A few cosmopolitan phylotypes dominate planktonic archaeal assemblages in widely different oceanic provinces

We compared the phylogenetic compositions of marine planktonic archaeal populations in different marine provinces. Samples from eight different environments were collected at two depths (surface and aphotic zone), and 16 genetic libraries of PCR-amplified archaeal 16S rRNA genes were constructed. The libraries were analyzed by using a three-step hierarchical approach. Membrane hybridization experiments revealed that most of the archaeal clones were affiliated with one of the two groups of marine archaea described previously, crenarchaeotal group I and euryarchaeotal group II. One of the 2,328 ribosomal DNA clones analyzed was related to a different euryarchaeal lineage, which was recently recovered from deep-water marine plankton. In temperate regions (Pacific Ocean, Atlantic Ocean, and Mediterranean Sea) both major groups were found at the two depths investigated; group II predominated at the surface, and group I predominated at depth. In Antarctic and subantarctic waters group II was practically absent. The clonal compositions of archaeal libraries were investigated by performing a restriction fragment length polymorphism (RFLP) analysis with two tetrameric restriction enzymes, which defined discrete operational taxonomic units (OTUs). The OTUs defined in this way were phylogenetically consistent; clones belonging to the same OTU were closely related. The clonal diversity as determined by the RFLP analysis was low, and most libraries were dominated by only one or two OTUs. Some OTUs were found in samples obtained from very distant places, indicating that some phylotypes were ubiquitous. A tree containing one example of each OTU detected was constructed, and this tree revealed that there were several clusters within archaeal group I and group II. The members of some of these clusters had different depth distributions.     

Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hilds, Eastern Antarctica

16S rDNA clone library analysis was used to examine the biodiversity and community structure within anoxic sediments of several marine-type salinity meromictic lakes and a coastal marine basin located in the Vestfolds Hills area of Eastern Antarctica. From 69 to 130 (555 total) 16S rDNA clones were analysed from each sediment sample, and restriction fragment length polymorphism (RFLP) and sequence analysis grouped the clones into 202 distinct phylotypes (a clone group with sequence similarity of > 0.98). A number of phylotypes and phylotype groups predominated in all libraries, with a group of 10 phylotypes (31% of clones) forming a novel deep branch within the low G + C Gram-positive division. Other abundant phylotypes detected in several different clone libraries grouped with Prochlorococcus cyanobacteria, diatom chloroplasts, delta proteobacteria ( Desulfosarcina group, Syntrophus and Geobacter / Pelobacter / Desulphuromonas group), order Chlamydiales (Parachlamydiaceae) and Spirochaetales (wall-less Antarctic spirochaetes). Most archaeal clones detected (3.1% of clones) belonged to a highly diverged group of Euryarchaeota clustering with clones previously detected in rice soil, aquifer sediments and hydrothermal vent material. Little similarity existed between the phylotypes detected in this study and other clone libraries based on marine sediment, suggesting that an enormous prokaryotic diversity occurs within marine and marine-derived sediments.     

Terminal-restriction fragment length polymorphism (T-RFLP) screening of a marine archaeal clone library to determine the different phylotypes

T-RFLP clone characterization (screening) was optimized for a fast and basepair-accurate characterization of clones from marine Archaea collected from the Eastern Mediterranean Sea. Because of the high sensitivity of T-RFLP fingerprinting, a protocol was developed where 10 initial PCR cycles gave detectable terminal fragments from clones. Additionally, forward and reverse primers for PCR were individually labeled and detected simultaneously to assess the suitability of the forward and reverse fragments for T-RFLP screening. Based on independent restriction digests with the tetrameric restriction enzymes HhaI, RsaI and HaeIII to characterize the 49 archaeal clones in our library, the clones were grouped into 13 T-RFLP operational taxonomic units (OTUs). Reverse fragments generally gave less heterogeneous fragments in size. The accuracy of T-RFLP screening was evaluated by sequencing representative clones. Closely related clones ( approximately 97% similarity) could only be resolved with multiple restriction digests where forward and reverse fragments were included in the analysis. All fragments from the clone library were detected in the T-RFLP fingerprint from the complex archaeal community. We found representatives of marine group I, II and III Archaea. Thus, the recently discovered low abundant marine group III Archaea could be clearly differentiated from the other clones in our library and comprised a considerable fraction of the clone library ( approximately 12%). Therefore, our T-RFLP screening approach proved successful in characterizing novel archaeal sequences from the marine environment.     

