     

Bacterial diversity in two coastal lagoons deduced from 16S rDNA PCR amplification and partial sequencing

Abstract: Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allows the study of microbial diversity in natural ecosystems without the need for cultivation. In this study this methodology has been applied to two coastal lagoons. Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prévost lagoon (located on the French Mediterranean coast) is more eutrophic than that of the Arcachon Bay (French Atlantic coast). Analysis of partial 16S rRNA gene sequences revealed that, in both environments, a relatively large number of clones related to Cytophaga/Flexibacter/Bacteroides as well as to α-Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied, in fact for most clones maximum similarity was below 95% for the approx. 200 bases sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Bacteria were obtained from Prévost lagoon; however only five branches were represented by the data from Arcachon. These findings indicated a higher bacterial phylogenetic diversity in the Prévost lagoon.     

一平浪盐矿古老岩盐沉积中可培养细菌的系统发育多样性研究

运用纯培养法和基于16S rRNA基因序列的系统发育分析对云南省一平浪盐矿古老岩盐沉积中可培养细菌的多样性进行了研究。用补充0.5~3.5mol/L NaCl的MBA和ISP2琼脂培养基从卤水、岩盐和盐土样品中分离到38株细菌,用细菌通用引物进行16S rRNA基因扩增和序列测定,用相关软件进行序列相似性搜索、比对和系统发育分析。结果表明,38个分离菌株可分为31个物种,属于4个大的系统发育类群(Proteobacteria,Bacteroidetes,Firmicutes,Actinobacteria)、17个科、24个属。多数菌株属于Proteobacteria门(18株,47.3%;Gamma-Proteobacteria,31.5%;Alpha-Proteobacteria,15.8%)和Firmicutes门(13株,34.2%)。这些分离菌株中,至少有3个菌株可能代表3个不同属的3个新物种:Y3、Y15和Y25分别代表Idiomarina属、Salinicoccus属和Saccharospirillum属的新物种;而菌株Y21有可能代表Staphylococcaceae科的一个新属。从以上结果可以看出,一平浪盐矿古老岩盐沉积中存在较为丰富的微生物物种多样性和系统发育多样性,并且潜藏着新的微生物资源。     

Actinobacterial diversity from marine sediments collected in Mexico

Seventeen different media known to support the growth and isolation of members of the class Actinobacteria were evaluated as selective isolation media for the recovery of this microbial group from marine sediments samples collected in the Gulf of California and the Gulf of Mexico. A general selective isolation procedure was employed for six sediments and nearly 300 actinomycetes were recovered from the selective isolation plates. Full 16S rRNA gene sequencing revealed that the isolates belonged to several actinobacterial taxa, notably to the genera Actinomadura, Dietzia, Gordonia, Micromonospora, Nonomuraea, Rhodococcus, Saccharomonospora, Saccharopolyspora, Salinispora, Streptomyces, “Solwaraspora” and Verrucosispora. Previous works on marine sediments have been restricted to the isolation of members of the genera Micromonospora, Rhodococcus and Streptomyces. This study provides further evidence that Actinobacteria present in marine habitats are not restricted to the Micromonospora-Rhodococcus-Streptomyces grouping. Indeed, this first systematic study shows the extent of actinobacterial diversity that can be found in marine sediments collected in Mexico and probably, worldwide. The 16S rRNA gene sequences of marine isolates A1, AA2, AA6, AB1, AB2, AG1, AI2, AK1, AL2, AO1, AO3, AR1, AW1, B1, BB1, BC1, C5, R1, R2, R3, AV1, AE1, AI1, AN1 and AP1 determined in this study have been deposited under GenBank accession numbers EU714241–EU714258 and FJ462359–FJ462365, respectively.     

