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.     

Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial community composition in deep-sea sediments of the South China Sea

The South China Sea, which is one of the largest marginal seas in the world, is predicted to have suitable accumulation conditions and exporting prospects for natural gas hydrate. The aim of this study was to explore the bacterial community composition of deep-sea sediments from such an ecosystem. DNA was extracted by five different methods and used as templates for PCR amplification of the V3 regions of the 16S rRNA gene. Denaturing gradient gel electrophoresis (DGGE) was used to separate the amplified products and analyse the 16S rRNA gene diversity of sediment samples. The results of DGGE indicated that the bacterial community composition is influenced by DNA extraction methods. Sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belong to Proteobacteria, Bacteroidetes, gram-positive bacteria and Archaea. Integrating different DNA extraction procedures are needed to analyse the actual bacterial diversity from environment when the amplification of 16S rRNA gene and construction of representative clone library were adopted.     

A rapid method for measuring ATP + ADP + AMP in marine sediment

In this report, I describe a method for rapid measurement of total adenylate (ATP + ADP + AMP) in marine sediment samples for estimating microbial biomass. A simple ‘boil and dilute’ method is described here, whereby adding boiled MilliQ water to sediments increases the detection limit for ATP + ADP + AMP up to 100-fold. The lowered detection limit of this method enabled the detection ATP + ADP + AMP in relatively low-biomass sub-seafloor sediment cores with 10⁴ 16S rRNA gene copies per gram. Concentrations of ATP + ADP + AMP correlated with 16S rRNA gene concentrations from bacteria and archaea across six different sites that range in water depth from 1 to 6000 m indicating that the ATP + ADP + AMP method can be used as an additional biomass proxy. In deep sea microbial communities, the ratio of ATP + ADP + AMP concentrations to 16S rRNA genes >1 m below seafloor was significantly lower compared to communities in the upper 30 cm of sediment, which may be due to reduced cell sizes and or lower ATP + ADP + AMP concentrations per cell in the deep sea sub-seafloor biosphere. The boil and dilute method for ATP + ADP + AMP is demonstrated here to have a detection limit sufficient for measuring low biomass communities from deep sea sub-seafloor cores. The method can be applied to frozen samples, enabling measurements of ATP + ADP + AMP from frozen sediment cores stored in core repositories from past and future international drilling campaigns.     

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.     

内参基因加标法定量土壤微生物目标基因绝对拷贝数

【目的】通过荧光定量PCR技术对土壤微生物目标基因进行绝对定量,其定量结果的准确性容易受到DNA提取得率以及腐殖酸抑制性的影响。【方法】采用内参基因加标法,利用构建的突变质粒DNA,对供试水稻土壤样品中的微生物16S r RNA目标基因的绝对拷贝数进行荧光定量PCR检测,用来表征该样品中细菌群落总体丰度。在定量前通过双向引物扩增方法验证突变质粒中的内参基因对供试土壤的特异性。【结果】不同水稻土壤样品的DNA提取量在样品间差异较大。通过内参基因加标法对DNA提取量进行校正,显著提高了16S r RNA基因绝对定量的精确度。不同水稻土壤样品间的变异系数为17.8,与未加标处理相比降低了66.7%。在此基础上,进一步通过内参基因加标法对土壤有机质和含水率均呈现典型空间特征差异的6处亚热带湿地土壤样品中的16S r RNA基因进行绝对定量。16S r RNA基因绝对拷贝数与土壤微生物生物量碳具有显著的线性相关性(R2=0.694,P0.001),表明内参校正后的16S r RNA基因绝对拷贝数可以准确反映单位质量土壤中微生物的丰度。【结论】内参基因加标法可以对DNA提取得率以及腐殖酸对PCR扩增的抑制性进行校正,从而提高绝对定量的准确性。基于内参基因加标法的目标基因绝对定量PCR检测,可作为土壤微生物生物量测量,以及微生物功能基因绝对丰度定量的一种核酸检测方法。     