Association of Marine Archaea with the Digestive Tracts of Two Marine Fish Species

Recent studies have shown that archaea which were always thought to live under strict anoxic or extreme environmental conditions are also present in cold, oxygenated seawater, soils, the digestive tract of a holothurian deep-sea-deposit feeder, and a marine sponge. In this study, we show, by using PCR-mediated screening in other marine eukaryotes, that marine archaea are also present in the digestive tracts of flounder and grey mullet, two fish species common in the North Sea, in fecal samples of flounder, and in suspended particulate matter of the North Sea water column. No marine archaea could be detected in the digestive tracts of mussels or the fecal pellets of a copepod species. The archaeal 16S ribosomal DNA clone libraries of feces of flounder and the contents of the digestive tracts of grey mullet and flounder were dominated by group II marine archaea. The marine archaeal clones derived from flounder and grey mullet digestive tracts and feces formed a distinct cluster within the group II marine archaea, with 76.7 to 89.8% similarity to previously described group II clones. Fingerprinting of the archaeal community of flounder digestive tract contents and feces by terminal restriction fragment length polymorphism of archaeal 16S rRNA genes after restriction with HhaI showed a dominant fragment at 249 bp, which is likely to be derived from group II marine archaea. Clones of marine archaea that were closely related to the fish-associated marine archaea clones were obtained from suspended particulate matter of the water column at two stations in the North Sea. Terminal restriction fragment length polymorphism fingerprinting of the archaeal community present in suspended particulate matter showed the same fragment pattern as was found for the archaeal community of the flounder digestive tract contents and feces. These data demonstrate that marine archaea are present in the digestive tracts and feces of very common marine fish. It is possible that the marine archaea associated with the digestive tracts of marine fish are liberated into the water column through the feces and subsequently contribute to the marine archaeal community of suspended particulate matter.     

Geomicrobiology of deep-sea deposits: estimating community diversity from low-temperature seafloor rocks and minerals

The role of deep‐sea microbial communities in the weathering of hydrothermal vent deposits is assessed using mineralogical and molecular biological techniques. The phylogenetic diversity of varied deep‐sea bare rock habitats associated with the oceanic spreading centre at the Juan de Fuca Ridge was accessed using restriction fragment length polymorphism (RFLP) and rDNA sequencing. The mineralogical composition of the deposits used for phylogenetic analysis was determined by X‐ray diffraction in order to determine the proportion and composition of sulphide minerals, and to determine degree of alteration associated with each sample. RFLP analyses resulted in 15 unique patterns, or Operational Taxonomic Units (OTUs). Most environments examined were dominated by only one or two OTUs, which often comprised approximately 60% of the rDNA clones generated from that environment. Only one environment, the Mound, had a representative rDNA clone from every OTU identified in this study. For one other environment, ODP sediments, rDNA clones were all contained in a single OTU. The diversity of the microbial community is found to decrease with decreasing reactivity of the sulphide component in the samples and with increasing presence of alteration products. Phylogenetic analyses reveal that OTUs contain representatives of the epsilon‐, beta‐ and gamma‐subdivisions of the Proteobacteria. OTU1, which dominates clone libraries from every environment and is increasingly dominant with increasing rock alteration, is closely related to a group of chemolithoautotrophic iron‐oxidizing bacteria that have been recently isolated from the deep sea. The apparent abundance and widespread distribution within the samples examined of the putative iron‐oxidizing bacteria that may be represented by OTU1 suggests that this physiological group could play an important role in rock‐weathering and carbon fixation at the seafloor.     

