Links between Geographic Location, Environmental Factors, and Microbial Community Composition in Sediments of the Eastern Mediterranean Sea

The bacterial community composition of marine surface sediments originating from various regions of the Eastern Mediterranean Sea (12 sampling sites) was compared by parallel use of three fingerprinting methods: analysis of 16S rRNA gene fragment heterogeneity by denaturing gradient electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), and analysis of phospholipid-linked fatty acid composition (PLFA). Sampling sites were located at variable depths (30–2860 m; water column depth above the sediments) and the sediments differed greatly also in their degree of petroleum contamination (0.4–18 μg g⁻¹), organic carbon (0.38–1.5%), and chlorophyll a content (0.01–7.7 μg g⁻¹). Despite a high degree of correlation between the three different community fingerprint methods, some major differences were observed. DGGE banding patterns showed a significant separation of sediment communities from the northern, more productive waters of the Thermaikos Gulf and the oligotrophic waters of the Cretan, S. Ionian, and Levantine Sea. T-RFLP analysis clearly separated the communities of deep sediments (>1494 m depth) from their shallow (<617 m) counterparts. PLFA analysis grouped a shallow station from the productive waters of the north with the deep oligotrophic sediments from the Ionian and Levantine Sea, with low concentrations of PLFAs, and hence low microbial biomass, as the common denominator. The degree of petroleum contamination was not significantly correlated to the apparent composition of the microbial communities for any of the three methods, whereas organic carbon content and sediment chlorophyll a were important in this regard.     

北极太平洋扇区海洋沉积物细菌多样性的系统发育分析

对北极太平洋扇区3个不同深度的海洋沉积物样品,采用PCR结合变性梯度凝胶电泳(DGGE)技术进行细菌16S rRNA基因V3区序列的系统发育分析。结果表明,同一个沉积物样品不同层次的DGGE电泳图谱不完全相同。从3个沉积物样品中共获得50条序列,大部分序列与从海洋环境尤其海洋沉积物获得的细菌16S rDNA序列相似性较高(88%~100%),归属于变形细菌(Proteobacteria)的gamma亚群、alpha亚群、beta亚群、epsilon亚群、delta亚群,Cytophaga_Flavobacterium_Bacteroides(CFB)群细菌和高G C含量的革兰氏阳性细菌等系统分类群,其中变形细菌(Proteobacteria)的gamma亚群为沉积物中的优势细菌类群。     

Bacterial diversity in sediments of the eutrophic Guanting Reservoir,China, estimated by analyses of 16S rDNA sequence

The bacterial diversity in different layers of sediment of the eutrophic Guanting Reservoir (China) was investigated using molecular ecological techniques. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA showed presence of different bacterial communities across depths of sediments. The trend was consistent with sedimentological layers as characterized by physical and chemical parameters. Sediments were sampled at the 4–6, 34–36, and 69–72 cm depths to represent upper, middle and lower layers and used to construct three 16S rDNA clone libraries. Out of a total of 760 positive clones obtained from the three sediment layers, 148 rDNA types were identified by amplified 16S rDNA restriction analysis (ARDRA) and grouped into 42 clusters or single lineages at the similarity of 70%. We used 16S rDNA sequencing to classify 60 clones representing different ARDRA clusters into nine phyla: Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Gemmatimonadetes, Nitrospirae, Proteobacteria and Verrucomicrobia. The diversity and distribution of rDNA types across depths were much different from the chemical profile of the sediment and pollution history of the reservoir.     

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

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

有机污染物对水体真细菌群落结构的影响

为了揭示有机污染物对环境真细菌组成和多样性的影响,应用末端限制性片段长度多态性(tRFLP)和16S rDNA文库技术并结合水质分析方法,比较分析了松花江流域内受不同程度有机污染的4个水体及其沉积物中真细菌的群落结构。tRFLP分析表明各水体及底泥均呈现较为复杂的群落结构模式,不同底泥群落形成的末端限制性片段(TRF)图谱具有很高的相似性,但随着污染程度的加强,部分类群明显富集,而且在水样组和泥样组内,群落结构的相似性同水质相似性是一致的,主成分分析(PCA)显示水样和泥样中的真细菌TRF形成不同的群。16SrDNA文库分析表明松花江哈尔滨段底泥中真细菌分布于10个门,Proteobacteria门占优势,达群落总数的21.92%(β-Proteobacteria亚门占10.96%),而有机染污物严重超标的生活污水排污道底泥中的微生物多样性较低,分布于7个门,Proteobacteria门为优势群,占群落的47.37%(α-Proteobacteria亚门占21.05%,δ/ε-Proteobacteria亚门占15.79%)。该研究表明向水体中长期排放高浓度有机物能使系统中微生物群落多样性降低,与污染物降解相关的功能微生物类群明显富集。     

Study of bacterial communities in Antarctic coastal waters by a combination of 16S rRNA and 16S rDNA sequencing