变性梯度凝胶电泳(DGGE)在微生物生态学中的应用

由于从环境样品中分离和培养细菌的困难,分子生物学方法已发展用来描述和鉴定微生物群落。近年来基于DNA方法的群落分析得到了迅速的发展,如PCR扩增技术,克隆文库法,荧光原位杂交法,限制性酶切片段长度多态性法,变性和温度梯度凝胶电泳法。DGGE已广泛用于分析自然环境中细菌、蓝细菌,古菌、微微型真核生物、真核生物和病毒群落的生物多样性。这一技术能够提供群落中优势种类信息和同时分析多个样品。具有可重复和容易操作等特点,适合于调查种群的时空变化,并且可通过对切下的带进行序列分析或与特异性探针杂交分析鉴定群落成员。DGGE分析微生物群落的一般步骤如下：一是核酸的提取,二是16S rRNA,18S rRNA或功能基因如可容性甲烷加单氧酶羟化酶基因(mmoX)和氨加单氧酶a一亚单位基因(amoA)片段的扩增,三是通过DGGE分析PCR产物。DGGE使用具有化学变性剂梯度的聚丙烯酰胺凝胶,该凝胶能够有区别的解链PCR扩增产物。由PCR产生的不同的DNA片段长度相同但核苷酸序列不同。因此不同的双链DNA片段由于沿着化学梯度的不同解链行为将在凝胶的不同位置上停止迁移。DNA解链行为的不同导致一个凝胶带图案,该图案是微生物群落中主要种类的一个轮廓。DGGE使用所有生物中保守的基因片段如细菌中的16S rRNA基因片段和真菌中的18S rRNA基因片段。然而同其他分子生物学方法一样,DGGE也有缺陷,其中之一是只能分离较小的片段,使用于系统发育分析比较和探针设计的序列信息量受到了限制。在某些情况下,由于所用基因的多拷贝导致一个种类多于一条带,因此不易鉴定群落结构到种的水平。此外,该技术具有内在的如单一细菌种类16S rDNA拷贝之间的异质性问题,可导致自然群落中微生物数量的过多估计。DGGE是分析微生物群落的一种有力的工具。不过为了减少DGGE和其它技术的缺陷,建议研究者结合DGGE和其它分子及微生物学方法以便更详细的观察微生物的群落结构和功能。     

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。     

Diversity of prokaryotes and methanogenesis in deep subsurface sediments from the Nankai Trough, Ocean Drilling Program Leg 190

Diversity of Bacteria and Archaea was studied in deep marine sediments by PCR amplification and sequence analysis of 16S rRNA and methyl co-enzyme M reductase (mcrA) genes. Samples analysed were from Ocean Drilling Program (ODP) Leg 190 deep subsurface sediments at three sites spanning the Nankai Trough in the Pacific Ocean off Shikoku Island, Japan. DNA was amplified, from three depths at site 1173 (4.15, 98.29 and 193.29 mbsf; metres below the sea floor), and phylogenetic analysis of clone libraries showed a wide variety of uncultured Bacteria and Archaea. Sequences of Bacteria were dominated by an uncultured and deeply branching 'deep sediment group' (53% of sequences). Archaeal 16S rRNA gene sequences were mainly within the uncultured clades of the Crenarchaeota. There was good agreement between sequences obtained independently by cloning and by denaturing gradient gel electrophoresis. These sequences were similar to others retrieved from marine sediment and other anoxic habitats, and so probably represent important indigenous bacteria. The mcrA gene analysis suggested limited methanogen diversity with only three gene clusters identified within the Methanosarcinales and Methanobacteriales. The cultivated members of the Methanobacteriales and some of the Methanosarcinales can use CO2 and H2 for methanogenesis. These substrates also gave the highest rates in 14C-radiotracer estimates of methanogenic activity, with rates comparable to those from other deep marine sediments. Thus, this research demonstrates the importance of the 'deep sediment group' of uncultured Bacteria and links limited diversity of methanogens to the dominance of CO2/H2 based methanogenesis in deep sub-seafloor sediments.     

Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis

Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.     