新疆沙湾冷泉沉积物中免培养古菌多样性初步研究

【目的】了解新疆沙湾冷泉沉积物的古菌组成及多样性。【方法】采用免培养法,液氮研磨提取冷泉沉积物总DNA,使用古菌通用引物进行16S rRNA基因扩增,构建16S rRNA基因文库。对阳性克隆进行HhaI限制性酶切分型,选出具有不同酶切图谱的序列进行测序,将所得序列与GenBank数据库中序列比对并构建16S rRNA基因系统发育树。【结果】从冷泉沉积物古菌16S rRNA基因文库中随机挑选了121个阳性克隆,共得到22个不同的可操作分类单元,BLAST结果表明全部克隆子归属于泉古菌门(Crenarchaeote)中免培养类群。系统发育分析归类为Soil-Freshwater-subsurface group和MarinegroupI,2个亚群并且各占整个文库的50%。其中40%左右的克隆子与具有无机碳和硝酸盐同化能力的泉古菌有高的相似性。此外还发现40%的克隆子与低温泉古菌类群具有很高的相似性。【结论】新疆沙湾冷泉沉积物中古菌类群多样性较低,但存有大量高度适应此低温、贫营养环境的泉古菌类群。     

Archaeal population dynamics during sequential reduction processes in rice field soil

The population dynamics of Archaea after flooding of an Italian rice field soil were studied over 17 days. Anoxically incubated rice field soil slurries exhibited a typical sequence of reduction processes characterized by reduction of nitrate, Fe(3+), and sulfate prior to the initiation of methane production. Archaeal population dynamics were followed using a dual approach involving molecular sequence retrieval and fingerprinting of small-subunit (SSU) rRNA genes. We retrieved archaeal sequences from four clone libraries (30 each) constructed for different time points (days 0, 1, 8, and 17) after flooding of the soil. The clones could be assigned to known methanogens (i.e., Methanosarcinaceae, Methanosaetaceae, Methanomicrobiaceae, and Methanobacteriaceae) and to novel euryarchaeotal (rice clusters I, II, and III) and crenarchaeotal (rice clusters IV and VI) lineages previously detected in anoxic rice field soil and on rice roots (R. Grosskopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983-4989, 1998). During the initiation of methanogenesis (days 0 to 17), we detected significant changes in the frequency of individual clones, especially of those affiliated with the Methanosaetaceae and Methanobacteriaceae. However, these findings could not be confirmed by terminal restriction fragment length polymorphism (T-RFLP) analysis of SSU rDNA amplicons. Most likely, the fluctuations in sequence composition of clone libraries resulted from cloning bias. Clonal SSU rRNA gene sequences were used to define operational taxonomic units (OTUs) for T-RFLP analysis, which were distinguished by group-specific TaqI restriction sites. Sequence analysis showed a high degree of conservation of TaqI restriction sites within the different archaeal lineages present in Italian rice field soil. Direct T-RFLP analysis of archaeal populations in rice field soil slurries revealed the presence of all archaeal lineages detected by cloning with a predominance of terminal restriction fragments characteristic of rice cluster I (389 bp), Methanosaetaceae (280 bp), and Methanosarcinaceae/rice cluster VI (182 bp). In general, the relative gene frequency of most detected OTUs remained rather constant over time during the first 17 days after flooding of the soil. Most minor OTUs (e.g., Methanomicrobiaceae and rice cluster III) and Methanosaetaceae did not change in relative frequency. Rice cluster I (37 to 30%) and to a lesser extent rice cluster IV as well as Methanobacteriaceae decreased over time. Only the relative abundance of Methanosarcinaceae (182 bp) increased, roughly doubling from 15 to 29% of total archaeal gene frequency within the first 11 days, which was positively correlated to the dynamics of acetate and formate concentrations. Our results indicate that a functionally dynamic ecosystem, a rice field soil after flooding, was linked to a relatively stable archaeal community structure.     

Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing.

Very small eukaryotic organisms (picoeukaryotes) are fundamental components of marine planktonic systems, often accounting for a significant fraction of the biomass and activity in a system. Their identity, however, has remained elusive, since the small cells lack morphological features for identification. We determined the diversity of marine picoeukaryotes by sequencing cloned 18S rRNA genes in five genetic libraries from North Atlantic, Southern Ocean, and Mediterranean Sea surface waters. Picoplankton were obtained by filter size fractionation, a step that excluded most large eukaryotes and recovered most picoeukaryotes. Genetic libraries of eukaryotic ribosomal DNA were screened by restriction fragment length polymorphism analysis, and at least one clone of each operational taxonomic unit (OTU) was partially sequenced. In general, the phylogenetic diversity in each library was rather great, and each library included many different OTUs and members of very distantly related phylogenetic groups. Of 225 eukaryotic clones, 126 were affiliated with algal classes, especially the Prasinophyceae, the Prymnesiophyceae, the Bacillariophyceae, and the Dinophyceae. A minor fraction (27 clones) was affiliated with clearly heterotrophic organisms, such as ciliates, the chrysomonad Paraphysomonas, cercomonads, and fungi. There were two relatively abundant novel lineages, novel stramenopiles (53 clones) and novel alveolates (19 clones). These lineages are very different from any organism that has been isolated, suggesting that there are previously unknown picoeukaryotes. Prasinophytes and novel stramenopile clones were very abundant in all of the libraries analyzed. These findings underscore the importance of attempts to grow the small eukaryotic plankton in pure culture.     

Diverse microbial communities inhabit Antarctic sponges

Genetic techniques were employed to investigate the archaeal, bacterial and eukaryotic communities associated with the Antarctic sponges Kirkpatrickia varialosa, Latrunculia apicalis, Homaxinella balfourensis, Mycale acerata and Sphaerotylus antarcticus. The phylogenetic affiliation of sponge-derived bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Denaturing gradient gel electrophoresis (DGGE) was used to determine the stability of bacterial associations within each sponge species and across spatial scales. Of the 150 archaeal clones from L. apicalis, K. varialosa and M. acerata screened by restriction fragment length polymorphism (RFLP) analysis, four unique operational taxonomic units (OTUs) were observed and all clustered closely together within the Crenarchaeota. Of the 250 sponge-derived bacterial clones screened by RFLP analysis, 61 were unique OTUs that were not detected during examination of 160 seawater-derived clones. Rarefaction analysis indicated that the clone libraries represented between 44 and 83% of the total estimated diversity. Phylogenetic analysis of sequence data revealed that the bacterial communities present in Antarctic sponges primarily clustered within the Gamma and Alpha proteobacteria and the Cytophaga/Flavobacterium of Bacteroidetes group. Bacterial DGGE analysis for replicate sponge and seawater samples at each Antarctic site revealed that bacterial communities were consistently detected within a particular species regardless of the collection site, with six bacterial bands exclusively associated with a single sponge species. Phylogenetic analysis of sequence data from eukaryotic DGGE analysis revealed that the communities present in Antarctic sponges fell into diatom and dinoflagellate clusters with many sequences having no known close relatives. In addition, seven eukaryotic sequences that were not detected in seawater samples or other sponge species were observed in K. varialosa.     

Phylogenetic diversity of planktonic archaea in the estuarine region of East China Sea

To examine the diversity and structure of archaeal communities in the Yangtze River estuarine region of East China Sea (ECS), the 16S rRNA gene clone libraries of two typical sites were constructed with the archaea-specific primers. In total, 71 clones randomly selected were screened by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and 21 clones with unique RFLP pattern were sequenced. All the sequences are clustered into the two groups of Marine Group I (MGI) and Marine Group II (MGII) which are affiliated with the phyla Crenarchaeota and Euryarchaeota, respectively. MGI clones dominate both libraries with 20 MGI sequences obtained. The majority of sequences are closely related to uncultured marine archaea except for two sequences of which the nearest neighbor is a newly identified isolate of nitrifying marine archaeon Nitrosopumilus maritimus (98% identity). The results indicate that ECS coastal waters are inhabited by archaeal community with low dominance and high diversity corresponding to the complex estuarine environments, suggesting that archaea may perform an important role in the estuarine ecosystem.     

Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel.

Newly described phylogenetic lineages within the domain Archaea have recently been found to be significant components of marine picoplankton assemblages. To better understand the ecology of these microorganisms, we investigated the relative abundance, distribution, and phylogenetic composition of Archaea in the Santa Barbara Channel. Significant amounts of archaeal rRNA and rDNA (genes coding for rRNA) were detected in all samples analyzed. The relative abundance of archaeal rRNA as measured by quantitative oligonucleotide hybridization experiments was low in surface waters but reached higher values (20 to 30% of prokaryotic rRNA) at depths below 100 m. Probes were developed for the two major groups of marine Archaea detected. rRNA originating from the euryarchaeal group (group II) was most abundant in surface waters, whereas rRNA from the crenarchaeal group (group I) dominated at depth. Clone libraries of PCR-amplified archaeal rRNA genes were constructed with samples from 0 and 200 m deep. Screening of libraries by hybridization with specific oligonucleotide probes, as well as subsequent sequencing of the cloned genes, indicated that virtually all archaeal rDNA clones recovered belonged to one of the two groups. The recovery of cloned rDNA sequence types in depth profiles exhibited the same trends as were observed in quantitative rRNA hybridization experiments. One representative of each of 18 distinct restriction fragment length polymorphism types was partially sequenced. Recovered sequences spanned most of the previously reported phylogenetic diversity detected in planktonic crenarchaeal and euryarchaeal groups. Several rDNA sequences appeared to be harbored in archaeal types which are widely distributed in marine coastal waters. In total, data suggest that marine planktonic crenarchaea and euryarchaea of temperate coastal habitats thrive in different zones of the water column. The relative rRNA abundance of the crenarchaeal group suggests that its members constitute a significant fraction of the prokaryotic biomass in subsurface coastal waters.     

Diversity of symbiotic archaeal communities in marine sponges from Korea

A molecular analysis of archaeal communities in eight sponges collected along the coast of Cheju Island, Korea was conducted using terminal-restriction fragment length polymorphism (T-RFLP) in conjunction with sequencing analysis of 16S rDNA clones. The terminal-restriction fragment (T-RF) profiles showed that each sponge had a simple archaeal community represented by a single major peak of the same size except for one unidentified sponge (01CJ20). In order to identify the components of the community, 170 archaeal 16S rDNA clones were recovered from sponges and analyzed by RFLP typing. Sequences of 19 representative clones for all RFLP types found in each sponge were determined and phylogenetic analysis was carried out. Seventeen of these archaeal 16S rDNA clones showed a high similarity to marine group I, belonging to the crenarchaeotes. In the phylogenetic tree, 15 archaeal clones were grouped into five sponge-associated archaeal clusters. In the unidentified sponge sample (01CJ20), one major T-RF peak was represented by a single RFLP type (40 clones), which implied a specific relationship between the sponge and its symbiotic archaeal components.     