Nitrate addition stimulates microbial decomposition of organic matter in salt marsh sediments

Salt marshes sequester carbon at rates more than an order of magnitude greater than their terrestrial counterparts, helping to mitigate climate change. As nitrogen loading to coastal waters continues, primarily in the form of nitrate, it is unclear what effect it will have on carbon storage capacity of these highly productive systems. This uncertainty is largely driven by the dual role nitrate can play in biological processes, where it can serve as a nutrient‐stimulating primary production or a thermodynamically favorable electron acceptor fueling heterotrophic metabolism. Here, we used a controlled flow‐through reactor experiment to test the role of nitrate as an electron acceptor, and its effect on organic matter decomposition and the associated microbial community in salt marsh sediments. Organic matter decomposition significantly increased in response to nitrate, even at sediment depths typically considered resistant to decomposition. The use of isotope tracers suggests that this pattern was largely driven by stimulated denitrification. Nitrate addition also significantly altered the microbial community and decreased alpha diversity, selecting for taxa belonging to groups known to reduce nitrate and oxidize more complex forms of organic matter. Fourier Transform‐Infrared Spectroscopy further supported these results, suggesting that nitrate facilitated decomposition of complex organic matter compounds into more bioavailable forms. Taken together, these results suggest the existence of organic matter pools that only become accessible with nitrate and would otherwise remain stabilized in the sediment. The existence of such pools could have important implications for carbon storage, since greater decomposition rates as N loading increases may result in less overall burial of organic‐rich sediment. Given the extent of nitrogen loading along our coastlines, it is imperative that we better understand the resilience of salt marsh systems to nutrient enrichment, especially if we hope to rely on salt marshes, and other blue carbon systems, for long‐term carbon storage.     

Diversity of cultivable actinobacteria in geographically widespread marine sediments

Reports describing actinobacteria isolated from marine environments have been dominated by Micromonospora, Rhodococcus and Streptomyces species. Recent culture-independent studies have shown that marine environments contain a high diversity of actinobacterial species that are rarely, if at all, recovered by cultivation-based methods. In this study, it is shown that cultivation-independent methods can be used to guide the application of selective isolation methods. The detection of marine-derived actinobacterial species that have previously only been reported from terrestrial habitats is highlighted. This study provides good evidence that the previously described low diversity of actinobacterial species isolated from marine environments does not reflect an actual low species diversity, and that the use of informed selective isolation procedures can aid in the isolation of members of novel taxa.     

Culture-independent nested PCR method reveals high diversity of actinobacteria associated with the marine sponges <Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> and <Emphasis Type="Italic">Sponge</Emphasis> sp.

A culture-independent nested polymerase chain reaction (PCR) technique was used to investigate the diversity of actinobacteria communities associated with the sponges Hymeniacidon perleve and Sponge sp. The phylogenetic affiliation of sponge-derived actinobacteria was then assessed by 16S rRNA sequencing of cloned DNA fragments. A total of 196 positive clones were screened by restriction fragment length polymorphism (RFLP) analysis; 48 unique operational taxonomic units (OTUs) were selected for sequencing. Rarefaction analysis indicated that the clone libraries represented 93% and 94% of the total estimated diversity for the two species, respectively. Phylogenetic analysis of sequence data revealed representatives of various phylogenetic divisions, which were related to the following ten actinobacterial genera: Acidimicrobium, Corynebacterium, Propionibacterium, Actinomyces, Micrococcus, Microbacterium, Streptomyces, Mycobacterium, Cellulosimicrobium, Sporichthya, and unidentified actinobacterial clones. A sponge-specific, previously uncultured actinobacteria community grouped within the subclass Acidimicrobidae was discovered from both H. perleve and Sponge sp. Sequences belonging to Acidimicrobium in the H. perleve and the Sponge sp. clone libraries represented 33% and 24% of the clones, respectively. In the Sponge sp. clone library Mycobacterium dominated, accounting for 70% of all clones. The presence of Acidimicrobium and mycobacteria within two sponges can lay the groundwork for attempts to culture these interesting bacteria for industrial applications.     