Analysis of Bacterial and Fungal Communities in Japanese- and Chinese-Fermented Soybean Pastes Using Nested PCR–DGGE

The microbial diversity of Japanese- and Chinese-fermented soybean pastes was investigated using nested PCR–denaturing gradient gel electrophoresis (DGGE). Five Japanese-fermented soybean paste samples and three Chinese-fermented soybean paste samples were analyzed for bacteria and fungi. Extracted DNA was used as a template for PCR to amplify 16S rRNA and 18S rRNA genes. The nearly complete 16S rRNA and 18S rRNA genes were amplified using universal primers, and the resulting products were subsequently used as a template in a nested PCR to obtain suitable fragments for DGGE. Tetragenococcus halophilus and Staphylococcus gallinarum were found to dominate the bacterial microbiota in Japanese samples, whereas Bacillus sp. was detected as the predominant species in Chinese samples. DGGE analysis of fungi in soybean pastes determined the presence of Aspergillus oryzae and Zygosaccharomyces rouxii in most of the Chinese and Japanese samples. Some differences were observed in the bacterial diversity of Japanese- and Chinese-fermented soybean pastes.     

16S rRNA基因在微生物生态学中的应用

16S rRNA(Small subunit ribosomal RNA)基因是对原核微生物进行系统进化分类研究时最常用的分子标志物(Biomarker),广泛应用于微生物生态学研究中。近些年来随着高通量测序技术及数据分析方法等的不断进步,大量基于16S rRNA基因的研究使得微生物生态学得到了快速发展,然而使用16S rRNA基因作为分子标志物时也存在诸多问题,比如水平基因转移、多拷贝的异质性、基因扩增效率的差异、数据分析方法的选择等,这些问题影响了微生物群落组成和多样性分析时的准确性。对当前使用16S rRNA基因分析微生物群落组成和多样性的进展情况做一总结,重点讨论当前存在的主要问题以及各种分析方法的发展,尤其是与高通量测序技术有关的实验和数据处理问题。     

狮子头热泉菌席样品环境总DNA提取方法的比较研究

通过对狮子头热泉7个环境菌席样品所提取的总DNA进行纯度检测、提取得率计算和DGGE分析,比较了3种直接和1种间接DNA提取方法。结果表明：综合利用多种裂解方式比单一裂解方式更能充分释放环境DNA;其中3种方法获得的DNA片段能够进行后续16S rDNA扩增;针对同一样品,不同方法提取的环境DNA,可获得不同DGGE群落指纹图谱;间接提取法提取的总DNA,能更好地反映狮子头热泉菌席的微生物多样性。     

Statistical assessment of DNA extraction methodology for culture-independent analysis of microbial community associated with diverse environmental samples

Cost-effectiveness, quality, time-effectiveness and ease of the methodology are the most crucial factors in isolating quality DNA from wide variety of samples. Thus, research efforts focusing on the development of an efficient DNA extraction protocol is the need of the hour. The present study therefore, focuses on development of an efficient, rapid and free of inhibitory substances based methodology for extracting metagenomic DNA from diverse environmental samples viz. anaerobic biogas digesta, ruminant stomach, human feces, soil, and microbial starter cultures used for preparation of fermented food. PCR–DGGE based analysis and quality metagenomic library preparation, using DNA extraction methodology, validates the developed protocol. The developed protocol is cost effective, capable of isolating DNA from small sample size (100–1000 µl), time efficient (1.5–2.0 h protocol) and results in significantly higher DNA yield (4–8 times increased yield) when compared to previously available DNA extraction method and a commercial DNA extraction kit. The DNA extracted from the samples using different protocols was evaluated based on its ability to identify diverse microbial species using PCR–DGGE profiles targeting variable region within the 16S rRNA gene. The results of microbial community analysis revealed comparability of the developed protocol to commercial kits, in effectively identifying dominant representatives of the microbial community in different samples. Using the DNA extracted from the presented methodology, metagenomic libraries were prepared, which were found suitable for sequencing on Illumina platform.     

