Stratification and seasonal stability of diverse bacterial communities in a Pinus merkusii (pine) forest soil in central Java,Indonesia

In Java, Indonesia, many nutrient-poor soils are intensively reforested with Pinus merkusii (pine). Information on nutrient cycles and microorganisms involved in these cycles will benefit the management of these important forests. Here, seasonal effects on the stratification of bacterial community structure in the soil profile of a tropical pine forest are described, and differences in bacterial communities are related to chemical and physical soil parameters. Culture-independent community profiles of litter, fragmented litter and mineral soil layers were made by denaturing gradient gel electrophoresis (DGGE) of 16S rDNA-specific polymerase chain reaction (PCR) fragments. The community profiles of the different soil layers clustered separately, correlating with significant differences in organic matter content between the three layers. The bacterial communities appeared to be stable during the wet season of 1998. The drought in 1997, caused by the El Ni?o climatic effect, did not influence the bacterial communities in fragmentation and mineral soil, although moisture content and other soil parameters were markedly lower than in the wet season. However, communities in litter were influenced by drought. In the litter layer, the moisture content was significantly lower than in the fragmentation and mineral layers during the dry season. A clone library was made from a litter sample taken during the wet season. Partial sequencing of 74 clones and linking the DGGE banding positions of these clones to bands in the DGGE profile of the sample from which the clone library was derived showed considerable bacterial diversity. Alpha-proteobacteria (40.5% of the clones, of which 57% belonged to the Rhizobium-Agrobacterium group) and high-G+C content, Gram-positive bacteria (36.5%) dominated the clone library.     

An advanced molecular strategy to identify bacterial communities on art objects

The application of culture-independent techniques based on molecular biological methods, especially on the PCR amplification of 16S rRNA genes, attempts to overcome some shortcomings of conventional cultivation methods and reveals far more complex bacterial communities on art objects than can be shown by cultivation methods. One of the major challenges of investigating microbial growth on art objects by molecular means is the extraction of DNA, due to small sample amounts and PCR inhibitors. In the present study, we introduce a DNA extraction protocol, which allowed the extraction of PCR-amplifiable DNA from samples derived from lime wall paintings and loamy soil underground. The DNA extracts were used to amplify 16S ribosomal fragments, which were subsequently analyzed by denaturing gradient gel electrophoresis (DGGE). In parallel with the DGGE analysis, clone libraries containing PCR fragments of the ribosomal gene were constructed and clones were screened by DGGE. Clone libraries allow the inclusion of the entire 16S rDNA sequence in the phylogenetic analyses of microorganisms, providing a more reliable phylogenetic identification of microorganisms than is obtained from sequence analyses of excised and directly sequenced DGGE bands.     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

Numerical Analysis of Grassland Bacterial Community Structure under Different Land Management Regimens by Using 16S Ribosomal DNA Sequence Data and Denaturing Gradient Gel Electrophoresis Banding Patterns

Bacterial diversity in unimproved and improved grassland soils was assessed by PCR amplification of bacterial 16S ribosomal DNA (rDNA) from directly extracted soil DNA, followed by sequencing of ~45 16S rDNA clones from each of three unimproved and three improved grassland samples (A. E. McCaig, L. A. Glover, and J. I. Prosser, Appl. Environ. Microbiol. 65:1721–1730, 1999) or by denaturing gradient gel electrophoresis (DGGE) of total amplification products. Semi-improved grassland soils were analyzed only by DGGE. No differences between communities were detected by calculation of diversity indices and similarity coefficients for clone data (possibly due to poor coverage). Differences were not observed between the diversities of individual unimproved and improved grassland DGGE profiles, although considerable spatial variation was observed among triplicate samples. Semi-improved grassland samples, however, were less diverse than the other grassland samples and had much lower within-group variation. DGGE banding profiles obtained from triplicate samples pooled prior to analysis indicated that there was less evenness in improved soils, suggesting that selection for specific bacterial groups occurred. Analysis of DGGE profiles by canonical variate analysis but not by principal-coordinate analysis, using unweighted data (considering only the presence and absence of bands) and weighted data (considering the relative intensity of each band), demonstrated that there were clear differences between grasslands, and the results were not affected by weighting of data. This study demonstrated that quantitative analysis of data obtained by community profiling methods, such as DGGE, can reveal differences between complex microbial communities.     

