属特异性T-RFLP技术用于乳酸杆菌的群落分析

【目的】设计乳酸杆菌属特异性T-RFLP技术(末端限制性片段长度多态性分析)对14株乳酸杆菌进行分型。【方法】采用源于16S-23S rRNA基因间隔区序列的乳酸杆菌属特异性引物LAB-rev,乳酸杆菌的属特异性引物,6-FAM荧光标记后结合16S上游通用引物7f用于乳酸杆菌的PCR扩增。【结果】选取HaeⅢ和HhaⅠ进行限制性酶切,最后对酶切后的产物末端测序得到T-RFLP峰谱图,该图谱能够快速准确地对不同种的乳酸杆菌进行定性、定量的分析。【结论】实验成功搭建T-RFLP技术用于微生态环境中乳酸杆菌检测的平台,对于在功能性食品、乳酸饮料和药物对肠道微生态的影响及菌种鉴定等领域有重大意义。     

Molecular analysis of microbial population transition associated with the start of denitrification in a wastewater treatment process

AIMS: The objective of this study is to determine the bacteria playing an important role in denitrification by monitoring the molecular dynamics accompanying the start of denitrification. METHODS AND RESULTS: cDNA reverse-transcribed from 16S rRNA was amplified with fluorescent labelled primer for terminal restriction fragment length polymorphism (T-RFLP) analysis and an unlabelled primer for cloning analysis. The terminal restriction fragments (T-RFs) that increased in association with the start of denitrification were determined. These T-RFs were identified by in silico analysis of 16S rRNA sequences obtained from cloning. As a result, it was clearly observed that the bacteria belonging to the genera Hydrogenophaga and Acidovorax increased in number after the start of denitrification. CONCLUSIONS: It was demonstrated that T-RFLP analysis targeting 16S rRNA is appropriate for the daily monitoring of a bacterial community to control wastewater treatment processes. Combination of the results of T-RFLP analysis and 16S rRNA clone library indicated that the bacteria belonging to the genera Hydrogenophaga and Acidovorax play an important role in denitrification. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide new insight to the 16S rRNA level of active denitrifying bacteria in wastewater treatment processes.     

Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial populations in human feces

New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.     

Application of New Primer-Enzyme Combinations to Terminal Restriction Fragment Length Polymorphism Profiling of Bacterial Populations in Human Feces

New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.     

Formation of pseudo-terminal restriction fragments,a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.     

Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.     

A novel strategy to extract specific phylogenetic sequence information from community T-RFLP

Cultivation-independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. Semi-automated terminal restriction fragment length polymorphism (T-RFLP) analyses yield high-resolution genetic profiles of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring. A serious limitation of T-RFLP analyses has been the inability to reliably affiliate observed terminal restriction fragments (T-RF) to phylogenetic groups. In the study presented here, we were able to overcome this limitation of T-RFLP. With a combination of adapter ligation, fragment size selection, and re-amplification with adapter site specific PCR, we were able to isolate a T-RF-fraction of a narrow size-range containing a T-RF that was significantly more abundant in heavy metal amended soils. Cloning the size-selected T-RF fraction allowed for the efficient isolation of clones containing this specific T-RF. Sequence determination and phylogenetic inference in RDP-II affiliated the sequence to unclassified cyanobacteria. Specific primer design and PCR amplification from bulk soil DNA allowed for independent confirmation of the results from bacterial T-RFLP and T-RF cloning. Our results show that specific T-RFs can be efficiently isolated and identified, and that the adapter ligation approach holds great potential for genetic profiling and for identification of community components of interest.     

Community structure analyses are more sensitive to differences in soil bacterial communities than anonymous diversity indices

Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities.     

Community Structure Analyses Are More Sensitive to Differences in Soil Bacterial Communities than Anonymous Diversity Indices

Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities.     

Novel bacterial lineages at the (sub)division level as detected by signature nucleotide-targeted recovery of 16S rRNA genes from bulk soil and rice roots of flooded rice microcosms

