Introduction of a novel 18S rDNA gene arrangement along with distinct ITS region in the saline water microalga Dunaliella

Background

Sundarban is the world's largest coastal sediment comprising of mangrove forest which covers about one million hectares in the south-eastern parts of India and southern parts of Bangladesh. The microbial diversity in this sediment is largely unknown till date. In the present study an attempt has been made to understand the microbial diversity in this sediment using a cultivation-independent molecular approach.

Results

Two 16 S rRNA gene libraries were constructed and partial sequencing of the selected clones was carried out to identify bacterial strains present in the sediment. Phylogenetic analysis of partially sequenced 16 S rRNA gene sequences revealed the diversity of bacterial strains in the Sundarban sediment. At least 8 different bacterial phyla were detected. The major divisions of detected bacterial phyla were Proteobacteria (alpha, beta, gamma, and delta), Flexibacteria (CFB group), Actinobacteria, Acidobacteria, Chloroflexi, Firmicutes, Planctomycetes and Gammatimonadates.

Conclusion

The gammaproteobacteria were found to be the most abundant bacterial group in Sundarban sediment. Many clones showed similarity with previously reported bacterial lineages recovered from various marine sediments. The present study indicates a probable hydrocarbon and oil contamination in this sediment. In the present study, a number of clones were identified that have shown similarity with bacterial clones or isolates responsible for the maintenance of the S-cycle in the saline environment.     

Composition of freshwater bacterial communities associated with cyanobacterial blooms in four Swedish lakes

The diversity of freshwater bacterioplankton communities has not been extensively studied despite their key role in foodwebs and the cycling of carbon and associated major elements. In order to explore and characterize the composition of bacterioplankton associated with cyanobacterial blooms, large 16S rRNA clone libraries from four lakes experiencing such blooms were analysed. The four libraries contained 1461 clones, of which 559 were prokaryotic sequences of non-cyanobacterial origin. These clones were classified into 158 operational taxonomic units affiliated mainly with bacterial divisions commonly found in freshwater systems, e.g. Proteobacteria, Bacteriodetes, Actinobacteria, Verrucomicrobia and Planctomycetes. Richness and evenness of non-cyanobacterial clones were similar to other clone libraries obtained for freshwater bacterioplankton, suggesting that bacterial communities accompanying cyanobacterial blooms are as diverse as non-bloom communities. Many of the identified operational taxonomic units grouped with known freshwater clusters but the libraries also contained novel clusters of bacterial sequences that may be characteristic for cyanobacterial blooms. About 25% of the operational taxonomic units were detected in more than one lake. Even so, 16S rRNA heterogeneity analysis demonstrated large differences in community composition between lakes regardless of their similar characteristics and close proximity. Hence even the similar environmental conditions created by different cyanobacterial blooms may foster very dissimilar bacterial communities, which could indicate that the genetic diversity in lake bacteria have been underestimated in the past.     

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based clone sequences showed that unclassified bacteria were the most abundant group, representing nearly 62% of all DNA sequences analyzed. Other phylogenetic groups identified included Proteobacteria (20%), Actinobacteria (9%), Cyanobacteria (4%), and Bacteroidetes (2%). The composition of RNA-based libraries (1122 sequences) was similar to the DNA-based libraries with a few notable exceptions: Proteobacteria were more dominant in the RNA clone libraries (i.e., 35% RNA; 20% DNA). Differences in the Proteobacteria composition were also observed; alpha-Proteobacteria was 22 times more abundant in the RNA-based clones while beta-Proteobacteria was eight times more abundant in the DNA libraries. Nearly twice as many DNA operational taxonomic units (OTUs) than RNA OTUs were observed at distance 0.03 (101 DNA; 53 RNA). Twenty-four OTUs were shared between all RNA- and DNA-based libraries (OTU_0.03) representing only 18% of the total OTUs, but 81% (1527/1883) of all sequences. Such differences between clone libraries demonstrate the necessity of generating both RNA- and DNA-derived clone libraries to compare these two different molecular approaches for community analyses.     

