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.     

广西水牛瘤胃中的细菌多样性

[目的]了解广西水牛瘤胃中细菌的组成及其可能的降解纤维素细菌的主要类群。[方法]提取水牛瘤胃内容物和高效降解滤纸的水牛瘤胃内容物的富集培养物的宏基因组DNA,以宏基因组DNA为模板,扩增16S rRNA基因序列,构建该两种样品的细菌的16S rRNA基因文库。通过对16S rRNA基因序列的分析,了解这两种样品的细菌群体种类及数量。 [结果] 水牛瘤胃内容物与其富集培养物中均主要含有LGCGPB (low G+C Gram-Positive Bacteria)、CFB (Cytophaga-Flexibacter-Bacteroides)两大类菌群和少数的螺旋体菌（Spirochaetes）,且LGCGPB所占的比例都是最高的,LGCGPB在水牛瘤胃内容物细菌中的比例为56.66%,而在富集培养物细菌中的比例升高为73.33%。在水牛瘤胃内容物中,丝状杆菌（Fibrobacteres）占3.33%,但在富集培养物中未被检测到。而在富集培养物中占13.33%的变形杆菌（Proteobacteria）,在水牛瘤胃内容物中未被检测到。本研究还发现了分类地位尚未明确的一菌群（R46）。[结论]细菌类群LGCGPB、Proteobacteria可能在水牛瘤胃中的纤维素降解过程中起重要作用。此外,水牛瘤胃中的细菌组成和牦牛、牛、羊瘤胃中的细菌组成较相似但比例有所不同。     

云南文山铝土矿区微生物多样性初步研究

目的 对云南文山铝土矿区矿石样品中的细菌多样性进行研究。方法 采集云南文山铝土矿矿石样品,利用16S rRNA高变基因序列构建细菌基因文库,建立系统发育树,初步探究该矿区细菌多样性。结果 文山铝土矿区矿石样品中细菌的主要菌群包括变形菌门、厚壁菌门、拟杆菌门、放线菌门和梭杆菌门,其中优势菌群为变形菌门的细菌。结果 云南省文山铝土矿区的矿石样品中存在丰富的细菌,且种群多样性较好。     

Bacterial community composition over a dry winter in meso- and eutrophic Portuguese water bodies

In order to investigate the bacterial diversity in a number of rivers, reservoirs and lakes in northern and central Portugal during the winter of 2004/5 (atypically dry), we applied molecular methodologies, namely denaturing gradient gel electrophoresis with primers targeting fractions of the bacterial 16S rRNA gene. Environmental parameters such as pH, conductivity, inorganic nutrients, total suspended solids and chlorophyll a were determined in order to characterize the trophic status of the studied water bodies. We found water bodies with oligotrophic to hypereutrophic characteristics. Organisms belonging to the Bacteroidetes and Alphaproteobacteria were found at the highest pH environment. Bacteroidetes were also related to high nutrient concentrations. Verrucomicrobia were associated with the most oligotrophic reservoir and low pH values. Actinobacteria were present in all samples from lakes and reservoirs, indicating its preference for lentic water bodies. Cyanobacteria dominance was related to high pH and conductivity levels. In general the conductivity values recorded in winter 2005 were the highest over recent years and chlorophyll a also reached very high levels. The data emphasize an enhanced risk of eutrophication for the studied water bodies, especially in the subsequent months when the temperature rises.     

Identification of culturable bacteria present in haemodialysis water and fluid

Water used to prepare haemodialysis fluid is not sterile, and its microbiological control is important for the prevention of haemodialysis-associated illness. Bacterial populations inhabiting a distribution system for haemodialysis water were studied over an 18-month period. 203 planktonic bacteria isolated on R2A medium were identified by restriction analysis and sequencing of 16S rRNA gene. A diverse bacterial community was detected, containing predominantly Gram-negative members of the Alphaproteobacteria and Betaproteobacteria, as well as representatives of the genus Mycobacterium. Ecological and clinical consequences are discussed: bacteria from the genera Novosphingobium, Pseudomonas and Sphingomonas have been described in the build-up of biofilms, and others like Acinetobacter, Mycobacterium or Brevibacterium may represent a health risk to patients under haemodialysis treatment.     