Using a newly developed 16S rRNA gene (rDNA)-targeted PCR assay with proposed group specificity for planctomycetes, we examined anoxic bulk soil of flooded rice microcosms for the presence of novel planctomycete-like diversity. For comparison, oxic rice roots were included as an additional sample in this investigation. The bacterial diversity detectable by this PCR assay was assessed by using a combined approach that included terminal restriction fragment length polymorphism (T-RFLP) analysis and comparative sequence analysis of cloned 16S rDNA. T-RFLP fingerprint patterns generated from rice roots contained 12 distinct terminal restriction fragments (T-RFs). In contrast, the T-RFLP fingerprint patterns obtained from the anoxic bulk soil contained 33 distinct T-RFs, a clearly higher level of complexity. A survey of 176 bulk soil 16S rDNA clone sequences permitted correlation of 20 T-RFs with phylogenetic information. The other 13 T-RFs remained unidentified. The predominant T-RFs obtained from rice roots could be assigned to members of the genus Pirellula within the Planctomycetales, while most of the T-RFs obtained from the bulk soil corresponded to novel lines of bacterial descent. Using a level of 16S rDNA sequence dissimilarity to cultured microorganisms of approximately 20% as a threshold value, we detected 11 distinct bacterial lineages for which pure-culture representatives are not known. Four of these lineages could be assigned to the order Planctomycetales, while one lineage was affiliated with the division Verrucomicrobia and one lineage was affiliated with the spirochetes. The other five lineages either could not be assigned to any of the main lines of bacterial descent or clearly expanded the known diversity of division level lineages WS3 and OP3. Our results indicate the presence of bacterial diversity at a subdivision and/or division level that has not been detected previously by the so-called universal 16S rDNA PCR assays.     

Post-amplification Klenow fragment treatment alleviates PCR bias caused by partially single-stranded amplicons

Partially single-stranded amplicons, formed during PCR amplification of single and mixed templates, are a potential source of bias in genetic diversity studies. The analysis of 16S rRNA gene diversity in mixed template samples by the fingerprinting technique terminal restriction fragment length polymorphism (T-RFLP) analysis can be biased by the occurrence of pseudo-T-RFs, i.e., restriction fragments occurring in addition to the expected terminal restriction fragments of single amplicons. This bias originates from PCR products, which are single-stranded at their terminal restriction site. Here we show that treatment of PCR amplicons with Klenow fragment prior to restriction digest and T-RFLP analysis minimized effectively the occurrence of pseudo-T-RFs. Klenow fragment activity filled in bases into the partially single-stranded amplicons and thereby restored the affected amplicons to complete double strands. Our method allowed to improve the assessment of genetic diversity and gene ratios from T-RFLP analysis of an original environmental sample. Since partially single-stranded amplicons might influence many PCR-based techniques, post-amplification treatment with Klenow fragment may be useful for a wide range of applications, which assess the composition of amplicon pools, e.g., the analysis of marker gene diversity in mixed template samples by fingerprinting techniques or the analysis of sequence diversity by cloning.     

Molecular analysis of bacterial communities associated with the roots of Douglas fir (Pseudotsuga menziesii) colonized by different ectomycorrhizal fungi

We studied the effect of ectomycorrhizal fungi on bacterial communities colonizing roots of Douglas fir (Pseudotsuga menziesii). Mycorrhizal tips were cleaned of soil and separated based on gross morphological characteristics. Sequencing of the internal transcribed spacers of the nuclear rRNA gene cluster indicated that the majority of the tips were colonized by fungi in the Russulaceae, with the genera Russula and Lactarius comprising 70% of the tips. Because coamplification of organellar 16S rRNA genes can interfere with bacterial community analysis of root tips, we developed and tested a new primer pair that permits amplification of bacterial 16S rRNA genes but discriminates more effectively against organellar sequences than commonly used bacterial primer sets. We then used terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of the 16S rRNA gene to examine differences in bacterial communities associated with the mycorrhizal tips. Cluster analysis of T-RFLP profiles indicated that there were different bacterial communities among the root tips; however, the communities did not seem to be affected by the taxonomic identity of the ectomycorrhizal fungi. Terminal restriction fragment profiling and sequencing of cloned partial 16S rRNA genes indicated that most bacteria on the ectomycorrhizal tips were related to the Alphaproteobacteria and the Bacteroidetes group.     

Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.     

Novel Bacterial Lineages at the (Sub)Division Level as Detected by Signature Nucleotide-Targeted Recovery of 16S rRNA Genes from Bulk Soil and Rice Roots of Flooded Rice Microcosms

