Phylogenetic diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis

In this study, for the first time the diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis collected from the Sousern Basin of Lake Baikal was investigated employing cultivation-independent approaches. In total, 102 bacterial 16S rRNA clones were screened using restriction fragment length polymorphism (RFLP) and 30 were selected for sequencing. BLASTN and phylogenetic analysis based on near full length 16S rDNA sequences showed that 22 operational taxonomic units (OTUs) were clustered in six known phyla: Actinobacteria (8 OTUs), alpha-Proteobacteria (4 OTUs), beta-Proteobacteria (4 OTUs), Verrucomicrobia (4 OTUs), Nitrospiracea (1 OTU) and Bacteroidetes (1 OTU). Remarkably all phylotypes were affiliated to uncultured microorganisms, however, all alpha-Proteobacteria sequences were closely related to bacteria derived from the freshwater sponge Spongilla lacustris. Our results reveal a high diversity in the L. baicalensis bacterial community and provide an insight into microbial ecology and diversity within freshwater sponges inhabiting the ancient Lake Baikal ecosystem.     

Microbial diversity in an in situ reactor system treating monochlorobenzene contaminated groundwater as revealed by 16S ribosomal DNA analysis

A molecular approach based on the construction of 16S ribosomal DNA clone libraries was used to investigate the microbial diversity of an underground in situ reactor system filled with the original aquifer sediments. After chemical steady state was reached in the monochlorobenzene concentration between the original inflowing groundwater and the reactor outflow, samples from different reactor locations and from inflowing and outflowing groundwater were taken for DNA extraction. Small-subunit rRNA genes were PCR-amplified with primers specific for Bacteria, subsequently cloned and screened for variation by restriction fragment length polymorphism (RFLP). A total of 87 bacterial 16S rDNA genes were sequenced and subjected to phylogenetic analysis. The original groundwater was found to be dominated by a bacterial consortium affiliated with various members of the class of Proteobacteria, by phylotypes not affiliated with currently recognized bacterial phyla, and also by sporulating and non-sporulating sulfate-reducing bacteria. The most occurring clone types obtained from the sediment samples of the reactor were related to the beta-Proteobacteria, dominated by sequences almost identical to the widespread bacterium Alcaligenes faecalis, to low G+C gram-positive bacteria and to Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) within the gamma subclass of Proteobacteria in the upper reactor sector. Although bacterial phylotypes originating from the groundwater outflow of the reactors also grouped within different subdivisions of Proteobacteria and low G+C gram-positive bacteria, most of the 16S rDNA sequences were not associated with the sequence types observed in the reactor samples. Our results suggest that the different environments were inhabited by distinct microbial communities in respect to their taxonomic diversity, particular pronounced between sediment attached microbial communities from the reactor samples and free-living bacteria from the groundwater in- and outflow.     

Microbial diversity in ostrich ceca as revealed by 16S ribosomal RNA gene clone library and detection of novel Fibrobacter species

The ostrich (Struthio camelus) is a herbivorous bird and although the hindgut is known as the site for fiber digestion, little is known about the microbial diversity in the ostrich hindgut. Our aim was to analyze the microbial diversity in ostrich ceca using a 16S ribosomal RNA gene (rDNA) clone library approach. A total of 310 clones were sequenced and phylogenetically analyzed and were classified into 110 operational taxonomy units (OTUs) based on a 98% similarity criterion. The similarity of the sequences ranged from 86 to 99% and 95 OTUs had less than 98% similarity to the sequences in the public databases. Coverage and the Shannon–Wiener index (H′) of the library were 83.9% and 4.29, respectively. The sequences were assigned to the following 6 phyla: Firmicutes (50.9% of the total number of sequences), Bacteroidetes (39.4%), Fibrobacteres (6.5%), Euryarchaeota (1.9%), Spirochaetes (1.0%), and Verrucomicrobia (0.3%); approximately 90% of the sequences were affiliated with Firmicutes and Bacteroidetes. The only OTU of Fibrobacteres (OTU 107), had 93 and 90% similarity to Fibrobacter succinogenes and F. intestinalis, respectively, suggesting a new species of Fibrobacter in ostrich ceca. Clostridium coccoides and C. leptum formed major groups within the Firmicutes. There was no OTU with high similarity (≥98%) to the 16S rDNA of cultivated fibrolytic bacteria in our library. Although two OTUs were affiliated with Euryarchaeota, no sequence was affiliated with methanogenic Archaea. This study presents the very complex ostrich cecal microbial community, in which the majority of the bacterial species have not yet been cultivated.     