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。     

Bacterial diversity in deep-sea sediment from northeastern Pacific Ocean

16S rDNA sequencing method is one of the effectively used culture-independent techniques in recent years. In this study, 16S rDNA sequencing method was used to investigate the bacterial diversity in deep-sea sediment from northeastern Pacific polymetallic nodule province. Total DNAs were extracted by using 2 different methods (chemical method and DNA extracting kit method). After purification, genomic DNA was amplified by using 2 universal primers (27F and 1492R). Clones were selected and sequenced randomly. After the sequences were checked by using the Chimera Check Program of the RDP database, a bacterial 16S rRNA gene library of 79 clones was established. Phylogenetic analysis indicated that 79 clones could be divided into 11 phylotypes. Gamma Proteobacteria (22.8%) and alpha Proteobacteria (16.5%) were the dominant components of the sediment bacterial community, followed by Planctomycetacia (7.6%), delta Proteobacteria (6.3%), Nitrospira (6.3%), Actinobacteria (6.3%), beta Proteobacteria (5%), Acidobacteria (5.1%), Sphingobacteria (3.8%), Firmicutes (2.5%) and uncultured bacteria (17.7%). Gamma Proteobacteria also dominated at slices 0–2 cm and 4–6 cm. Different slices had different types of bacteria, alpha Proteobacteria, gamma Proteobacteria, delta Proteobacteria, Planctomycetacia, Nitrospira, Actinobacteria and Acidobacteria, however, appeared in all slices. Pseudomonas is common in many different deep-sea environments. In this study, it accounted for 22.2% of the total gamma Proteobacteria.     

Diversity of microorganisms in decaying maize stalks revealed by a molecular method

Microbial diversity in decaying maize stalk was characterized by constructing and analyzing rRNA gene clone library. Total 47 OTUs were obtained from 82 bacterial clones, including Proteobacteria (64.6%), Actinobacteria (30.5%), Bacteroidetes (2.4%) and Firmicutes (2.4%). Most proteobacterial clones were members of Rhizobium, Pseudomonas and Stenotrophomonas. Eighty-four percent of Actinobacteria was related to Microbacterium. Only 14 OTUs were identified from 124 fungal clones, including Ascomycota (88%) and Basidiomycota (12%). Sixty percent of Ascomycota were members of Eupenicillium and Paecilomyces but all Basidiomycota were close to Kurtzmanomyces nectairei.     

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).     

Culture-independent analysis of fecal microbiota in cattle

The phylogenetic diversity of the fecal bacterial community in Holstein cattle was determined by 16S ribosomal RNA gene sequence analysis. The sequences were affiliated with the following phyla: Firmicutes (81.3%), Bacteroidetes (14.4%), Actinobacteria (2.5%), and Proteobacteria (1.4%). The Clostridium leptum subgroup was the most phylogenetically diverse group in cattle feces. In addition, a number of previously uncharacterized and unidentified bacteria were recognized in clone libraries.     

Molecular characterization of bacterial diversity from British Columbia forest soils subjected to disturbance

Bacteria from forest soils were characterized by DNA sequence analysis of cloned 16S rRNA gene fragments (16S clones). Surface organic matter and mineral soil samples from a British Columbia Ministry of Forests Long-Term Soil Productivity (LTSP) installation were collected during winter and summer from two disturbance treatments: whole-tree harvesting with no soil compaction (plot N) and whole-tree harvesting plus complete surface organic matter removal with heavy soil compaction (plot S). Phylogenetic analyses revealed that 87% of 580 16S clones were classified as Proteobacteria, Actinobacteria, Acidobacterium, Verrucomicrobia, Bacillus/Clostridium group, Cytophaga-Flexibacter-Bacteroides group, green nonsulfur bacteria, Planctomyces, and candidate divisions TM6 and OP10. Seventy-five 16S clones could not be classified into known bacterial divisions, and five 16S clones were related to chloroplast DNA. Members of Proteobacteria represented 46% of the clone library. A higher proportion of 16S clones affiliated with y-Proteobacteria were from plot N compared with plot S. 16S rRNA gene fragments amplified with Pseudomonas-specific primers and cloned (Ps clones) were examined from mineral-soil samples from plots N and S from three LTSP installations. A significantly greater proportion of sequenced Ps clones from plot N contained Pseudomonas 16S rRNA gene fragments compared with Ps clones from plot S.     