普通和稀释培养基研究太湖沉积物可培养细菌的多样性

采用普通牛肉汁培养基和 10倍稀释的普通牛肉汁培养基 (以下简称稀释培养基 )研究太湖沉积物中细菌多样性 ,发现在稀释培养基上生长的细菌数量普遍是在普通牛肉汁琼脂培养基上生长的细菌数量的 3～ 5倍。分离得到纯培养物的 16SrDNA部分序列 (5′端约 5 0 0bp)分析表明 ,不同培养基上生长的优势细菌类群存在差别 :普通培养基生长的细菌主要为γ_Proteobacteria(35. 1% ) ,其次为Actinobacteria(2 4 5 % )和Firmicutes(2 2 . 3% )等类群 ,其中大部分细菌与假单胞菌属 (Pseudomoas)、芽孢杆菌属 (Bacillus)和节杆菌属 (Archrobacter)细菌的系统关系密切 ;稀释培养基生长的细菌则主要为Actinobacteria(2 7. 1% )、Firmicutes(2 5 . 7% )、α_Proteobacteria(2 1. 4 % )和γ_Proteobacteria(15. 7% )等类群 ,与芽孢杆菌属 (Bacillus) (2 5. 7% )发育系统关系密切的细菌为优势属。研究结果表明同时采用两种培养基有助于从太湖沉积物中分离到更多种微生物。     

云南兰坪铅锌矿区细菌多样性研究

目的研究云南省兰坪铅锌矿区矿石样品中的细菌群落结构。方法构建细菌16S rRNA高变区基因文库。结果矿石内部细菌种类丰富多样,文库测序结果表明矿石样品中细菌分属于4个门,27个属;其中变形菌门（Proteobacteria）类群是优势种群,占所有细菌总克隆数的51.7%,而且大多数属于γ-变形菌纲亚群。各克隆序列分析结果表明,其中部分细菌序列与已报道物种的相似性较低。结论表明云南省兰坪铅锌矿矿石样品中可能存在新的细菌种类。     

山西左云县采煤区人工湿地冬季沉积物细菌群落多样性

【背景】人工湿地处理煤炭矿井废水效果显著,而关于人工湿地沉积物的细菌群落结构和特征研究较少。【目的】阐明人工湿地净化采煤区废水的效应和细菌群落结构及特征。【方法】对山西省左云县采煤区人工湿地不同采样点水质进行监测,利用高通量测序技术对人工湿地沉积物中细菌16S rRNA基因V3-V4区进行测序,分析沉积物中细菌群落组成。【结果】采煤区废水流经人工湿地后水质得到有效改善,其中生化需氧量(Biochemical oxygen demand,BOD5)、化学需氧量(Chemical oxygen demand,CODCr)、总氮(Total nitrogen,TN)、总磷(Total phosphorus,TP)去除率分别达到76.2%、93.4%、73.4%和99.3%;测序共获得2 832个操作分类单元(Operational taxonomic units,OTUs),共753个属,分布于51个门的150个纲;4个采样点中优势菌门为变形菌门(Proteobacteria,50%-64.7%)和拟杆菌门(Bacteroidetes,15.8%-21.2%),变形菌门中优势菌纲为β-变形菌纲和γ-变形菌纲,其中M1、M2、M3中富含氨氮氧化细菌的β-变形菌纲和γ-变形菌纲丰度最高;M4沉积物细菌OTU最多,多样性最高;沉积物特有优势菌属Sulfurimonas、Sulfuricurvum的相对丰度与NH4+-N的含量显著负相关,Azoarcus菌属相对丰度与TN存在显著正相关,Novosphingobium菌属相对丰度与TP含量存在显著正相关。【结论】煤矿废水流经人工湿地可得到有效的净化,细菌多样性在人工湿地生态功能中起到了决定作用。     