How stable is stable? Function versus community composition.

The microbial community dynamics of a functionally stable, well-mixed, methanogenic reactor fed with glucose were analyzed over a 605-day period. The reactor maintained constant pH and chemical oxygen demand removal during this period. Thirty-six rrn clones from each of seven sampling events were analyzed by amplified ribosomal DNA restriction analysis (ARDRA) for the Bacteria and Archaea domains and by sequence analysis of dominant members of the community. Operational taxonomic units (OTUs), distinguished as unique ARDRA patterns, showed reproducible distribution for three sample replicates. The highest diversity was observed in the Bacteria domain. The 16S ribosomal DNA Bacteria clone library contained 75 OTUs, with the dominant OTU accounting for 13% of the total clones, but just 21 Archaea OTUs were found, and the most prominent OTU represented 50% of the clones from the respective library. Succession in methanogenic populations was observed, and two periods were distinguished: in the first, Methanobacterium formicicum was dominant, and in the second, Methanosarcina mazei and a Methanobacterium bryantii-related organism were dominant. Higher variability in Bacteria populations was detected, and the temporal OTU distribution suggested a chaotic pattern. Although dominant OTUs were constantly replaced from one sampling point to the next, phylogenetic analysis indicated that inferred physiologic changes in the community were not as dramatic as were genetic changes. Seven of eight dominant OTUs during the first period clustered with the spirochete group, although a cyclic pattern of substitution occurred among members within this order. A more flexible community structure characterized the second period, since a sequential replacement of a Eubacterium-related organism by an unrelated deep-branched organism and finally by a Propionibacterium-like species was observed. Metabolic differences among the dominant fermenters detected suggest that changes in carbon and electron flow occurred during the stable performance and indicate that an extremely dynamic community can maintain a stable ecosystem function.     

Microbial diversity and heterogeneity in sandy subsurface soils

Microbial community diversity and heterogeneity in saturated and unsaturated subsurface soils from Abbott's Pit in Virginia (1.57, 3.25, and 4.05 m below surface) and Dover Air Force Base in Delaware (6.00 and 7.50 m below surface) were analyzed using a culture-independent small-subunit (SSU) rRNA gene (rDNA)-based cloning approach. Four to six dominant operational taxonomic units (OTUs) were identified in 33 to 100 unique SSU rDNA clones (constituting about 40 to 50% of the total number of SSU rDNA clones in the clone library) from the saturated subsurface samples, whereas no dominant OTUs were observed in the unsaturated subsurface sample. Less than 10% of the clones among samples from different depths at the same location were identical, and the proportion of overlapping OTUs was lower for the samples that were vertically far apart than for adjacent samples. In addition, no OTUs were shared between the Abbott's Pit and Dover samples. The majority of the clones (80%) had sequences that were less than 5% different from those in the current databases. Phylogenetic analysis indicated that most of the bacterial clones were affiliated with members of the Proteobacteria family (90%), gram-positive bacteria (3%), and members of the Acidobacteria family (3%). Principal component analysis revealed that samples from different geographic locations were well separated and that samples from the same location were closely grouped together. In addition, the nonsaturated subsurface samples from Abbott's Pit clustered together and were well separated from the saturated subsurface soil sample. Finally, the overall diversity of the subsurface samples was much lower than that of the corresponding surface soil samples.     