Phylogenetic diversity of sediment bacteria from the southern Cretan margin,Eastern Mediterranean Sea

This study is the first culture-independent report on the regional variability of bacterial diversity in oxic sediments from the unexplored southern Cretan margin (SCM). Three main deep basins (water column depths: 2670–3603 m), located at the mouth of two submarine canyons (Samaria Gorge and Paximades Channel) and an adjacent slope system, as well as two shallow upper-slope stations (water column depths: 215 and 520 m), were sampled. A total of 454 clones were sequenced and the bacterial richness, estimated through five clone libraries using rarefaction analysis, ranged from 71 to 296 unique phylotypes. The average sequence identity of the retrieved Cretan margin sequences compared to the >1,000,000 known rRNA sequences was only 93.5%. A diverse range of prokaryotes was found in the sediments, which were represented by 15 different taxonomic groups at the phylum level. The phylogenetic analysis revealed that these new sequences grouped with the phyla Acidobacteria, Planctomycetes, Actinobacteria, Gamma-, Alpha- and Delta-proteobacteria. Only a few bacterial clones were affiliated with Chloroflexi, Bacteroidetes, Firmicutes, Gemmatimonadetes, Verrucomicrobia, Nitrospirae, Beta-proteobacteria, Lentisphaerae and Dictyoglomi. A large fraction of the retrieved sequences (12%) did not fall into any taxonomic division previously characterized by molecular criteria, whereas four novel division-level lineages, termed candidate division SCMs, were identified. Bacterial community composition demonstrated significant differences in comparison to previous phylogenetic studies. This divergence was mainly triggered by the dominance of Acidobacteria and Actinobacteria and reflected a bacterial community different from that currently known for oxic and pristine marine sediments.     

新疆塔里木盆地可培养嗜盐放线菌系统发育多样性

应用纯培养手段和基于16S rRNA基因序列的系统发育分析,对从塔里木盆地高盐环境土壤样品中分离的18株可培养嗜盐放线菌多样性进行了研究.实验结果表明,18株嗜盐放线菌可3个(GlycomycetaceaePseudonocardineae和Nocardiopsaceae),在有效发表的5个属的嗜盐放线菌中有4个属的嗜盐放线菌被分离到.多数菌株属于Actinopolyspora属(38.9%),Nocardiopsis属(27.8%)和Streptomonospora属(22.2%),是塔里木盆地高盐环境中嗜盐放线菌的优势类群.这些分离菌株中,菌株YIM 92370与最近种的相似性为92%,在Glycomycetaceae科内形成一个独立的分支,极有可能代表Glycomycetaceae科的一个新属.研究结果表明塔里木盆地高盐环境中存在有较为丰富的嗜盐放线菌系统发育多样性,并且潜藏着新类型的放线菌资源.     

宜宾浓香型白酒酿造过程中可培养细菌的系统发育多样性

【目的】为较系统地了解宜宾浓香型白酒酿造过程中可培养细菌的多样性,得到一些潜在的微生物资源。【方法】采用改良的NA培养基和高氏I号培养基分离、去除冗余,测定所得细菌纯培养物的16S rRNA基因,进行系统发育分析。【结果】分离得到603株细菌,4株菌的序列与GenBank中典型菌株序列相似性低于97%,代表着潜在新类群;599株菌与GenBank中34个属、101个种的典型菌株序列相似性大于97%,其中以Bacillus为绝对优势菌(315株),Streptomyces(121株)、Lysinibacillus(35株)、Staphylococcus(45株)为次优势菌,其余各属菌株均在10株以下。而且有16个属均只检测到1株菌。【结论】宜宾浓香型白酒发酵过程中的细菌呈现出较为丰富的多样性和一定的稳定性。     

新疆顿巴斯他乌盐湖沉积物免培养古菌多样性

【目的】了解新疆顿巴斯他乌盐湖沉积物免培养古菌组成及多样性。【方法】利用免培养法直接从顿巴斯他乌盐湖沉积物样品中提取环境总DNA,采用古菌通用引物对16S rRNA基因进行扩增,构建基因克隆文库。对随机挑选的59个阳性克隆进行HaeⅢ限制性酶切分型并测序、BLAST比对及构建16S rRNA基因系统发育树。【结果】文库覆盖率为89%,Shannon-Wiener指数为2.69,共得到21个不同的可操作分类单元,分属于广古菌门(Euryarchaeota,92%)和泉古菌门(Crenarchaeota,8%),其中多数为盐杆菌科(Halobacteriaceae,88%)的盐杆菌属(Halobacterium,24%)、盐盒菌属(Haloarcula,18%)、盐碱红菌属(Natronorubrum,14%)、盐红菌属(Halorubrum,8%)等,与海盐环境(thalassohaline)获得的16S rRNA基因序列相似性最高(﹥95%);整个文库中约11%的克隆与可培养古菌多个属的相似性小于97%。【结论】顿巴斯他乌盐湖古菌多样性略低于同类高盐环境,组成较为一致,只是各类群所占百分比稍有不同,且可能存在一些潜在新物种或新类群。     