Microbial community structure in moraine lakes and glacial meltwaters, Mount Everest

The bacterial diversity and abundance in two moraine lakes and two glacial meltwaters (5140, 5152, 5800 and 6350 m above sea level, respectively) in the remote Mount Everest region were examined through 16S rRNA gene clone library and flow cytometry approaches. In total, 247 clones were screened by RFLP and 60 16S rRNA gene sequences were obtained, belonging to the following groups: Proteobacteria (8% alpha subdivision, 21% beta subdivision, and 1% gamma subdivision), Cytophaga-Flavobacteria-Bacteroides (CFB) (54%), Actinobacteria (4%), Planctomycetes (2%), Verrucomicrobia (2%), Fibrobacteres (1%) and Eukaryotic chroloplast (3%), respectively. The high dominance of CFB distinguished the Mount Everest waters from other mountain lakes. The highest bacterial abundance and diversity occurred in the open moraine lake at 5152 m, and the lowest in the glacial meltwater at 6350 m. Low temperature at high altitude is considered to be critical for component dominancy. At the same altitude, nutrient availability plays a role in regulating population structure. Our results also show that the bacteria in Mount Everest may be derived from different sources.     

Molecular-phylogenetic characterization of microbial communities imbalances in the small intestine of dogs with inflammatory bowel disease

An association between luminal commensal bacteria and inflammatory bowel disease (IBD) has been suggested in humans, but studies investigating the intestinal microbial communities of dogs with IBD have not been published. The aim of this study was to characterize differences of the small intestinal microbial communities between dogs with IBD and healthy control dogs. Duodenal brush cytology samples were endoscopically collected from 10 dogs with IBD and nine healthy control dogs. DNA was extracted and 16S rRNA gene was amplified using universal bacterial primers. Constructed 16S rRNA gene clone libraries were compared between groups. From a total of 1240 selected clones, 156 unique 16S rRNA gene sequences were identified, belonging to six phyla: Firmicutes (53.4%), Proteobacteria (28.4%), Bacteroidetes (7.0%), Spirochaetes (5.2%), Fusobacteria (3.4%), Actinobacteria (1.1%), and Incertae sedis (1.5%). Species richness was significantly lower in the IBD group (P=0.038). Principal component analysis indicated that the small intestinal microbial communities of IBD and control dogs are composed of distinct microbial communities. The most profound difference involved enrichment of the IBD dogs with members of the Enterobacteriaceae family. However, differences involving members of other families, such as Clostridiaceae, Bacteroidetes and Spirochaetes, were also identified. In conclusion, canine IBD is associated with altered duodenal microbial communities compared with healthy controls.     

Phylogenetic diversity of bacteria in an earth-cave in Guizhou province, southwest of China

The objective of this study was to analyze the phylogenetic composition of bacterial community in the soil of an earth-cave (Niu Cave) using a culture-independent molecular approach. 16S rRNA genes were amplified directly from soil DNA with universally conserved and Bacteria-specific rRNA gene primers and cloned. The clone library was screened by restriction fragment length polymorphism (RFLP), and representative rRNA gene sequences were determined. A total of 115 bacterial sequence types were found in 190 analyzed clones. Phylogenetic sequence analyses revealed novel 16S rRNA gene sequence types and a high diversity of putative bacterial community. Members of these bacteria included Proteobacteria (42.6%), Acidobacteria (18.6%), Planctomycetes (9.0%), Chloroflexi (Green nonsulfur bacteria, 7.5%), Bacteroidetes (2.1%), Gemmatimonadetes (2.7%), Nitrospirae (8.0%), Actinobacteria (High G+C Gram-positive bacteria, 6.4%) and candidate divisions (including the OP3, GN08, and SBR1093, 3.2%). Thirty-five clones were affiliated with bacteria that were related to nitrogen, sulfur, iron or manganese cycles. The comparison of the present data with the data obtained previously from caves based on 16S rRNA gene analysis revealed similarities in the bacterial community components, especially in the high abundance of Proteobacteria and Acidobacteria. Furthermore, this study provided the novel evidence for presence of Gemmatimonadetes, Nitrosomonadales, Oceanospirillales, and Rubrobacterales in a karstic hypogean environment.     