Bacterial diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis

Bacterial communities in buffalo rumen were characterized using a culture-independent approach for a pooled sample of rumen fluid from 3 adult Surti buffaloes. Buffalo rumen is likely to include species of various bacterial phyla, so 16S rDNA sequences were amplified and cloned from the sample. A total of 191 clones were sequenced and similarities to known 16S rDNA sequences were examined. About 62.82% sequences (120 clones) had >90% similarity to the 16S rDNA database sequences. Furthermore, about 34.03% of the sequences (65 clones) were 85–89% similar to 16S rDNA database sequences. For the remaining 3.14%, the similarity was lower than 85%. Phylogenetic analyses were also used to infer the makeup of bacterial communities in the rumen of Surti buffalo. As a result, we distinguished 42 operational taxonomic units (OTUs) based on unique 16S r DNA sequences: 19 OTUs affiliated to an unidentified group (45.23% of total OTUs), 11 OTUs of the phylum Firmicutes, also known as the low G+C group (26.19%), 7 OTUs of theCytophaga-Flexibacter-Bacteroides phylum (16.66%), 4 OTUs of Spirochaetes (9.52%), and 1 OTU of Actinobacteria (2.38%). These include 10 single-clone OTUs, so Good’s coverage (94.76%) of 16S rRNA libraries indicated that sequences identified in the libraries represent the majority of bacterial diversity present in rumen.     

Microbial Diversity and Heterogeneity in Sandy Subsurface Soils

Microbial community diversity and heterogeneity in saturated and unsaturated subsurface soils from Abbott's Pit in Virginia (1.57, 3.25, and 4.05 m below surface) and Dover Air Force Base in Delaware (6.00 and 7.50 m below surface) were analyzed using a culture-independent small-subunit (SSU) rRNA gene (rDNA)-based cloning approach. Four to six dominant operational taxonomic units (OTUs) were identified in 33 to 100 unique SSU rDNA clones (constituting about 40 to 50% of the total number of SSU rDNA clones in the clone library) from the saturated subsurface samples, whereas no dominant OTUs were observed in the unsaturated subsurface sample. Less than 10% of the clones among samples from different depths at the same location were identical, and the proportion of overlapping OTUs was lower for the samples that were vertically far apart than for adjacent samples. In addition, no OTUs were shared between the Abbott's Pit and Dover samples. The majority of the clones (80%) had sequences that were less than 5% different from those in the current databases. Phylogenetic analysis indicated that most of the bacterial clones were affiliated with members of the Proteobacteria family (90%), gram-positive bacteria (3%), and members of the Acidobacteria family (3%). Principal component analysis revealed that samples from different geographic locations were well separated and that samples from the same location were closely grouped together. In addition, the nonsaturated subsurface samples from Abbott's Pit clustered together and were well separated from the saturated subsurface soil sample. Finally, the overall diversity of the subsurface samples was much lower than that of the corresponding surface soil samples.     

采用未培养技术对荷斯坦奶牛瘤胃细菌多样性进行初步分析

采用未培养(Culture independent)技术直接从荷斯坦奶牛瘤胃液中提取瘤胃细菌微生物混合DNA(也叫元基因组DNA),利用细菌16SrDNA通用引物27F与1492R,扩增瘤胃混合微生物的16SrDNA,根据16SrDNA序列对瘤胃细菌多样性进行初步分析。通过16SrDNA序列同源性分析,发现有多于一半以上的序列与可培养的菌株的同源性小于90%,属于不可培养的菌株。选用45条测得序列与已知序列构建系统发育树,分析结果表明,它们分属于两大类LGCGPB(the lowG CGram positivebac teria)和CFB(Cytophaga_Flexibacter $CBacteroides group),剩下的克隆尚难确定其分类地位,可能是代表新属和种的序列,这些序列已向GenBank提交并得到序列号(AY986777_AY986791)。     

Novel predominant archaeal and bacterial groups revealed by molecular analysis of an anaerobic sludge digester