Bacterial Diversity in Linglong Gold Mine,China

Bacteria have been actively regulating cycles of various elements in the environment. To explore the potential bacterial role in gold biogeochemical cycling, this study analyzed the bacterial diversity of mine rock (MR) and surface soil (SS) samples from Linglong gold mine using 16S rRNA gene clone library analysis and cultivation method. From MR, 24 operational taxonomic units (OTUs) were identified from MR, covering 3 phyla and 18 genera. Meanwhile, 24 OTUs were identified from SS, including 4 phyla and 18 genera. Compared with 16S rRNA gene clone library analysis, 28 aerobic and 34 anaerobic isolates were obtained, whereas 26 aerobic and 71 anaerobic strains were isolated from SS. The cultivable bacteria were affiliated with Firmicutes, Proteobacteria and Actinobacteria phyla, and dominated by Firmicutes. These results underscore the high level of bacterial diversity in the gold mine. Our study provides information on the microbial diversity in Linglong gold mine and sheds light on the existence and potential function of bacteria in the gold biogeochemical cycling.     

Impact of chromium-contaminated wastewaters on the microbial community of a river

The influence of chromium on the microbial community structure was analyzed in a river system subjected to long-term chromium contamination, by plating and by sequencing 16S rRNA genes cloned from DNA extracted from the river sediments. We also analyzed the influence of chromium on the ability of the microbial community to resist and reduce Cr(VI) and on its resistance to antibiotics. Shifts in the microbial community structure were analyzed by amplified ribosomal DNA restriction analysis fingerprinting. The isolates obtained were phylogenetically related to Actinobacteria, Firmicutes, Bacteroidetes and Proteobacteria, whereas Acidobacteria and Deltaproteobacteria were only revealed by clone analyses. Cr(VI)-resistant and Cr(VI)-reducing strains were isolated in all sites examined. However, each sample site had a microbial community with a different antibiotic resistance pattern. Our study seems to indicate that in this river ecosystem chromium influenced the microbial communities, altering some of their functional characteristics, such as the percentage of the microbial community able to resist or to reduce Cr(VI) and the phylogenetic groups isolated, but it did not affect the structural diversity. Furthermore, the concentration of Cr(VI) in the sediments could not be correlated with a lower number of bacteria or lower index of generic diversity, neither with the ability of the microbial community to resist or to reduce higher Cr(VI) concentrations.     

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.     

Identification of mixed bacterial DNA contamination in broad-range PCR amplification of 16S rDNA V1 and V3 variable regions by pyrosequencing of cloned amplicons

Using a sensitive and rapid method combining broad-range PCR amplification of bacterial 16S rDNA fragments and pyrosequencing for detection, identification and typing, we have found contaminating bacterial DNA in our reagents used for PCR. Identified bacteria are the water-borne bacterial genera Pseudomonas, Stenotrophomonas, Xanthomonas, Ralstonia and Bacillus. Our results are in concordance with recent reports of contaminated industrial water systems. In light of this conclusion, we believe that there is a need for increased awareness of possible contamination in uncertified widely used molecular biology reagents, including ultra-pure water. Since sequence-based 16S rDNA techniques are used in a variety of settings for bacterial typing and the characterization of microbial communities, we feel that future certification of molecular biology reagents, as free of nucleic acids, would be advantageous.     

The Relationship between Phytoplankton Diversity and Community Function in a Coastal Lagoon

The decrease of biodiversity related to the phenomena of global climate change is stimulating the scientific community towards a better understanding of the relationships between biodiversity and ecosystem functioning. In ecosystems where marked biodiversity changes occur at seasonal time scales, it is easier to relate them with ecosystem functioning. The objective of this work is to analyse the relationship between phytoplankton diversity and primary production in St. André coastal lagoon – SW Portugal. This lagoon is artificially opened to the sea every year in early spring, exhibiting a shift from a marine dominated to a low salinity ecosystem in winter. Data on salinity, temperature, nutrients, phytoplankton species composition, chlorophyll a (Chl a) concentration and primary production were analysed over a year. Modelling studies based on production-irradiance curves were also conducted. A total of 19 taxa were identified among diatoms, dinoflagellates and euglenophyceans, the less abundant group. Lowest diversities (Shannon–Wiener index) were observed just before the opening to the sea. Results show a negative correlation (p<0.05) between diversity and chlorophyll a (Chl a) concentration (0.2–40.3 mg Chl a m⁻³). Higher Chl a values corresponded to periods when the community was dominated by the dinoflagellate Prorocentrum minimum (>90% of cell abundance) and production was maximal (up to 234.8 mg C m⁻³ h⁻¹). Maximal photosynthetic rates (P_max) (2.0–22.5 mg C mg Chl a⁻¹ h⁻¹) were higher under lower Chl a concentrations. The results of this work suggest that decreases in diversity are associated with increases in biomass and production, whereas increases correspond to opposite trends. It is suggested that these trends, contrary to those observed in terrestrial and in some benthic ecosystems, may be a result of low habitat diversity in the water column and resulting competitive pressure. The occurrence of the highest photosynthetic rates when Chl a is low, under some of the highest diversities, suggests a more efficient use of irradiance under low biomass–high diversity conditions. Results suggest that this increased efficiency is not explained by potential reductions in nutrient limitation and intraspecific competition under lower biomasses and may be a result of niche complementarity.     