Phylogenetic diversity of winter bacterioplankton of eutrophic siberian reservoirs as revealed by 16S rRNA gene sequence

Using 16S rRNA gene sequence analyses we investigated the bacterial diversity of winter bacterioplankton of two eutrophic Siberian reservoirs. These reservoirs show similarity in phytoplankton community composition in spring and autumn but tend to differ in summer in exhibiting cyanobacterial bloom. Forty-eight unique partial 16S RNA gene sequences retrieved from two libraries were mostly affiliated with the class Actinobacteria, b subdivision of the class Proteobacteria, and the phylum Cytophaga-Flavobacterium-Bacteroides. The clone library of the pond exhibiting summer cyanobacterial bloom showed more diversity in sequence composition. A significant number of bacterial 16S rRNA gene clones were closely related to freshwater bacteria previously found in different aquatic ecosystems. This finding confirms the assumption that some bacterial clades are globally distributed.     

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.     

Diversity analyses of microbial communities in petroleum samples from Brazilian oil fields

Recent studies of oil fields have shown that the microbial diversity is represented by bacteria and archaea of wide distribution, and that many of these organisms have potential to metabolize organic and inorganic compounds. Biodegradation processes in oil industry are of great relevance, since it may be related with the loss of petroleum quality and can bring problems during production. The aim of this study was to compare the microbial communities present in biodegraded (GMR75) and non-biodegraded (PTS1) terrestrial oils from the Potiguar Basin (RN, Brazil) by using cultivation (microbial enrichments and isolation) and molecular approaches (16S rRNA gene libraries). The cultivated microorganisms recovered were affiliated with the phyla Actinobacteria, Firmicutes and Proteobacteria. Both bacterial 16S rRNA gene libraries revealed a great diversity, encompassing representatives from 8 different phyla (Actinobacteria, Bacteroidetes, Deferribacteres, Spirochaetes, Firmicutes, Proteobacteria, Thermotogae and Synergistetes) for the GMR75 sample, and from 5 different phyla (Actinobacteria, Chloroflexi, Firmicutes, Proteobacteria and Thermotoga) for the PTS1 sample. The archaeal 16S rRNA gene library was obtained only for GMR75 oil and all phylotypes were affiliated with the family Methanomicrobiaceae. Diversity results suggest that methanogenesis is the dominant terminal process for hydrocarbon degradation in GMR oil field, driven by anaerobic biodegradation.     

Molecular and culture-based analyses of prokaryotic communities from an agricultural soil and the burrows and casts of the earthworm Lumbricus rubellus

The microbial populations in no-till agricultural soil and casts of the earthworm Lumbricus rubellus were examined by culturing and molecular methods. Clone libraries of the 16S rRNA genes were prepared from DNA isolated directly from the soil and earthworm casts. Although no single phylum dominated the soil library of 95 clones, the largest numbers of clones were from Acidobacteria (14%), Cytophagales (13%), Chloroflexi (8%), and gamma-Proteobacteria (8%). While the cast clone library of 102 clones was similar to the soil library, the abundances of several taxa were different. Representatives of the Pseudomonas genus as well as the Actinobacteria and Firmicutes increased in number, and one group of unclassified organisms found in the soil library was absent in the cast library. Likewise, soil and cast archaeal 16S rRNA gene libraries were similar, although the abundances of some groups were different. Two hundred and thirty aerobic bacteria were also isolated on general heterotrophic media from casts, burrows, and soil. The cast isolates were both phenotypically and genotypically different from the soil isolates. The cast isolates were more likely to reduce nitrate, grow on acetate and Casamino Acids, and utilize fewer sugars than the soil isolates. On the basis of their ribotypes, the cast isolates were dominated by Aeromonas spp. (28%), which were not found in the soil isolates, and other gamma-Proteobacteria (49%). In contrast, the soil isolates were mostly Actinobacteria (53%), Firmicutes (16%), and gamma-Proteobacteria (19%). Isolates obtained from the sides of earthworm burrows were not different from the soil isolates. Diversity indices for the collections of isolates as well as rRNA gene libraries indicated that the species richness and evenness were decreased in the casts from their levels in the soil. These results were consistent with a model where a large portion of the microbial population in soil passes through the gastrointestinal tract of the earthworm unchanged while representatives of some phyla increase in abundance.     