An ecological study on distribution of Antarctic bacterial communities was determined by 16S-based phylogenetic analyses of clone libraries derived from RNA and DNA extracted from two different marine areas and compared between each other. Superficial seawater samples were collected from four stations in Ross Sea, three of them located in Rod Bay and one in Evans Cove; for each station two clone libraries (16S rDNA and 16S rRNA) were prepared and evident divergences between DNA and RNA libraries of each site were obtained. Of all phylotypes 93.6% were found in RNA libraries; in contrast, only 31 phylotypes (70.5%) were retrieved from total microbial community (DNA libraries). DNA and RNA sequences related to gamma-Proteobacteria and Bacteroidetes groups, typical for Antarctic sea-ice bacterial communities, were detected in analysed sites. 16S rDNA and rRNA libraries derived from the two different areas were enriched by picophytoplanktonic 16S sequences of plastid and mitochondrion origins, reflecting that the algal blooms occurred during sampling (Antarctic summer 2003). The finding in Rod Bay libraries of high percentage of DNA clones apparently affiliated with beta-Proteobacteria typical for activated sludges and well water could be explained by the presence of a sewage depuration system at this site. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA gene sequencing is preferred approach to have a more reliable vision on the composition of microbial communities.     

High bacterial diversity of a waste gas-degrading community in an industrial biofilter as shown by a 16S rDNA clone library

The bacterial diversity of an industrial biofilter used for waste gas abatement in an animal-rendering plant was investigated. A 16S rDNA clone library was generated and 444 clones were screened using computer-aided amplified ribosomal DNA restriction analysis (ARDRA). Of the screened clones, 60.8% showed unique ARDRA patterns and the remaining 174 clones were clustered into 65 groups. Almost full-length 16S rDNA sequences of 106 clones were determined and 90.5% of the clones were affiliated with the two phyla Proteobacteria and Bacteroidetes. Alpha-, Beta-, and Gammaproteobacteria accounted for 22.1, 17.6 and 18.6% respectively. Minor portions were affiliated with the Actinobacteria (2.0%), Firmicutes and Verrucomicrobia (both 1.0%), and the Deltaproteobacteria and Thermomicrobia (each 0.5%). Only six out of the 106 16S rDNA sequences exhibited similarities of more than 97% to classified bacterial species indicating that a substantial fraction of the clone sequences were derived from unknown taxa. It was also evaluated whether a database containing 281 computer-simulated bacterial rDNA fragment patterns generated from published reference sequences can be used for identification purposes. The data analysis demonstrated that this was possible only for a small number of clones, which were closely related to described bacterial strains. Rarefaction analysis of ARDRA clusters demonstrated that the 444 clones screened are insufficient to describe the entire diversity of the clone library.     

Prokaryotic diversity in continuous cropping and rotational cropping soybean soil

In spite of the techniques based on the amplification of 16S rRNA genes (16S rDNA) to compare bacterial communities that are now widely in use in microbial ecology, little is known about the composition of the soybean continuous cropping (CC) and rotational cropping (RC) soil microbial community. To address this, we compared the levels of bacterial community diversity in RC and 5-year CC rhizosphere soil samples. We selected 407 clones in RC and 490 clones in CC for restriction fragment length polymorphism analysis. A total of 123 phylotypes were identified among the 16S rDNA clones, while 78 unique and 21 common phylotypes were identified among the CC soil isolates. Analysis of sequences from a subset of the phylotypes showed that at least 11 bacterial divisions were represented in the clone libraries. The phylotype richness, frequency distribution (evenness), and composition of the two clone libraries were investigated using a variety of diversity indices. Although the analysis of diversity indices and LIBSHUFF comparisons revealed that the compared libraries were not significantly different ( P =0.05) between the RC vs. CC soils, some differences could be observed in terms of specific phyla and groups. We concluded that the group variance was not determined immediately by the cropping system's induction, but was a long-term and slow process.     

Diversity and distribution of DNA sequences with affinity to ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in the Arctic Ocean

The spatial distribution and diversity of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria (hereinafter referred to as ammonia oxidizers) in the Arctic Ocean were determined. The presence of ammonia oxidizers was detected by PCR amplification of 16S rRNA genes using a primer set specific for this group of organisms (nitA and nitB, which amplifies a 1.1-kb fragment between positions 137 and 1234, corresponding to Escherichia coli 16S rDNA numbering). We analyzed 246 samples collected from the upper water column (5 to 235 m) during March and April 1995, September and October 1996, and September 1997. Ammonia oxidizers were detected in 25% of the samples from 5 m, 80% of the samples from 55 m, 88% of the samples from 133 m, and 50% of the samples from 235 m. Analysis of nitA-nitB PCR product by nested PCR-denaturing gradient gel electrophoresis (DGGE) showed that all positive samples contained the same major band (band A), indicating the presence of a dominant, ubiquitous ammonia oxidizer in the Arctic Ocean basin. Twenty-two percent of the samples contained additional major bands. These samples were restricted to the Chukchi Sea shelf break, the Chukchi cap, and the Canada basin; areas likely influenced by Pacific inflow. The nucleotide sequence of the 1.1-kb nitA-nitB PCR product from a sample that contained only band A grouped with sequences designated group 1 marine Nitrosospira-like sequences. PCR-DGGE analysis of 122 clones from four libraries revealed that 67 to 71% of the inserts contained sequences with the same mobility as band A. Nucleotide sequences (1.1 kb) of another distinct group of clones, found only in 1995 samples (25%), fell into the group 5 marine Nitrosomonas-like sequences. Our results suggest that the Arctic Ocean beta-proteobacterial ammonia oxidizers have low diversity and are dominated by marine Nitrosospira-like organisms. Diversity appears to be higher in Western Arctic Ocean regions influenced by inflow from the Pacific Ocean through the Bering and Chukchi seas.     