A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anaerobic sludge digester. Two 16S rRNA gene libraries were constructed using total genomic DNA, and amplified by polymerase chain reaction (PCR) using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 246 and 579 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was performed using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota and Crenarchaeota. Interestingly, we detected a novel monophyletic group of 164 clones representing 66.6% of the archaeal library. Culture enrichment and probe hybridization show that this group grows better under formate or H2-CO2. Within the bacterial library 95.6% of the operational taxonomic units (OTUs) represent novel putative phylotypes never described before, and affiliated with eight divisions. The Bacteroidetes phylum is the most abundant and diversified phylogenetic group representing 38.8% of the OTUs, followed by the gram-positives (27.7%) and the Proteobacteria (21.3%). Sequences affiliated with phylogenetic divisions represented by few cultivated representatives such as the Chloroflexi, Synergistes, Thermotogales or candidate divisions such as OP9 and OP8 are represented by <5% of the total OTUs. A comprehensive set of 15 16S and 23S rRNA-targeted oligonucleotide hybridization probes was used to quantify these major groups by dot blot hybridization within 12 digester samples. In contrast to the clone library, Firmicutes and Actinobacteria together accounted for 21.8 +/- 14.9% representing the most abundant phyla. They were surprisingly followed by the Chloroflexi representing 20.2 +/- 4.6% of the total 16S rRNA. The Proteobacteria and the Bacteroidetes group accounted for 14.4 +/- 4.9% and 14.5 +/- 4.3%, respectively, WWE1, a novel lineage, accounted for 11.9 +/- 3.1% while Planctomycetes and Synergistes represented <2% each. Using the novel set of probes we extended the coverage of bacterial populations from 52% to 85.3% of the total rRNA within the digester samples.     

Effect of process temperature on bacterial and archaeal communities in two methanogenic bioreactors treating organic household waste

The bacterial and archaeal community structure was examined in two methanogenic anaerobic digestion processes degrading organic household waste at mesophilic (37 degrees C) and thermophilic (55 degrees C) temperatures. Analysis of bacterial clone libraries revealed a predominance of Bacteroidetes (34% of total clones) and Chloroflexi (27%) at the mesophilic temperature. In contrast, in the thermophilic clone library, the major group of clones were affiliated with Thermotogae (61%). Within the domain Archaea, the phyla Euryarchaeota and Crenarchaeota were both represented, the latter only at the mesophilic temperature. The dominating archaeons grouped with Methanospirillum and Methanosarcina species at the mesophilic and thermophilic temperature, respectively. Generally, there was a higher frequency of different sequences at the lower temperature, suggesting a higher diversity compared to the community present at the thermophilic temperature. Furthermore, it was not only the species richness that was affected by temperature, but also the phylogenetic distribution of the microbial populations.     

Characterisation of the microbial diversity in a pig manure storage pit using small subunit rDNA sequence analysis

The microbial community structure of pig manure slurry (PMS) was determined with comparative analysis of 202 bacterial, 44 archaeal and 33 eukaryotic small subunit (SSU) rDNA partial sequences. Based on a criterion of 97% of sequence similarity, the phylogenetic analyses revealed a total of 108, eight and five phylotypes for the Bacteria, Archaea and Eukarya lineages, respectively. Only 36% of the bacterial phylotypes were closely related (>or=97% similarity) to any previously known sequence in databases. The bacterial groups most often represented in terms of phylotype and clone abundance were the Eubacterium (22% of total sequences), the Clostridium (15% of sequences), the Bacillus-Lactobacillus-Streptococcus subdivision (20% of sequences), theMycoplasma and relatives (10% of sequences) and the Flexibacter-Cytophaga-Bacteroides (20% of sequences). The global microbial community structure and phylotype diversity show a close relationship to the pig gastrointestinal tract ecosystem whereas phylotypes from the Acholeplasma-Anaeroplasma and the Clostridium purinolyticum groups appear to be better represented in manure. Archaeal diversity was dominated by three phylotypes clustering with a group of uncultured microorganisms of unknown activity and only distantly related to the Thermoplasmales and relatives. Other Archaea were methanogenic H2/CO2 utilisers. No known acetoclastic Archaea methanogen was found. Eukaryotic diversity was represented by a pluricellular nematode, two Alveolata, a Blastocystis and an Entamoebidae. Manure slurry physico-chemical characteristics were analysed. Possible inhibitory effects of acetate, sulphide and ammonia concentrations on the microbial anaerobic ecosystem are discussed.     

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.     