Molecular diversity of nitrogen-fixing bacteria associated with Chrysopogon zizanioides (L.) Roberty (vetiver), an essential oil producer plant

The essential oil produced by vetiver can vary in amount and composition depending on the bacterial community associated with its roots. Some of these bacteria could also promote plant growth by fixing nitrogen. This study aimed to analyze the diversity of diazotrophic bacteria tightly associated with roots of different vetiver genotypes. nifH-based PCR-denaturing gradient gel electrophoresis (DGGE) and clone libraries were used. DGGE profiles obtained from bulk and rhizosphere soils and root DNA amplified with nifH primers showed that samples from rhizosphere soil and root were separated at 68% similarity. Twelve bands were excised from the DGGE and sequenced. High similarity with nifH sequences of Bradyrhizobium sp., Pseudacidovorax sp. and Xanthobacter sp. was observed. Moreover, three nifH clone libraries were generated using polF/polR-primers from root DNA samples obtained from vetiver genotypes UFS-VET001, UFS-VET003 and UFS-VET004. In UFS-VET001, 24.2% of 95 clones were affiliated with sequences of Mesorhizobium loti while in UFS-VET003 41.5% of 89 clones were affiliated with Sphingomonas azotifigens, and in UFS-VET004 36.4% of 85 clones were affiliated with Klebsiella pneumoniae. The data obtained can be used to guide the isolation of diazotrophic bacteria, which may contribute to plant growth promotion and improvement of the production of essential oil in vetiver.     

Numerical analysis of grassland bacterial community structure under different land management regimens by using 16S ribosomal DNA sequence data and denaturing gradient gel electrophoresis banding patterns

Bacterial diversity in unimproved and improved grassland soils was assessed by PCR amplification of bacterial 16S ribosomal DNA (rDNA) from directly extracted soil DNA, followed by sequencing of ~45 16S rDNA clones from each of three unimproved and three improved grassland samples (A. E. McCaig, L. A. Glover, and J. I. Prosser, Appl. Environ. Microbiol. 65:1721-1730, 1999) or by denaturing gradient gel electrophoresis (DGGE) of total amplification products. Semi-improved grassland soils were analyzed only by DGGE. No differences between communities were detected by calculation of diversity indices and similarity coefficients for clone data (possibly due to poor coverage). Differences were not observed between the diversities of individual unimproved and improved grassland DGGE profiles, although considerable spatial variation was observed among triplicate samples. Semi-improved grassland samples, however, were less diverse than the other grassland samples and had much lower within-group variation. DGGE banding profiles obtained from triplicate samples pooled prior to analysis indicated that there was less evenness in improved soils, suggesting that selection for specific bacterial groups occurred. Analysis of DGGE profiles by canonical variate analysis but not by principal-coordinate analysis, using unweighted data (considering only the presence and absence of bands) and weighted data (considering the relative intensity of each band), demonstrated that there were clear differences between grasslands, and the results were not affected by weighting of data. This study demonstrated that quantitative analysis of data obtained by community profiling methods, such as DGGE, can reveal differences between complex microbial communities.     

Microbial community profiling in cis- and trans-dichloroethene enrichment systems using denaturing gradient gel electrophoresis

The effective and accurate assessment of the total microbial community diversity is one of the primary challenges in modem microbial ecology, especially for the detection and characterization of unculturable populations and populations with a low abundance. Accordingly, this study was undertaken to investigate the diversity of the microbial community during the biodegradation of cis- and trans-dichloroethenes in soil and wastewater enrichment cultures. Community profiling using PCR targeting the 16S rRNA gene and denaturing gradient gel electrophoresis (PCR-DGGE) revealed an alteration in the bacterial community profiles with time. Exposure to cis- and trans-dichloroethenes led to the disappearance of certain genospecies that were initially observed in the untreated samples. A cluster analysis of the bacterial DGGE community profiles at various sampling times during the degradation process indicated that the community profile became stable after day 10 of the enrichment. DNA sequencing and phylogenetic analysis of selected DGGE bands revealed that the genera Acinetobacter, Pseudomonas, Bacillus, Comamonas, and Arthrobacter, plus several other important uncultured bacterial phylotypes, dominated the enrichment cultures. Thus, the identified dominant phylotypes may play an important role in the degradation of cis- and trans-dichloroethenes.     