南海深海沉积物放线菌多样性分析

【目的】免培养和纯培养相结合分析南海深海沉积物放线菌多样性。【方法】免培养方法通过提取沉积物宏基因组DNA,利用放线菌门特异性引物扩增放线菌16S r RNA基因序列,构建放线菌16S r RNA基因克隆文库,文库经RFLP(Restriction fragment length polymorphism)分析后挑选代表序列测序并进行多样性指数分析和系统发育分析。可培养方法利用8种培养基进行菌株分离,对排重后的菌株进行16S r RNA基因序列多样性分析。【结果】构建的两个深海位点的16S r RNA基因克隆文库在放线菌门的放线菌纲(Actinobacteria)、酸微菌纲(Acidimicrobiia)、腈基降解菌纲(Nitriliruptoria)和嗜热油菌纲(Thermoleophilia)4个纲中均有分布;两个位点中的种群结构有差异,N40-4位点的优势种群是放线菌纲的链霉菌目(Streptomycetales);N63-4位点的优势种群是腈基降解菌纲的腈基降解菌目(Nitriliruptorales)。8种培养基共分离出41株放线菌,根据形态特征排重后得到的19株菌分布于10个不同的属,12个不同的种,其中稀有放线菌属比例较高,菌株OAct400为潜在的微杆菌属(Microbacterium)新种。【结论】南海深海沉积物蕴含着丰富的放线菌物种资源及大量未知种群,具有进一步研究的价值。     

High bacterial diversity in permanently cold marine sediments.

A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with Myxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the gamma subclass of the class Proteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity within the groups and within the clone library. Rarefaction analysis suggested that the total diversity assessed by 16S rDNA analysis was very high in these permanently cold sediments and was only partially revealed by screening of 353 clones.     

Diversity and Community Structure of Archaea in Deep Subsurface Sediments from the Tropical Western Pacific

Archaeal 16S rRNA gene clone libraries using PCR amplicons from eight different layers of the MD06-3051 core were obtained from the tropical Western Pacific sediments. A total of 768 clones were randomly selected, and 264 representative clones were sequenced by restriction fragment length polymorphism. Finally, 719 valid clones and 104 operational taxonomic units were identified after chimera-check and ≥97% similarity analysis. The phylogenetic analysis of 16S rDNA sequences obtained from sediment samples were very diverse and showed stratification with depth. Majority of the members were most closely related to uncultivated groups and physiologically uncharacterized assemblages. All phylotypes were affiliated with Crenarchaeota (76%) and Euryarchaeota (24%), respectively. Deep-sea archaeal group (DSAG, 41% of total clones) and miscellaneous crenarchaeotic group (MCG, 29% of total clones) belonging to Crenarchaeota were the most predominant archaeal 16S rDNA phylotypes in clone libraries. Phylotypes in this study shared high similarity with those in subsurface sediments from Peru Margin sites, which indicated that different geographical zones might host similar members of archaeal populations based on similar sedimentary environments. In our study, members of DSAG and MCG seemed to dominate certain layers of the nonhydrate sediments, suggesting a wide ecophysiological adaptation than previously appreciated. The spatial distribution and community structure of these groups might vary with the different geochemical gradients of the environment.     

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.     

Molecular characterization of sulfate-reducing bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.     

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.     

Microbial diversity in surface sediments of the Xisha Trough, the South China Sea

Microbial communities were obtained from the surface sediments of the Xisha Trough using the culture-independent technique. The characteristics of the 16S rDNA gene amplified from the sediments indicated that archaeal clones could be grouped into Euryarchaeota and Crenarchaeota, respectively. Two archaeal groups, Marine Crenarchaeotic GroupI and Terrestrial Miscellaneous Euryarchaeotal Group, were the most dominant archaeal 16S rDNA gene components in the sediments. The remaining components were related to the members of Marine Benthic Group B, Marine Benthic Group A, Marine Benthic Group D, Novel Euryarchaeotic Group and C3. The bacterial clones exhibited greater diversity than the archaeal clones with the 16S rDNA gene sequences from the members of Proteobacteria, Planctomycetes, Actinobacteria, Firmicutes, Chloroflexi, Acidobacteria, candidate division OP8, Bacterioidetes/Chlorobi and Verrucomicrobia. Most of these lineages represented uncultured microorganisms. The result suggests that a vast amount of microbial resource in the surface sediments of the South China Sea has not been known.     

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.