滨海盐沼湿地有机碳的沉积与埋藏研究进展

滨海盐沼湿地有着较高的碳沉积速率和固碳能力,在缓解全球变暖方面发挥着重要作用,而盐渍土壤是滨海盐沼湿地碳收支研究中最大的有机碳库,研究其碳沉积与埋藏对于理解滨海湿地碳收支有着重要的意义.本文从滨海盐沼湿地土壤有机碳的来源、土壤有机碳库与沉积速率、盐沼湿地有机碳的埋藏机制、全球变化与滨海盐沼湿地碳封存等几方面对滨海盐沼湿地有机碳沉积与埋藏的相关研究进行综述.今后研究应侧重:1)加强对控制滨海盐沼湿地碳储存变异的基本因素的迸一步研究;2)对测量滨海盐沼湿地沉积物碳储量和沉积碳埋藏速率的方法进行标准化;3)对潮汐影响下滨海盐沼湿地碳与邻近生态系统之间的横向交换通量进行量化;4)探明全球变暖的影响和生产力的提高是否可以抵消因呼吸增强而造成的有机碳降解速率的升高.确定固碳速率变化驱动因子,理解气候变化和人类活动对碳埋藏的影响机制,有助于提升我国滨海盐沼湿地的固碳能力.     

海岸盐沼湿地土壤硫循环中的微生物及其作用

硫及硫化合物的动态循环是海岸盐沼湿地的重要组成部分,硫酸盐还原菌(SRB)和硫氧化菌(SOB)是推动硫循环的重要微生物。硫酸盐还原菌把硫酸盐还原为硫化物,同时消耗土壤中的有机物质;硫氧化菌把还原性硫化合物氧化为硫酸盐,缓解土壤中硫化物的积累,它们共同维持硫循环的动态平衡。本文综述了海岸盐沼湿地土壤中硫的存在形式、硫的地球化学循环以及在硫循环过程中扮演重要角色的硫酸盐还原菌和硫氧化菌的生物多样性、活性测定方法及其生态学意义等的最新研究进展,并提出了存在的问题及研究展望。     

滨海盐沼湿地有机碳的沉积与埋藏研究进展

滨海盐沼湿地有着较高的碳沉积速率和固碳能力,在缓解全球变暖方面发挥着重要作用,而盐渍土壤是滨海盐沼湿地碳收支研究中最大的有机碳库,研究其碳沉积与埋藏对于理解滨海湿地碳收支有着重要的意义.本文从滨海盐沼湿地土壤有机碳的来源、土壤有机碳库与沉积速率、盐沼湿地有机碳的埋藏机制、全球变化与滨海盐沼湿地碳封存等几方面对滨海盐沼湿地有机碳沉积与埋藏的相关研究进行综述.今后研究应侧重:1)加强对控制滨海盐沼湿地碳储存变异的基本因素的进一步研究;2)对测量滨海盐沼湿地沉积物碳储量和沉积碳埋藏速率的方法进行标准化;3)对潮汐影响下滨海盐沼湿地碳与邻近生态系统之间的横向交换通量进行量化;4)探明全球变暖的影响和生产力的提高是否可以抵消因呼吸增强而造成的有机碳降解速率的升高.确定固碳速率变化驱动因子,理解气候变化和人类活动对碳埋藏的影响机制,有助于提升我国滨海盐沼湿地的固碳能力.     