昆明盐矿古老岩盐沉积中的原核生物多样性

应用PCR-DGGE和rRNA分析法研究了昆明盐矿古老岩盐沉积中的原核生物多样性。样品的细菌DGGE分析得到27条带,古菌得到18条带。样品与纯培养得到的19个属菌株的DGGE图谱对比分析发现,细菌18个属菌株,只有1个属菌株与样品中的1条带迁移位置都不一致;古菌1个属的菌株不与样品中任何条带迁移位置一致。表明纯培养所得菌株并非该环境中的优势类群。同时,建立了样品细菌和古菌的16S rDNA克隆文库,从中分别挑取36个细菌克隆和20个古菌克隆进行ARDRA分析。细菌可分为10个OTUs,其中3个OTUs是优势类群,分别占38.9%,25.0%,16.7%,其余7个OTUs各含有1个克隆。古菌分为8个OTUs,没有明显的优势类群。每个OTU的代表克隆16S rDNA序列分析表明,细菌分属3大类群:α-Proteobacteria,γ-Proteobacteria和Actinobacteria,以Pseudomonas属菌为优势,含有其它岩盐沉积中没有发现的Actinobacteria。古菌主要是Halorubrum属、Haloterrigena属菌和未培养古菌。本研究表明,昆明盐矿古老岩盐沉积具有较丰富的原核生物多样性,含有大量未知的、未培养或不可培养的原核生物,但在原核生物物种组成和丰度上,免培养与此前的纯培养研究结果存在一定差异。因此,结合使用两类方法才能较全面地认识高盐极端环境微生物的多样性。     

Variation of Bacterial Community Diversity in Rhizosphere Soil of Sole-Cropped versus Intercropped Wheat Field after Harvest

As the major crops in north China, spring crops are usually planted from April through May every spring and harvested in fall. Wheat is also a very common crop traditionally planted in fall or spring and harvested in summer year by year. This continuous cropping system exhibited the disadvantages of reducing the fertility of soil through decreasing microbial diversity. Thus, management of microbial diversity in the rhizosphere plays a vital role in sustainable crop production. In this study, ten common spring crops in north China were chosen sole-cropped and four were chosen intercropped with peanut in wheat fields after harvest. Denaturing gradient gel electrophoresis (DGGE) and DNA sequencing of one 16S rDNA fragment were used to analyze the bacterial diversity and species identification. DGGE profiles showed the bacterial community diversity in rhizosphere soil samples varied among various crops under different cropping systems, more diverse under intercropping system than under sole-cropping. Some intercropping-specific bands in DGGE profiles suggested that several bacterial species were stimulated by intercropping systems specifically. Furthermore, the identification of these dominant and functional bacteria by DNA sequencing indicated that intercropping systems are more beneficial to improve soil fertility. Compared to intercropping systems, we also observed changes in microbial community of rhizosphere soil under sole-crops. The rhizosphere bacterial community structure in spring crops showed a strong crop species-specific pattern. More importantly, Empedobacter brevis, a typical plant pathogen, was only found in the carrot rhizosphere, suggesting carrot should be sown prudently. In conclusion, our study demonstrated that crop species and cropping systems had significant effects on bacterial community diversity in the rhizosphere soils. We strongly suggest sorghum, glutinous millet and buckwheat could be taken into account as intercropping crops with peanut; while hulled oat, mung bean or foxtail millet could be considered for sowing in wheat fields after harvest in North China.     