16S rRNA sequences and differences in bacteria isolated from the Muztag Ata glacier at increasing depths

Small subunit 16S rRNA sequences, growth temperatures, and phylogenetic relationships have been established for 129 bacterial isolates recovered under aerobic growth conditions from different regions of a 22-m ice core from the Muztag Ata Mountain glacier on the Pamirs Plateau (China). Only 11% were psychrophiles (grew at 2 degrees C or -2 degrees C up to approximately 20 degrees C), although the majority (82%) were psychrotolerant (grew at 2 degrees C or -2 degrees C up to 37 degrees C). The majority of the isolates had 16S rRNA sequences similar to previously determined sequences, ranging from 85% to 100% identical to database sequences. Based on their 16S rRNA sequences, 42.6% of the isolates were high-G+C (HGC) gram-positive bacteria, 23.3% were gamma-Proteobacteria, 14.7% were alpha-Proteobacteria, 14.7% were Flavobacteria, and 4.7% were low-G+C (LGC) gram-positive bacteria. There were clear differences in the depth distribution, with Proteobacteria, HGC/Cytophaga-Flavobacterium-Bacteroides (CFB), Proteobacteria, LGC/CFB/HGC, Cryobacterium psychrophilum, HGC/CFB, Proteobacteria/HGC/CFB, and HGC/CFB being the predominant isolates from ice that originated from 2.7 to 3.8, 6.2, 7.5, 8.3, 9.0, 9.7, 12.5, and 15.3 m below the surface, respectively. This layered distribution of bacterial isolates presumably reflects both differences in bacteria inhabiting the glacier's surface, differences in bacteria deposited serendipitously on the glacier's surface by wind and snowfall, and nutrient availability within the ice.     

新疆艾比湖湿地博乐河入口处土壤细菌多样性分析

【目的】了解新疆艾比湖湿地国家级自然保护区非培养土壤细菌群落组成及多样性。【方法】采用非培养法直接从湿地土壤提取总DNA进行16S r RNA基因扩增,构建细菌16S r RNA基因克隆文库。使用MspⅠ和AfaⅠ限制性内切酶对阳性克隆进行16S r RNA基因扩增片段的限制性酶切分析(Amplified r DNA restriction analysis,ARDRA),挑取具有不同双酶切图谱的克隆进行测序,序列比对并构建16S r RNA基因系统发育树。【结果】从土壤细菌的16S r RNA基因文库中随机挑取75个不同谱型的克隆子,共得到58个OTUs,系统发育归类为8个细菌类群:绿弯菌门(Chloroflexi)、蓝藻门(Cyanobacteria)、变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrob)和芽单胞菌门(Gemmatimonadetes)。其中,变形菌门为第一优势菌群,拟杆菌门为第二优势菌群,两者约占总克隆的65%。【结论】艾比湖湿地博乐河入口处土壤细菌多样性丰富,且存在一定数量的潜在微生物新种。     

Bacterial diversity in the sediment from polymetallic nodule fields of the Clarion-Clipperton Fracture Zone

The Clarion-Clipperton Fracture Zone (CCFZ) is located in the northeastern equatorial Pacific and contains abundant polymetallic nodules. To investigate its bacterial diversity, four libraries of 16S rRNA genes were constructed from sediments of four stations in different areas of the CCFZ. In total, 313 clones sequenced from the 4 libraries were assigned into 14 phylogenetic groups and 1 group of 28 unclassified bacteria. High bacterial diversity was predicted by the rarefaction analysis. The most dominant group overall was Proteobacteria, but there was variation in each library: Gammaproteobacteria was the most dominant group in two libraries, E2005-01 and ES0502, while Alphaproteobacteria and Deltaproteobacteria were the most dominant groups in libraries EP2005-03 and WS0505, respectively. Seven groups, including Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Betaproteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes, were common to all four libraries. The remaining minor groups were distributed in libraries with different patterns. Most clones sequenced in this study were clustered with uncultured bacteria obtained from the environment, such as the ocean crust and marine sediment, but only distantly related to isolates. Bacteria involved in the cycling of metals, sulfur and nitrogen were detected, and their relationship with their habitat was discussed. This study sheds light on the bacterial communities associated with polymetallic nodules in the CCFZ and provides primary data on the bacterial diversity of this area.