利用小亚基核糖体RNA 技术分析温室黄瓜近根土壤古菌和真菌多样性

土壤古菌和真菌在温室生态系统是仅次于细菌的微生物,具有类似于细菌的重要生态功能。通过构建古菌16S rRNA和真菌18S rRNA基因克隆文库,分析温室黄瓜近根土壤古菌和真菌群落结构组成,为开发利用温室这一特殊的生态环境中丰富的微生物资源以及理解微生物与植物间的互作提供参考依据。采用研磨-冻融-溶菌酶-蛋白酶K-SDS热处理以及CTAB处理等理化方法,提取和纯化微生物总DNA,构建古菌16S rRNA和真菌18S rRNA基因克隆文库。利用DOTUR软件将古菌和真菌序列按照相似性97％的标准分成若干个可操作分类单元 (OTUs)。土壤古菌克隆文库主要包括泉古菌门和未分类的古菌两大类,并有少部分广域古菌类群,所有泉古菌均属于热变形菌纲,共45个OTUs;真菌克隆文库包括真菌门的大多数亚门真菌,共24个OTUs,未发现担子菌亚门真菌。古菌多样性比较丰富,且发现少量的广域古菌 (甲烷菌),这一情况可能与温室长期高温高湿,高有机质含量,土壤处于缺氧环境有关;土壤真菌的优势种群为子囊菌,占到土壤真菌的80％以上,这可能与绝大多数植物真菌性病害属于土传病害,通过菌丝体、菌核或子囊壳在土壤病残体中越冬有一定的关系。     

Archaeal Population Dynamics during Sequential Reduction Processes in Rice Field Soil

The population dynamics of Archaea after flooding of an Italian rice field soil were studied over 17 days. Anoxically incubated rice field soil slurries exhibited a typical sequence of reduction processes characterized by reduction of nitrate, Fe³⁺, and sulfate prior to the initiation of methane production. Archaeal population dynamics were followed using a dual approach involving molecular sequence retrieval and fingerprinting of small-subunit (SSU) rRNA genes. We retrieved archaeal sequences from four clone libraries (30 each) constructed for different time points (days 0, 1, 8, and 17) after flooding of the soil. The clones could be assigned to known methanogens (i.e., Methanosarcinaceae, Methanosaetaceae, Methanomicrobiaceae, and Methanobacteriaceae) and to novel euryarchaeotal (rice clusters I, II, and III) and crenarchaeotal (rice clusters IV and VI) lineages previously detected in anoxic rice field soil and on rice roots (R. Grosskopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983–4989, 1998). During the initiation of methanogenesis (days 0 to 17), we detected significant changes in the frequency of individual clones, especially of those affiliated with the Methanosaetaceae and Methanobacteriaceae. However, these findings could not be confirmed by terminal restriction fragment length polymorphism (T-RFLP) analysis of SSU rDNA amplicons. Most likely, the fluctuations in sequence composition of clone libraries resulted from cloning bias. Clonal SSU rRNA gene sequences were used to define operational taxonomic units (OTUs) for T-RFLP analysis, which were distinguished by group-specific TaqI restriction sites. Sequence analysis showed a high degree of conservation of TaqI restriction sites within the different archaeal lineages present in Italian rice field soil. Direct T-RFLP analysis of archaeal populations in rice field soil slurries revealed the presence of all archaeal lineages detected by cloning with a predominance of terminal restriction fragments characteristic of rice cluster I (389 bp), Methanosaetaceae (280 bp), and Methanosarcinaceae/rice cluster VI (182 bp). In general, the relative gene frequency of most detected OTUs remained rather constant over time during the first 17 days after flooding of the soil. Most minor OTUs (e.g., Methanomicrobiaceae and rice cluster III) and Methanosaetaceae did not change in relative frequency. Rice cluster I (37 to 30%) and to a lesser extent rice cluster IV as well as Methanobacteriaceae decreased over time. Only the relative abundance of Methanosarcinaceae (182 bp) increased, roughly doubling from 15 to 29% of total archaeal gene frequency within the first 11 days, which was positively correlated to the dynamics of acetate and formate concentrations. Our results indicate that a functionally dynamic ecosystem, a rice field soil after flooding, was linked to a relatively stable archaeal community structure.     