Phylogenetic diversity, composition and distribution of bacterioplankton community in the Dongjiang River, China

Bacterioplankton community compositions in the Dongjiang River were characterized using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library construction. Water samples in nine different sites were taken along the mainstem and three tributaries. In total, 24 bands from DGGE gels and 406 clones from the libraries were selected and sequenced, subsequently analyzed for the bacterial diversity and composition of those microbial communities. Bacterial 16S rRNA gene sequences from freshwater bacteria exhibited board phylogenetic diversity, including sequences representing the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteriodetes, Verrucomicrobia, and candidate division TM7. Members of Betaproteobacteria group were the most dominant in all sampling sites, followed by Gammaproteobacteria, Alphaproteobacteria, and Actinobacteria. DGGE profiles and the ∫-LIBSHUFF analysis revealed similar patterns of bacterial diversity among most sampling sites, while spatial distribution variances existed in all sites along the river basin. Statistical analysis showed that bacterial species distribution strongly correlated with environmental variables, such as nitrate and ammonia, suggesting that nitrogen nutrients may shape the microbial community structure and composition in the Dongjiang River. This study had important implications for the comparison with other rivers elsewhere and contributed to the growing data set on the factors that structure bacterial communities in freshwater ecosystems.     

Efficiency of DNA extraction methods on the evaluation of soil microeukaryotic diversity

The evaluation of microbial molecular diversity has been mainly based on the extraction of total DNA from environmental samples. The indirect extraction methods, which have been used for prokaryotes, have never been used to recover soil microeukaryotic DNA. We evaluated the efficiency of an improved indirect DNA extraction protocol developed herein and the direct lysis (the sodium dodecyl sulfate (SDS)-based method and commercial DNA extraction kit) on estimating the molecular diversity of soil microbial eukaryotes. DNA quality and quantity as well as denaturing gradient gel electrophoresis (DGGE) profiles were determined using three soil samples from different stations. The indirect method detected the highest DGGE bands in spite of the low DNA yield. The commercial kit detected a lower number of DGGE bands than the indirect method. The SDS-based method produced the lowest DGGE bands and DNA purity but the highest yield. Using the indirect method, we further evaluated the effect of freezing and air-dried preservations on estimating the microeukaryotic diversity. In spite of the low DNA yield obtained from the air-dried preservation, no significant differences were found in either the number of DGGE bands or the DNA purity between two manners. Our results indicate that the improved indirect method could obtain a high purity of intracellular DNA and high efficiency in the estimation of molecular diversity of soil microbial eukaryotes.     

Impact of fumigants on soil microbial communities.

Agricultural soils are typically fumigated to provide effective control of nematodes, soilborne pathogens, and weeds in preparation for planting of high-value cash crops. The ability of soil microbial communities to recover after treatment with fumigants was examined using culture-dependent (Biolog) and culture-independent (phospholipid fatty acid [PLFA] analysis and denaturing gradient gel electrophoresis [DGGE] of 16S ribosomal DNA [rDNA] fragments amplified directly from soil DNA) approaches. Changes in soil microbial community structure were examined in a microcosm experiment following the application of methyl bromide (MeBr), methyl isothiocyanate, 1,3-dichloropropene (1,3-D), and chloropicrin. Variations among Biolog fingerprints showed that the effect of MeBr on heterotrophic microbial activities was most severe in the first week and that thereafter the effects of MeBr and the other fumigants were expressed at much lower levels. The results of PLFA analysis demonstrated a community shift in all treatments to a community dominated by gram-positive bacterial biomass. Different 16S rDNA profiles from fumigated soils were quantified by analyzing the DGGE band patterns. The Shannon-Weaver index of diversity, H, was calculated for each fumigated soil sample. High diversity indices were maintained between the control soil and the fumigant-treated soils, except for MeBr (H decreased from 1.14 to 0.13). After 12 weeks of incubation, H increased to 0.73 in the MeBr-treated samples. Sequence analysis of clones generated from unique bands showed the presence of taxonomically unique clones that had emerged from the MeBr-treated samples and were dominated by clones closely related to Bacillus spp. and Heliothrix oregonensis. Variations in the data were much higher in the Biolog assay than in the PLFA and DGGE assays, suggesting a high sensitivity of PLFA analysis and DGGE in monitoring the effects of fumigants on soil community composition and structure. Our results indicate that MeBr has the greatest impact on soil microbial communities and that 1,3-D has the least impact.     

Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGE

AIM: To evaluate the rpoB gene as a biomarker for PCR-DGGE microbial analyses using soil DNA from the Cerrado, Brazil. METHODS: DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. RESULTS: The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE.     

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.     

Soil microbial community structure across a thermal gradient following a geothermal heating event

In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity. Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65 degrees C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees. Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the Bacteria or Archaea domain. The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils. Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the Acidobacterium (51%) and Planctomyces (18%) divisions. Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50 degrees C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil. A soil microcosm laboratory experiment simulated the geothermal heating event. As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50 degrees C occurred within 1 week and appeared to stabilize after 3 weeks. The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them.     

Effects of site and plant species on rhizosphere community structure as revealed by molecular analysis of microbial guilds

The bacterial and fungal rhizosphere communities of strawberry (Fragaria ananassa Duch.) and oilseed rape (Brassica napus L.) were analysed using molecular fingerprints. We aimed to determine to what extent the structure of different microbial groups in the rhizosphere is influenced by plant species and sampling site. Total community DNA was extracted from bulk and rhizosphere soil taken from three sites in Germany in two consecutive years. Bacterial, fungal and group-specific (Alphaproteobacteria, Betaproteobacteria and Actinobacteria) primers were used to PCR-amplify 16S rRNA and 18S rRNA gene fragments from community DNA prior to denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial fingerprints of soil DNA revealed a high number of equally abundant faint bands, while rhizosphere fingerprints displayed a higher proportion of dominant bands and reduced richness, suggesting selection of bacterial populations in this environment. Plant specificity was detected in the rhizosphere by bacterial and group-specific DGGE profiles. Different bulk soil community fingerprints were revealed for each sampling site. The plant species was a determinant factor in shaping similar actinobacterial communities in the strawberry rhizosphere from different sites in both years. Higher heterogeneity of DGGE profiles within soil and rhizosphere replicates was observed for the fungi. Plant-specific composition of fungal communities in the rhizosphere could also be detected, but not in all cases. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Rostock site revealed that Streptomyces sp. and Rhizobium sp. were among the dominant ribotypes in the strawberry rhizosphere, while sequences from Arthrobacter sp. corresponded to dominant bands from oilseed rape bacterial fingerprints.     

Impact of Fumigants on Soil Microbial Communities

Agricultural soils are typically fumigated to provide effective control of nematodes, soilborne pathogens, and weeds in preparation for planting of high-value cash crops. The ability of soil microbial communities to recover after treatment with fumigants was examined using culture-dependent (Biolog) and culture-independent (phospholipid fatty acid [PLFA] analysis and denaturing gradient gel electrophoresis [DGGE] of 16S ribosomal DNA [rDNA] fragments amplified directly from soil DNA) approaches. Changes in soil microbial community structure were examined in a microcosm experiment following the application of methyl bromide (MeBr), methyl isothiocyanate, 1,3-dichloropropene (1,3-D), and chloropicrin. Variations among Biolog fingerprints showed that the effect of MeBr on heterotrophic microbial activities was most severe in the first week and that thereafter the effects of MeBr and the other fumigants were expressed at much lower levels. The results of PLFA analysis demonstrated a community shift in all treatments to a community dominated by gram-positive bacterial biomass. Different 16S rDNA profiles from fumigated soils were quantified by analyzing the DGGE band patterns. The Shannon-Weaver index of diversity, H, was calculated for each fumigated soil sample. High diversity indices were maintained between the control soil and the fumigant-treated soils, except for MeBr (H decreased from 1.14 to 0.13). After 12 weeks of incubation, H increased to 0.73 in the MeBr-treated samples. Sequence analysis of clones generated from unique bands showed the presence of taxonomically unique clones that had emerged from the MeBr-treated samples and were dominated by clones closely related to Bacillus spp. and Heliothrix oregonensis. Variations in the data were much higher in the Biolog assay than in the PLFA and DGGE assays, suggesting a high sensitivity of PLFA analysis and DGGE in monitoring the effects of fumigants on soil community composition and structure. Our results indicate that MeBr has the greatest impact on soil microbial communities and that 1,3-D has the least impact.     