基于12S和16S rRNA序列的湍蛙属部分物种的系统发育关系

测定了湍蛙属 6个种共 10个种群 ,以及 4个外群种的线粒体 12S和 16SrRNA基因片段 ,比对后有94 0bp序列 ,发现 35 2个变异位点、 186个简约性位点。运用NJ法、MP法、ML法构建了系统关系树 ,各系统树一致表明内群为一单系群 ,分为两组 :第一组中 ,四川湍蛙两种群先聚合 ,再和棕点湍蛙聚为一支 ;第二组中 ,香港湍蛙和戴云湍蛙聚为一支 ,而香港大屿山离岛湍蛙种群首先与华南湍蛙相聚 ,再与武夷湍蛙构成姐妹支。研究结果表明 :香港地区增加 1种湍蛙分布 ;戴云湍蛙是一有效种 ;四川湍蛙的石棉和洪雅种群间遗传差异达到或超过其他种间的分歧水平。     

我国南北大豆产区慢生大豆根瘤菌的遗传多样性和系统发育研究

利用16S rRNA基因RFLP、16S rRNA基因序列分析以及16S-23S rRNA IGS PCR RFLP技术对分离自我国南北大豆产区的慢生大豆根瘤菌进行了群体遗传多样性和系统发育研究。16S rRNA基因PCR RFLP分析以及16S rRNA基因序列分析结果表明:所有供试慢生大豆根瘤菌可分为B.japonicum和B.elkanii两个类群,其中属于B.japonicum的为优势种群,占供试菌株的91%,属于B.elkanii的仅占9%,多样性水平较低。16S-23S rRNA IGS PCRRFLP研究结果表明:属于B.japonicum的慢生根瘤菌具有较丰富的遗传多样性,在69%的相似性水平上可分为群Ⅰ和群Ⅱ两大类群。群I的菌株以分离自黑龙江和河北等北部区域的菌株为代表,群Ⅱ的菌株以分离自广西和江苏等南部地域的菌株为代表,反映出明显的地域特征。两群菌株在系统发育上均与USDA6、USDA110和USDA122等B.japonicum的模式或代表菌株有差异。     

新疆艾比湖和伊吾湖可培养嗜盐古菌多样性

新疆地区盐湖密布,蕴藏着丰富的微生物资源。为保护和利用微生物物种与基因资源,作者从新疆准噶尔盆地的艾比湖和天山山间盆地的伊吾湖分离纯化嗜盐微生物。采用PCR方法扩增其中65株嗜盐古菌16SrRNA基因序列。序列分析表明,分离的嗜盐古菌分属6个属,艾比湖以Haloterrigena和Natrinema属的菌株为主,伊吾湖由Haloarcula和Halorubrum两个属的菌株构成。通过多样性指数、丰富度指数、均匀度指数和物种相对多度模型对分离的菌株进行多样性分析和比较,结果表明,盐湖嗜盐古菌的多样性指数、丰富度指数和均匀度指数具有一定相关性,艾比湖可培养嗜盐古菌的多样性高于伊吾湖。研究发现了一些新的物种资源,表明新疆盐湖中孕育的特色微生物资源亟待保护与利用。     

孟加拉虎粪便放线菌的分离及其多样性

【目的】建立有效分离动物粪便放线菌的方法,认识孟加拉虎粪便放线菌的多样性。【方法】从预处理、抑制剂、培养基三个方面考虑,采用平板稀释的分离方法。用细菌通用引物扩增获得放线菌菌株的16SrRNA基因,根据系统发育分析进行鉴定。【结果】孟加拉虎粪便样品中,可培养放线菌的菌落平均数量为1.10×108cfu/g(粪便湿重);对分离到的110株纯培养放线菌的16S rRNA基因部分序列分析表明:它们分布于10个科、12个属,主要是一些菌丝分化程度低的放线菌,如:Arthrobacter、Dietzia、Kocuria、Corynebacterium、Microbacterium等;产丝的放线菌主要以Streptomyces占优势,约占分离到放线菌总数的64%。【结论】干热处理粪便样品可以大大提高放线菌的出菌率;添加多种抑制剂及不含天然成分的组合培养基较适合粪便放线菌的分离;孟加拉虎粪便中可培养放线菌的多样性较丰富。本研究提供的分离动物粪便放线菌的有效方法,为动物粪便放线菌资源的研究和开发利用提供了借鉴作用。     