四种红树植物根际土壤微生物Ⅰ型和Ⅱ型PKS基因的检测与多样性分析

[目的]探究红树林土壤中聚酮合酶(Polyketide synthase,PKS)基因的多样性和新颖性.[方法]用Ⅰ型和Ⅱ型PKS基因酮基合成酶(Ketosynthase,KS)域的简并引物对海南清澜港红树林海莲、黄槿、银叶、老鼠簕4种红树根际土壤样品中DNA进行PCR扩增,之后利用PCR-限制性酶切片段多样性(PCR-RFLP)和测序分析法对Ⅰ型和Ⅱ型PKS基因的多样性进行探讨.[结果]对得到的72条Ⅰ型PKS基因的酮基合成酶(Ketosynthase,KS)域DNA序列进行PCR-RFLP分析,共得到51个可操作分类单元(Operational taxonomic unit,OTUs),其中37个OTUs为单克隆产生,没有明显的优势OTU.选取了26个代表不同OTU的克隆进行测序分析,这些序列与GenBank中已知序列的最大相似率均未超过85％. KS域氨基酸序列的系统发育分析显示,所得KS域来源广泛,包括蓝细菌门(Cyanobacteria)、变形杆菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和一些未可培养细菌;对55条PKSⅡ基因KS域DNA序列的PCR-RFLP分析后共得到25个OTUs,有两个明显的优势OTUs,代表的克隆子数所占比例超过10％.[结论]PCR-RFLP分析表明红树林根际土壤中存在着丰富多样的Ⅰ型和Ⅱ型PKS基因,且前者多样性更高;低的序列相似度表明所获得的PKSⅠ基因KS域序列独特;系统发育分析表明得到的PKSⅠ基因来源广泛.     

Quantitatively evaluating mistaken clone assignments by RFLP analysis of 16S rRNA genes: a case study

We quantitatively evaluated the errors of clone assignment based on the restriction fragment length polymorphism (RFLP) pattern of 16S rRNA genes. Eighty clones were randomly selected from a 16S rRNA gene library and were categorized into 35 operational taxonomic units (OTU) based on their indistinguishable enzyme restriction patterns of 3 tetrameric restriction enzymes RsaI, BsuRI, and HinfI. All of these clones were then sequenced and were reassigned into 36-53 OTUs using the DOTUR program when sequence similarities of 95%-100% were used. The number of the identically assigned clones ranged from 53 to 61 and the percentage varied from 66.3% to 76.3%. The Shannon-Weaver index for the bacterial community observed by RFLP analysis was 2.75, equal to that estimated by DOTUR at a 97% sequence similarity. Compared with clones assigned with the DOTUR program at a 97% sequence similarity, only 61 clones (76.3%) were correctly assigned by RFLP analysis. Six clones (7.5%) were assigned mistakenly at the phylum level, and the positions of 13 clones (16.2%) were phylogenetically different at a lower taxonomic rank.     

Bacterial diversity at different depths in lead-zinc mine tailings as revealed by 16S rRNA gene libraries

Bacterial communities at 10 cm, 100 cm, and 200 cm depths in a 100-year-old lead-zinc tailing heap were evaluated by constructing 16S rRNA gene libraries. In total, 98 operational taxonomic units (OTUs) were identified from 193 clones at a 3% sequence difference level. The OTU number and species richness decreased with the depth. Species composition was significantly different between the three libraries. Fifty-seven percent of the examined clones were Acidobacteria and 27% belonged to Proteobacteria. Other sequences included Chloroflexi, Firmicutes, Chlamydiae, Actinobacteria, Gemmatimonadetes, Nitrospira, and three unclassified OTUs. Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were mainly distributed in the rhizosphere of naturally colonizing plants; however, Deltaproteobacteria, Acidobacteria, and Chloroflexi tended to inhabit the deeper tailings (below the 100 cm-depth).     