克罗恩病患者肠道菌群多样性变化研究初探

目的 探讨克罗恩病（CD）患者肠道菌群多样性变化。方法 收集8例确诊CD患者的粪便标本（CD组）,以同期8例健康人群粪便作为对照组（CN组）。收集新鲜粪便样本提取DNA,并将样本进行PCR扩增,最终进行16S rRNA数据分析。结果 测序共获得990 890条高质量序列,CD组和CN组多样性指数Shannon 和Simpson比较差异有统计学意义（t=‒3.802和t=‒2.886,P<0.05）。OTUs（可操作分类学单位）比较韦恩图显示CD组共有7 437株菌种,而CN组共有7 744株菌种,其中3 438株菌种重叠。LDA差异贡献分析图提示CD患者肠道菌群以芽胞杆菌、变形菌、乳杆菌目、放线菌目、异常球菌纲、栖热菌目、假单胞菌目和交替单胞菌目等为主要特征差异细菌菌类,而CN组则以双歧杆菌目及双歧杆菌科,Odoribacteraceae、Christensenellaceae、弧菌目及弧菌科等为主要特征差异细菌菌类。据物种进化树的样本菌落分布图显示：两组样本厚壁菌门、放线菌门、拟杆菌门、变形菌门、疣微菌门、酸杆菌门和螺旋菌门占主要门类分布。CD组及CN组细菌科类分析结果：毛螺菌科、韦荣球菌科、瘤胃菌科、丹毒丝菌科、红蝽菌科、紫单胞菌科、双歧杆菌科、普雷沃菌科、拟杆菌科、产碱菌科和链球菌科在两组样本中共同存在。同时从该进化树可发现,CD组中红蝽菌科、产碱菌科、丹毒丝菌科和链球菌科相对于CN组优势丰度分布,而普雷沃菌科、拟杆菌科和双歧杆菌科比CN组丰度明显减少。此外,Dethiosulfovibrionaceae、丙酸杆菌科、盐单胞菌科、希万菌科等为CD组特有菌种。结论 CD组的菌群多样性明显少于CN组,CD组有其特有优势菌群。     

Metagenomic Investigation of Bacterial and Archaeal Diversity of Hammam Essalihine Hot Spring from Khenchela,Algeria

Abstract

Hot springs are natural environments where hot groundwater comes out from the earth. Exploring the microbial diversity present in hot springs is important first to determine the microorganisms able to proliferate there and to understand their role in biogeochemical cycles. In Algeria, research concerning microbial populations in those ecosystems is limited. This study describes bacterial and archaeal diversity of the ‘Hammam Essalihine’ hot spring in Khenchela province in north-east Algeria using a culture-independent approach. This is the first microbial diversity investigation in the ‘Hammam Essalihine’ hot spring using next-generation sequencing techniques to assess the species classification of thermophilic microorganisms. Genomic DNA was extracted from water samples and the V4–V5 region of 16S rRNA gene were amplified, sequenced, and analyzed. The average temperature of water varies from 68 to 70?°C. High-throughput sequencing analysis revealed the presence of 21 bacterial phyla, including an unknown phylum and distributed across 42 families and 39 genera. The majority of the sequences were observed to belong to the kingdom Bacteria. The bacterial community from this hot spring is dominated by Proteobacteria (41.52%), Chloroflexi (7.62%), and Bacteroidetes (7.62%), whereas the community of Archaea is scarcely present in the study site and the two identified operational taxonomic units (OTUs) are far from what is known in the GenBank database. The study shows several uncharacterized sequences, indicating that the water of ‘Hammam Essalihine’ hot spring contains undescribed microorganisms. This study is thought to add to the understanding of thermophile diversity and ecology of ‘Hammam Essalihine’ hot spring.     