Molecular phylogenetic diversity of bacteria associated with the leachate of a closed municipal solid waste landfill

A 16S rDNA-based molecular study was performed to determine the nature of the bacterial constituents of the leachate from a closed municipal solid waste landfill. Total community DNA was extracted and bacterial 16S rRNA genes were subsequently amplified and cloned. Recombinant rDNA clones in the library were randomly selected, and they were sequenced for a single run and then grouped. A total of 76 sequence types representing 138 randomly selected nonchimeric clones were identified. Full-length sequencing and phylogenetic analysis of the sequence types revealed that more than 90% of the screened clones were affiliated with low-G+C gram-positive bacteria (38.4%), Proteobacteria (35.5%), the Cytophaga Flexibacter Bacteroides group (11.6%), and Spirochaetes (5.1%). Minor portions were affiliated with Verrucomicrobia (2.9%), candidate division OP11 (2.2%), and the green nonsulfur bacteria, Cyanobacteria and the Deinococcus Thermus group (each <1.0%). Although some rDNA sequences clustered with genera or taxa that were classically identified within anaerobic treatment systems and expected with known functions, a substantial fraction of the clone sequences showed relatively low levels of similarity with any other reported rDNA sequences and thus were derived from unknown taxa. These results suggest that bacterial communities in landfill environment are far more complex than previously expected and remain largely unexplored.     

Analysis of the bacterial diversity in the fecal material of the endangered Yangtze finless porpoise, <Emphasis Type="Italic">Neophocaena phocaenoides asiaeorientalis</Emphasis>

The aim of this study was to determine the bacteria present in the fecal material of the endangered Yangtze finless porpoise, Neophocaena phocaenoides asiaeorientalis. Fecal samples were collected from 12 Yangtze finless porpoises living in the wild at Poyang Lake, located in Jiangxi Province, China. To determine the bacterial diversity, a 16S rRNA gene clone library using the bacterial PCR primers fD1 and rP2, was prepared. A total of 138 near-full-length sequences were analyzed and 39 operational taxonomic units (OTUs) were identified. Sequences showing ≥97% similarity were grouped together as an OTU. Six different phyla were identified in which 38 OTUs were classified. Most of the OTUs contained sequences belonged to the phylum Firmicutes (51.3%), followed by Tenericutes (17.9%), Proteobacteria (15.4%), Actinobacteria (7.7%), Deinococcus-Thermus (2.6%) and Cyanobacteria (2.6%). A phylum could not be assigned for one clone within one OTU (2.6%). It appears that the Yangtze finless porpoise has a more diverse range of bacteria compared to other aquatic mammals, such as seals.     

Prokaryotic diversity in Zostera noltii-colonized marine sediments

The diversity of microorganisms present in a sediment colonized by the phanerogam Zostera noltii has been analyzed. Microbial DNA was extracted and used for constructing two 16S rDNA clone libraries for Bacteria and Archaea. Bacterial diversity was very high in these samples, since 57 different sequences were found among the 60 clones analyzed. Eight major lineages of the Domain Bacteria were represented in the library. The most frequently retrieved bacterial group (36% of the clones) was delta-Proteobacteria related to sulfate-reducing bacteria. The second most abundant group (27%) was gamma-Proteobacteria, including five clones closely related to S-oxidizing endosymbionts. The archaeal clone library included members of Crenarchaeota and Euryarchaeota, with nine different sequences among the 15 analyzed clones, indicating less diversity when compared to the Bacteria organisms. None of these sequences was closely related to cultured Archaea organisms.     

Comparison of bacterioplankton communities in three mariculture ponds farming different commercial animals in subtropical Chinese coast

In order to explore the responses of the bacterioplankton community to different types of aquaculture environments, three mariculture ponds comprised of groupers (Epinephelus diacanthus, ED), prawns (Penaeus vannamei, PV), and abalone (Haliotis diversicolor supertexta, HDS) in southeast, coastal China were investigated. The free-living bacterial diversity was analyzed through the construction of 16S rDNA clone library. A total of 203 16S rDNA sequences from three clone libraries were classified into 118 operational taxonomic units (OTUs), of which 51, 31, and 42 OTUs were distributed in the ED, PV, and HDS pond, respectively, with Bacteroidetes (30.6%), Actinobacteria (55.2%), and Cyanobacteria (32.8%) as the dominant division in the respective ponds. Meanwhile, each pond occupied some unique OTUs that were affiliated with uncommon (sub-)phyla, such as candidate OP11 division, Acidobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia. Bacterial diversity in the ED pond was the richest, followed by the HDS and the PV pond. OTUs of 61.9% and 94.9% have less than 90% and 97% similarity to their nearest neighbors in public databases, respectively. All OTUs were grouped into 67 clusters, covering 11 (sub-)phyla. The OTUs only from single pond distributed in 53 clusters (79.1%), the OTUs shared by two ponds were affiliated with 14 clusters (20.9%), and none of clusters was formed by the OTUs which commonly originated from the three pond libraries, suggesting that the composition of bacterial populations in these ponds were significantly different. These results indicate that the aquatic environment created by different mariculture animals may foster very special and complex bacterial communities. Handling editor: David Philip Hamilton     

Archaea and the prokaryote-to-eukaryote transition.