Genetic Diversity of Eukaryotic Microorganisms in Lake Taihu,a Large Shallow Subtropical Lake in China

We investigated the genetic diversity of eukaryotic microorganisms (0.8-20 mum) by sequencing cloned 18S rRNA genes in six genetic libraries constructed from six locations in Lake Taihu, a large shallow subtropical lake in China. Genetic libraries of eukaryotic ribosomal RNA were screened by restriction fragment length polymorphism (RFLP) analysis, and one clone representative of each RFLP pattern was partially sequenced. A total of 528 clones were clustered into 165 RFLP patterns and finally into 131 operational taxonomic unit (OTUs). Phylogenetic analysis revealed that each library included many unique OTUs, as well as members of distantly related phylogenetic groups. A majority of the clones were from alveolates, stramenopiles, cercozoa, cryptophytes, chlorophytes, and fungi, with members of choanoflagellida, euglenida, centroheliozoa, ancyromonadidae, ichthyosporea, and kathablepharid representing a minor fraction of the library. Six OTUs (15 clones) were not related to any known eukaryotic group. Canonical correspondence analysis suggested that the differences in eukaryotic microorganism community composition of in the six regions were partially related to trophic status, sediment resuspension, and top-down regulation by metazooplankton.     

Microbial community in a geothermal aquifer associated with the subsurface of the Great Artesian Basin, Australia

To investigate the biomass and phylogenetic diversity of the microbial community inhabiting the deep aquifer of the Great Artesian Basin (GAB), geothermal groundwater gushing out from the aquifer was sampled and analyzed. Microbial cells in the groundwater were stained with acridine orange and directly counted by epifluorescence microscopy. Microbial cells were present at a density of 10⁸–10⁹ cells per liter of groundwater. Archaeal and bacterial small-subunit rRNA genes (rDNAs) were amplified by PCR with Archaea- and Bacteria-specific primer sets, and clone libraries were constructed separately. A total of 59 clones were analyzed in archaeal and bacterial 16S rDNA libraries, respectively. The archaeal 16S rDNA clones were divided into nine operated taxonomic units (OTUs) by restriction fragment length polymorphism. These OTUs were closely related to the methanogenic genera Methanospirillum and Methanosaeta, the heterotrophic genus Thermoplasma, or miscellaneous crenarchaeota group. More than one-half of the archaeal clones (59% of total 59 clones) were placed beside phylogenetic clusters of methanogens. The majority of the methanogen-related clones (83%) was closely related to a group of hydrogenotrophic methanogens (genus Methanospirillum). The bacterial OTUs branched into seven phylogenetic clusters related to hydrogen-oxidizing thermophiles in the genera Hydrogenobacter and Hydrogenophilus, a sulfate-reducing thermophile in the genus Thermodesulfovibrio, chemoheterotropic bacteria in the genera Thermus and Aquaspirillum, or the candidate division OP10. Clones closely related to the thermophilic hydrogen-oxidizers in the genera Hydrogenobacter and Hydrogenophilus were dominant in the bacterial clone library (37% of a total of 59 clones). The dominancy of hydrogen-users strongly suggested that H₂ plays an important role as a primary substrate in the microbial ecosystem of this deep geothermal aquifer.     

High 16S rDNA bacterial diversity in glacial meltwater lake sediment,Bratina Island,Antarctica

The microbial diversity in maritime meltwater pond sediments from Bratina Island, Ross Sea, Antarctica was investigated by 16S rDNA-dependent molecular phylogeny. Investigations of the vertical distribution, phylogenetic composition, and spatial variability of Bacteria and Archaea in the sediment were carried out. Results revealed the presence of a highly diverse bacterial population and a significantly depth-related composition. Assessment of 173 partial 16S rDNA clones analyzed by amplified rDNA restriction analysis (ARDRA) using tetrameric restriction enzymes (HinP1I 5'G/CGC3'and Msp I. 5'C/CGG3', BioLabs) revealed 153 different bacterial OTUs (operational taxonomic units). However, only seven archaeal OTUs were detected, indicating low archaeal diversity. Based on ARDRA results, 30 bacterial clones were selected for sequencing and the sequenced clones fell into seven major lineages of the domain Bacteria; the alpha, gamma, and delta subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides, the Spirochaetaceae, and the Actinobacteria. All of the archaeal clones sequenced belonged to the group Crenarchaeota and phylogenetic analysis revealed close relationships with members of the deep-branching Group 1 Marine Archaea.