胜利油藏不同时间细菌群落结构的比较

利用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)和构建16S rRNA基因克隆文库2种方法,对孤岛油田两口井(注水井G和采油井L)在相距9个月的2个时间点(A和B)所采集样品的细菌群落结构进行了比较。DGGE图谱聚类分析表明注水井在2个时间点的微生物群落结构相似性为48.1%,而采油井的相似性只有28.7%。16S rRNA基因克隆文库结果表明,A时间点样品G中的优势菌群为Betaproteobacteria、Gammaproteobacteria,还有Deferribacteres、Firmicutes、Bacteroidetes等;而样品L中,Gammaproteobacteria中的Moraxellaceae含量达到97%。B时间点G中除了优势菌Betaproteobacteria之外,Deferribacteres的数量显著增加,成为优势菌;而L在B时间点优势菌除Gammaproteobacteria外,还有Betaproteobacteria和Firmicutes。采油井中的微生物群落结构随时间发生了显著改变,而注水井变化不显著。这一结果部分揭示了微生物采油过程中地层微生物群落的变化规律,有助于进一步阐明微生物驱油的机理。     

Molecular assessment on impact of uranium ore contamination in soil bacterial diversity

Impact of uranium (U) ore and soluble uranium (at pH 4.0) contamination on agricultural soil bacterial diversity was assessed by using laboratory microcosms for one year. Diversity and abundance of metabolically active bacterial populations in periodically collected microcosm’s samples were analyzed by extracting total RNA and preparation of cDNA followed by analysis of 16S rRNA gene by DGGE and real time PCR. DGGE analysis revealed prominent shift of soil bacterial population due to uranium ore contamination within 12 months while uranium ore along with soluble U completely destroyed the soil bacterial diversity within first six months. Real time PCR based analysis indicated 100–200 folds increase in 16S rRNA gene copies of total as well as individual bacterial taxa in both U ore amended and unamended soils in first six months while increase in incubation period upto 12 months showed reduction of the same only in U ore amended soil. Antagonistic effect of U ore contamination on soil bacterial diversity indicated the severe impact of U mining likely to have on nearby ecosystems. Role of U at acidic pH in destroying the diversity completely is noteworthy as it corroborated the disastrous consequence of acid mine drainage generated from U mine sites.     

Analysis of the composition of bacterial communities in oil reservoirs from a southern offshore Brazilian basin

The aim of this study was to characterize and compare the bacterial community structure of two distinct oil samples from a petroleum field in Brazil by using both molecular, based on the construction of 16S rRNA gene libraries, and cultivation methods. Statistical comparisons of libraries based on Amplified Ribosomal DNA Restriction Analysis (ARDRA) data revealed no significant differences between the communities recovered in the non-biodegraded (NBD) and highly biodegraded oils (HBD). BlastN analysis of the 16S rRNA gene sequences representative of distinct ribotypes from both oils showed the presence of nine different bacterial genera in these samples, encompassing members of the genera Arcobacter, Halanaerobium, Marinobacter, Propionibacterium, Streptomyces, Leuconostoc, Acinetobacter, Bacillus and Streptococcus. Enrichments obtained using oil as inoculum and sole carbon source yielded bacterial isolates showing high 16S rRNA gene sequence similarity with Achromobacter xylosoxidans, Bacillus subtilis, Brevibacillus sp., Dietzia sp. and Methylobacterium sp. Comparison between the data obtained using cultivation-independent and enrichment cultures suggests that different selection of community members may occur when using distinct approaches. All the organisms found, except for Leuconostoc sp. and Streptococus sp., have been previously reported in the literature as hydrocarbon degraders and/or associated to oil field environments.