Phylogenetic 16S rRNA analysis reveals the presence of complex and partly unknown bacterial communities in Tito Bustillo cave,Spain, and on its Palaeolithic paintings

Tito Bustillo cave (Ribadesella, Spain) contains valuable Palaeolithic paintings, which date back 15 000-20 000 years. Since 1969, the cave has been open to the public. Rock wall surfaces, spelaeothems and soils are covered by apparent biofilms of phototrophic microorganisms, which develop under artificial lighting. In addition, rock surfaces present conspicuous bacterial growth in the form of round colonies of different colours and about 1-2 mm in diameter. Even the famous Paintings Panel shows some evident microbial growth. In the present study, bacterial communities on the paintings and on the rock surfaces near the paintings were analysed by culture-independent techniques, including polymerase chain reaction (PCR) amplification of bacterial 16S rRNA genes (16S rDNA), phylogenetic sequence analyses and genetic community fingerprinting by denaturing gradient gel electrophoresis (DGGE). DGGE fingerprints showed complex bacterial community patterns. Forty-one clones matching DGGE bands of the community fingerprints were sequenced, representing about 39% of DNA fragments in the DGGE patterns. Phylogenetic sequence analyses revealed a high number of phylogenetically novel 16S rDNA sequence types and a high diversity of putatively chemotrophic and heterotrophic bacteria. Sequences were phylogenetically most closely related to the Proteobacteria (20 clones), green non-sulphur bacteria (three clones), Planctomycetales order (one clone), Cytophaga-Flexibacter- Bacteroides division (one clone) and the Actinobacteria (four clones). Furthermore, we report the presence of members of the Acidobacterium division (12 clones) in a karstic hypogean environment. Members of this phylum have not so far been detected in these particular environments.     

红树林土壤细菌群落16S rDNA V3片段PCR产物的DGGE分析

从土壤中抽提微生物总DNA ,直接扩增 16SrDNAV3片段 ,应用变性梯度凝胶电泳 (DGGE)和分子克隆技术分析 16SrDNAV3片段的多态性 ,发现地域因素和红树品种都是影响土壤细菌群落结构的因素。通过对杯萼海桑土壤 16SrDNAV3片段PCR产物两个DGGE条带进行分子克隆、序列测定和Blast分析 ,发现每个DGGE条带包含着许多不同的 16SrDNAV3片段 ,并且其中多数为NCBI未收录的序列。这表明DGGE和克隆技术相结合的方法是研究土壤微生物群落结构的一种可行方法。     

PCR-DGGE detection of the bacterial community structure in the Inner Mongolia steppe with two different DNA extraction methods

Compared with conventional methods, molecular biological technique, such as PCR-DGGE (denaturing gradient gel electrophoresis), is informative in examining the structure of the soil bacterial community through the extraction of microbial DNA from soil and generation of bacterial community profiles by PCR amplification of 16S rRNA genes. Extraction efficiency of soil microbial DNA is the most important step in these methods. At present, the frozen-thawing method and bead-beating method are most widely used in genomic extraction. Nevertheless, comparison of these two methods has not been conducted in different soil types, especially in humus-rich soil. In this study, extraction efficiencies of the two methods were compared in humus-rich steppe soil in Inner Mongolia based on the PCR-DGGE analysis of bacterial community structure. The results indicated that the bead-beating method is better than the frozen-thawing method in genomic DNA extraction efficiency. In addition, 21 bands in the DGGE pattern with the bead-beating method were further selected, cloned and sequenced. Based on similarity matching, all the sequences formed five major clusters: Actinobacteria; α-, β-, γ-, Proteobacteria ; Bacteriodetes ; Gemmatimonadetes and Acidobacteria . Of the 21 clones obtained from DGGE patterns, YC4 showed 99.7% similarity to Pseudomonas sp. (DQ339153); YC5, YC18 and YC19 showed 99.9 % similarity to Gram-positive bacterium (AB008510), Virgisporangium ochraceum (AB006162) and Micromonospora chalcea (X92613), respectively.