Phylogenetic diversity of endolithic bacteria in Bole granite rock in Xinjiang

The endolithic environment is a ubiquitous microbial habitat for microorganisms, such as lichens, Cyanobacteria and fungi, and it provides mineral nutrients and growth surfaces. In extremely environments, such as hot and cold desert, endolithic communities are often the main form of life. More recently, endolithic microbial communities have been observed inhabiting a variety of rock types ranging from hard granite to porous rocks such as basalt, dolomite, limestone, sandstone and granites. Regardless of geographic location and rock type, each of these habitats is characterized by a subsurface microclimate that prevents endolithic microorganisms growth. Photosynthesis-based endolithic microbial communities commonly inhabit the outer millimeters to centimeters of rocks exposed to the surface. The ability to fix carbon dioxide and in some cases atmospheric dinitrogen, gives the Cyanobacteria a clear competitive advantage over heterotrophic bacteria, so it is been called the main primary producer. Light quality and intensity appear to be the main determinant of the maximum depth to which growth occurs in endolithic phototrophic communities. Valleys of Fantastic Rocks in Bole is close to Alashankou Port of Xinjiang which belongs to extreme continental climate. In order to investigate the structure, composition and diversity of endolithic bacterial community in exposed granitic porphyry in the Valleys of Fantastic Rocks, environmental DNA was directly extracted from granite rock, the 16S rRNA genes were amplified from the total DNA by PCR with bacterial-specific primers, and an endolithic bacterial clone library was constructed. Positive clones were randomly selected from the library and identified by Restriction Fragment Length Polymorphism (RFLP). The unique rRNA types clones were sequenced, analysised and then constructed phylogenetic tree. In total, 129 positive clones were screened and grouped into 46 operational taxonomic unites (OTUs). The clone coverage C value was 89.15%, indicating that most of the estimated endolithic bacterial diversity was sampled. BLAST analysis indicated that 46 OTUs were divided into seven phyla (Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Planctomycetes, Proteobacteria) and five unknown groups. Cyanobacteria (43%), especially the Gp I, form the functional basis for an endolithic bacteria community which contain a wide spectrum species of chemotrophic bacteria (33%) with mainly Actinobacteria, α-Proteobacteria, Acidobacteria. Additionally, most clones that derived from the endolithic bacteria clone library showed high similarity to the sequence deposited in GenBank database with 97%–99%. Besides, 35% of the clones showed less than 97% of sequence similarity, of which 12% sequences were affiliated to genus Rubrobacter. The results suggested that endolithic bacteria in Valleys of Fantastic Rocks in Xinjiang were highly diverse in species richness, and maybe have a diversity of potential novel species and lineages.     

青藏高原阿里、那曲和海西地区土壤可培养放线菌的多样性

【目的】为了研究青藏高原北部地区土壤可培养放线菌的多样性,并比较不同选择性分离培养基对高原土壤放线菌的分离效果。【方法】使用9种分离培养基,并尝试添加藤黄微球菌发酵液,对采集自阿里、那曲和海西地区的14份土壤样品中的放线菌进行选择性分离。通过16S r RNA基因序列分析对分离菌株进行初步分类鉴定,并在不同分类水平上统计所分离得到的放线菌多样性。【结果】分离得到去重复后的放线菌255株,分布于放线菌门的8个目,14个科,23个属,包含94个可能的物种。其中至少25个物种可能为新种,分布于13个属。链霉菌属的菌株108株,可能的物种28个,是最主要的优势菌属。分离培养基中添加藤黄微球菌发酵液明显增加了放线菌分离菌株的数量和多样性,稀释的葡萄糖酵母麦芽汁培养基适合分离链霉菌,淀粉甘油脯氨酸培养基、丙酸钠酪蛋白培养基等则适合分离稀有放线菌。【结论】青藏高原北部土壤放线菌多样性非常丰富,并且存在较多的新颖放线菌类群;添加藤黄微球菌发酵液是提高放线菌分离效率的有效手段。