Since the late 1970s, determining the phylogenetic relationships among the contemporary domains of life, the Archaea (archaebacteria), Bacteria (eubacteria), and Eucarya (eukaryotes), has been central to the study of early cellular evolution. The two salient issues surrounding the universal tree of life are whether all three domains are monophyletic (i.e., all equivalent in taxanomic rank) and where the root of the universal tree lies. Evaluation of the status of the Archaea has become key to answering these questions. This review considers our cumulative knowledge about the Archaea in relationship to the Bacteria and Eucarya. Particular attention is paid to the recent use of molecular phylogenetic approaches to reconstructing the tree of life. In this regard, the phylogenetic analyses of more than 60 proteins are reviewed and presented in the context of their participation in major biochemical pathways. Although many gene trees are incongruent, the majority do suggest a sisterhood between Archaea and Eucarya. Altering this general pattern of gene evolution are two kinds of potential interdomain gene transferrals. One horizontal gene exchange might have involved the gram-positive Bacteria and the Archaea, while the other might have occurred between proteobacteria and eukaryotes and might have been mediated by endosymbiosis.     

Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods

A combination of culture-dependent and culture-independent methodologies (Bacteria and Archaea 16S rRNA gene clone library analyses) was used to determine the microbial diversity present within a geographically distinct high Arctic permafrost sample. Culturable Bacteria isolates, identified by 16S rRNA gene sequencing, belonged to the phyla Firmicutes, Actinobacteria and Proteobacteria with spore-forming Firmicutes being the most abundant; the majority of the isolates (19/23) were psychrotolerant, some (11/23) were halotolerant, and three isolates grew at -5 degrees C. A Bacteria 16S rRNA gene library containing 101 clones was composed of 42 phylotypes related to diverse phylogenetic groups including the Actinobacteria, Proteobacteria, Firmicutes, Cytophaga - Flavobacteria - Bacteroides, Planctomyces and Gemmatimonadetes; the bacterial 16S rRNA gene phylotypes were dominated by Actinobacteria- and Proteobacteria-related sequences. An Archaea 16S rRNA gene clone library containing 56 clones was made up of 11 phylotypes and contained sequences related to both of the major Archaea domains (Euryarchaeota and Crenarchaeota); the majority of sequences in the Archaea library were related to halophilic Archaea. Characterization of the microbial diversity existing within permafrost environments is important as it will lead to a better understanding of how microorganisms function and survive in such extreme cryoenvironments.     

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.     

新疆沙湾冷泉沉积物的细菌系统发育多样性

为了解新疆沙湾冷泉沉积物的细菌群落组成与类群多样性,利用免培养方法直接从沙湾冷泉沉积物中提取环境总DNA,构建细菌16S rRNA基因文库。对随机挑选的241个细菌阳性克隆子进行HaeIII酶切分型得到86个可操作分类单元(OTUs),系统发育分析将其归为11个门:放线菌门(Actinobacteria),酸杆菌门(Acidobacteria),拟杆菌门(Bacteroidetes),绿菌门(Chlorobi),蓝细菌门(Cyanobacteria),厚壁菌门(Firmicutes),芽单胞菌门(Gemmatimonadetes),硝化螺旋菌门(Nitrospirae),变形菌门(Proteobacteria),浮霉菌门(Planctomycetes),疣微菌门(Verrucomicrobia)。其中酸杆菌门和变型菌门为优势类群,分别占细菌克隆文库的48%和25%。超过1/3的OTUs序列与GenBank中已存序列具有较低相似性(相似性小于95%)。此外20%左右的克隆子与固氮细菌和硝酸盐氧化细菌相关。研究结果表明,新疆沙湾冷泉沉积物中细菌种类丰富,代谢类型多样而且存在大量未知类群。