Reconstruction of ribosomal RNA genes from metagenomic data

Direct sequencing of environmental DNA (metagenomics) has a great potential for describing the 16S rRNA gene diversity of microbial communities. However current approaches using this 16S rRNA gene information to describe community diversity suffer from low taxonomic resolution or chimera problems. Here we describe a new strategy that involves stringent assembly and data filtering to reconstruct full-length 16S rRNA genes from metagenomicpyrosequencing data. Simulations showed that reconstructed 16S rRNA genes provided a true picture of the community diversity, had minimal rates of chimera formation and gave taxonomic resolution down to genus level. The strategy was furthermore compared to PCR-based methods to determine the microbial diversity in two marine sponges. This showed that about 30% of the abundant phylotypes reconstructed from metagenomic data failed to be amplified by PCR. Our approach is readily applicable to existing metagenomic datasets and is expected to lead to the discovery of new microbial phylotypes.     

Matching molecular diversity and ecophysiology of benthic cyanobacteria and diatoms in communities along a salinity gradient

The phylogenetic diversity of oxygenic phototrophic microorganisms in hypersaline microbial mats and their distribution along a salinity gradient were investigated and compared with the halotolerances of closely related cultivated strains. Segments of 16S rRNA genes from cyanobacteria and diatom plastids were retrieved from mat samples by DNA extraction and polymerase chain reaction (PCR), and subsequently analysed by denaturing gradient gel electrophoresis (DGGE). Sequence analyses of DNA from individual DGGE bands suggested that the majority of these organisms was related to cultivated strains at levels that had previously been demonstrated to correlate with characteristic salinity responses. Proportional abundances of amplified 16S rRNA gene segments from phylogenetic groupings of cyanobacteria and diatoms were estimated by image analysis of DGGE gels and were generally found to correspond to abundances of the respective morphotypes determined by microscopic analyses. The results indicated that diatoms accounted for low proportions of cells throughout, that the cyanobacterium Microcoleus chthonoplastes and close relatives dominated the communities up to a salinity of 11% and that, at a salinity of 14%, the most abundant cyanobacteria were related to highly halotolerant cultivated cyanobacteria, such as the recently established phylogenetic clusters of Euhalothece and Halospirulina . Although these organisms in cultures had previously demonstrated their ability to grow with close to optimal rates over a wide range of salinities, their occurrence in the field was restricted to the highest salinities investigated.     

Bacterial Diversity of the Broadbalk ‘Classical’ Winter Wheat Experiment in Relation to Long-Term Fertilizer Inputs

With more than 160 years of contrasting fertilizer regimes, the Broadbalk winter wheat experiment represents a unique experimental resource for studying the effects of long-term fertilizer application on microbial population diversity. Using DGGE and clone library analysis, we report here on eubacterial species diversity (16S rRNA gene) and diversity within two sets of gene products associated with microbial N acquisition: NifH (nitrogen fixation) and AmtB (ammonium transport). Comparisons were made within and between soils treated with mineral N fertilizer, farmyard manure or receiving no fertilizer. Analysis of 16S rRNA gene DGGE profiles showed no clear patterns to qualitatively distinguish bacterial community structure between the three different treatments (P > 0.05), with all samples containing a range of eubacterial taxa similar to those that are characteristic of soil bacteria reported elsewhere. Intra-plot heterogeneity was high and of a similar magnitude to that between treatments. This lack of qualitative between plot differences was echoed in the representative sequences of 16S rRNA, nifH, and amtB genes in the various samples. Taken together, both phylogenetic and functional gene analyses showed bacterial communities in the Broadbalk-trial soil were very stable and relatively non-responsive to long-term management of balanced fertilizer inputs.     

Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE

The analysis of microbial communities in environmental samples requires accurate and reproducible methods for extraction of DNA from sample matrices that have different physical and chemical characteristics. Even with the same sample type, variations in laboratory methods can result in different DNA yields. To circumvent this problem, we have developed an easy and inexpensive way to normalize the quantities of DNA that involves the addition of an internal standard prepared from plasmid DNA. The method was evaluated by comparing DNA yields using different DNA extraction procedures, after which the DNA was used for microbial community analysis by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S ribosomal RNA (rRNA) and for quantification of 16S rRNA gene copy numbers in environmental samples by real-time PCR. Our results show that use of the internal standard allows normalization of the resulting data and more accurate quantification of gene copy numbers in soil samples. These methods should also have broad application for various other types of environmental samples.     

Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied.     

Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods.     

Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.