Using a newly developed 16S rRNA gene (rDNA)-targeted PCR assay with proposed group specificity for planctomycetes, we examined anoxic bulk soil of flooded rice microcosms for the presence of novel planctomycete-like diversity. For comparison, oxic rice roots were included as an additional sample in this investigation. The bacterial diversity detectable by this PCR assay was assessed by using a combined approach that included terminal restriction fragment length polymorphism (T-RFLP) analysis and comparative sequence analysis of cloned 16S rDNA. T-RFLP fingerprint patterns generated from rice roots contained 12 distinct terminal restriction fragments (T-RFs). In contrast, the T-RFLP fingerprint patterns obtained from the anoxic bulk soil contained 33 distinct T-RFs, a clearly higher level of complexity. A survey of 176 bulk soil 16S rDNA clone sequences permitted correlation of 20 T-RFs with phylogenetic information. The other 13 T-RFs remained unidentified. The predominant T-RFs obtained from rice roots could be assigned to members of the genus Pirellula within the Planctomycetales, while most of the T-RFs obtained from the bulk soil corresponded to novel lines of bacterial descent. Using a level of 16S rDNA sequence dissimilarity to cultured microorganisms of approximately 20% as a threshold value, we detected 11 distinct bacterial lineages for which pure-culture representatives are not known. Four of these lineages could be assigned to the order Planctomycetales, while one lineage was affiliated with the division Verrucomicrobia and one lineage was affiliated with the spirochetes. The other five lineages either could not be assigned to any of the main lines of bacterial descent or clearly expanded the known diversity of division level lineages WS3 and OP3. Our results indicate the presence of bacterial diversity at a subdivision and/or division level that has not been detected previously by the so-called universal 16S rDNA PCR assays.     

Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.     

Development and application of a T-RFLP data analysis method using correlation coefficient matrices

Environmental microbiology studies commonly use terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes, for example, to analyze changes in community structure in relation to changing physicochemical and biological conditions over space and time. Although T-RFLP is most useful for comparing samples from different environments, a large number of samples makes effective analysis difficult using the Web-based tools that are currently available. To resolve this dilemma, we used a new approach for calculating data from multiple T-RFLP samples by estimating terminal fragment combinations, then applying a correlation analysis using two different fluorescent dyes generated simultaneously from all samples. This calculation was based on the expectation that the proportions of two terminal fragments from one full-length polymerase chain reaction fragment would be nearly the same in each analysis. Using this program, the oral microflora in 73 human saliva samples were analyzed, and 24 bacterial groups, with peak areas of at least 0.5% and correlation coefficients of 0.55 or greater, were identified from the T-RFs within 40 s.     

Bacterial communities in Arctic fjelds of Finnish Lapland are stable but highly pH-dependent

The seasonal and spatial variations of microbial communities in Arctic fjelds of Finnish Lapland were studied. Phospholipid fatty acid analysis (PLFA) and terminal restriction fragment analysis (T-RFLP) of amplified 16S rRNA genes were used to assess the effect of soil conditions and vegetation on microbial community structures along different altitudes of two fjelds, Saana and Jehkas. Terminal restriction fragments were additionally analysed from c. 160 cloned sequences and isolated bacterial strains and matched with those of soil DNA samples. T-RFLP and PLFA analyses indicated relatively similar microbial communities at various altitudes and under different vegetation of the two fjelds. However, soil pH had a major influence on microbial community composition. Members of the phylum Acidobacteria dominated especially in the low pH soils (pH 4.6-5.2), but above pH 5.5, the relative amount of terminal restriction fragments corresponding to acidobacterial clones was substantially lower. Both T-RFLP and PLFA analysis indicated stable microbial communities as the DNA and fatty acid profiles were similar in spring and late summer samples sampled over 3 years. These results indicate that differences in microbial community composition could be explained primarily by variation in the bedrock materials that cause variation in the soil pH.     

Analysis of the endophytic actinobacterial population in the roots of wheat (Triticum aestivum L.) by terminal restriction fragment length polymorphism and sequencing of 16S rRNA clones

The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.     

Evaluation of three different forward primers by terminal restriction fragment length polymorphism analysis for determination of fecal bifidobacterium spp. in healthy subjects

The 27F forward primer is frequently used in 16S rRNA gene libraries and T-RFLP analysis. However, Bifidobacterium spp. were barely detected with this primer in human fecal samples. In this study, fecal microbiota were analyzed using the T-RFLP method with three different forward primers (27F, 35F, and 529F) in conjunction with one reverse primer (1492R). T-RFLP analysis of fecal microbiota using 35F and 529F detected higher proportions of the terminal restriction fragment (T-RF) corresponding to Bifidobacterium spp. than that using 27F. 27F is in imperfect agreement while 35F and 529F are in good concordance with the 16S rRNA gene sequences of Bifidobacterium spp., and the latter primers allowed for the detection of T-RFs of Bifidobacterium spp. in fecal samples from five healthy subjects. The T-RFs presumed to be Bifidobacterium spp. were cloned and sequenced, and found to match the 16S rRNA gene sequences of Bifidobacterium spp. Among the five fecal samples, two samples with low frequencies of T-RFs of Bifidobacterium spp. were detected using these forward primers. This probably reflects a low prevalence of Bifidobacterium spp. in these two samples. Our study emphasizes the importance of selecting a suitable forward primer for detection of Bifidobacterium spp.