Statistical methods for characterizing diversity of microbial communities by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes

The analysis of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes has proven to be a facile means to compare microbial communities and presumptively identify abundant members. The method provides data that can be used to compare different communities based on similarity or distance measures. Once communities have been clustered into groups, clone libraries can be prepared from sample(s) that are representative of each group in order to determine the phylogeny of the numerically abundant populations in a community. In this paper methods are introduced for the statistical analysis of T-RFLP data that include objective methods for (i) determining a baseline so that 'true' peaks in electropherograms can be identified; (ii) a means to compare electropherograms and bin fragments of similar size; (iii) clustering algorithms that can be used to identify communities that are similar to one another; and (iv) a means to select samples that are representative of a cluster that can be used to construct 16S rRNA gene clone libraries. The methods for data analysis were tested using simulated data with assumptions and parameters that corresponded to actual data. The simulation results demonstrated the usefulness of these methods in their ability to recover the true microbial community structure generated under the assumptions made. Software for implementing these methods is available at http://www.ibest.uidaho.edu/tools/trflp_stats/index.php.     

Characterization of microbial communities found in the human vagina by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes

To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for the isolation of genomic DNA and an optimal strategy for T-RFLP analyses. The various genera in the model community could best be resolved by digesting amplicons made using bacterial primers 8f and 926r with HaeIII; fewer strains could be resolved using other primer-enzyme combinations, and no combination successfully distinguished certain species of the same genus. To demonstrate the utility of the approach, samples from five women that had been collected over a 2-month period were analyzed. Differences and similarities among the vaginal microbial communities of the women were readily apparent. The T-RFLP data suggest that the communities of three women were dominated by a single phylotype, most likely species of Lactobacillus. In contrast, the communities of two other women included numerically abundant populations that differed from Lactobacillus strains whose 16S rRNA genes had been previously determined. The T-RFLP profiles of samples from all the women were largely invariant over time, indicating that the kinds and abundances of the numerically dominant populations were relatively stable throughout two menstrual cycles. These findings show that T-RFLP of 16S rRNA genes can be used to compare vaginal microbial communities and gain information about the numerically dominant populations that are present.     

Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the Proteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.     

Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.     

Haloacetic acid-degrading bacterial communities in drinking water systems as determined by cultivation and by terminal restriction fragment length polymorphism of PCR-amplified haloacid dehalogenase gene fragments

Aims: To characterize the HAA‐degrading bacteria in drinking water systems. Methods and Results: Haloacetic acid (HAA)‐degrading bacteria were analysed in drinking water systems by cultivation and by a novel application of terminal restriction fragment length polymorphism (tRFLP). Substantial similarities were observed among the tRFLP patterns of dehI and dehII gene fragments in drinking water samples obtained from three different cities (Minneapolis, MN; St Paul, MN; Bucharest, Romania) and from one biologically active granular activated carbon filter (Hershey, PA). The dominant fragment in the tRFLP profiles of dehI genes from the drinking water samples matched the pattern from an Afipia sp. that was previously isolated from drinking water. In contrast, the dominant fragment in the tRFLP profiles of dehII genes did not match any previously characterized dehII gene fragment. PCR cloning was used to characterize this gene fragment, which had <65% nucleotide sequence identity with any previously characterized dehII gene. Conclusions: Afipia spp. are an appropriate model organism for studying the biodegradation of HAAs in drinking water distribution systems as encoded by dehI genes; the organism that harbours the most prominent dehII gene in drinking water has yet to be cultivated and identified. Significance and Impact of the Study: The development of a novel application of tRFLP targeting dehI and dehII genes could be broadly useful in understanding HAA‐degrading bacteria in numerous environments.     

基于T-RFLP方法的连栽杉木根际土壤细菌群落变化研究

运用末端限制性片段长度多态性技术(T-RFLP),研究福建省尤溪国有林场的多代连栽杉木根际土壤细菌群落变化特征。结果表明:杉木多代连栽后根际微生态失衡,表现为碳循环菌和氮循环菌等有益菌数量减少,病原菌数量增加。在不同代数杉木土壤中,4种内切酶消化后产生的末端限制性片段(T-RFs)数量具有显著差异,第一代杉木(first Chinese fir rotation plantation,FCP)最高,第二代杉木(second Chinese fir rotation plantation,SCP)次之,第三代杉木(third Chinese fir rotation plantation,TCP)最少。土壤理化性质结果表明连栽导致土壤养分逐代降低,除土壤碳氮比与细菌群落多样性呈显著负相关外,土壤养分含量指标与细菌群落多样性呈显著正相关。T-RFLP实验共鉴定出细菌门类12门,其中厚壁菌门、蓝藻门、放线菌门和变形菌门是主要优势菌群。随栽植代数的增加,变形菌门细菌比例递增,而厚壁菌门细菌比例递减。土壤细菌群落多样性指数,表现为随栽植代数增长而减少,呈现FCPSCPTCP趋势,与T-RFs片段数量变化趋势基本一致,表明多代连栽降低了杉木根际土壤细菌群落多样性。依据功能性不同,可将鉴定出的细菌划分为5类:碳循环功能菌、硫循环功能菌、氮循环功能菌、纤维降解菌及病原菌。连栽导致碳循环菌和氮循环菌相对含量降低,硫循环菌和纤维降解菌相对含量提高,病原菌相对含量随栽植代数增加而上升。     

Diversity of uncultured microorganisms associated with the seagrass Halophila stipulacea estimated by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes.

The diversity of microorganisms associated with the leaves of the seagrass Halophila stipulacea in the northern Gulf of Elat was examined by culture-independent analysis. Microorganisms were harvested by a sonication treatment for total-community genomic DNA isolation. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used for PCR amplification. The 16S rDNA PCR products were subcloned and further characterized by a restriction fragment length analysis termed ARDRA (amplified rDNA restriction analysis). These analyses were carried out after reamplifying the cloned fragments with two primers binding symmetrically to the plasmid immediately on both sides of the cloned insert. Computer-aided clustering was performed after separate restriction analysis with enzymes HinfI and HpaII. By this method, 103 cloned 16S rDNA fragments were clustered into a total of 58 different groups. Sequence analysis of clones with an identical ARDRA pattern confirmed that members of an ARDRA group were closely related to each other. The sequenced clones were found to be affiliated with a marine snow-associated plastid-like rRNA clone and with a marine Hyphomonas strain, respectively. The method applied in this study could be useful for the routine study of other microbial communities of interest.     

Identification of acetic acid bacteria by restriction fragment length polymorphism analysis of a PCR-amplified fragment of the gene coding for 16S rRNA

Acetic acid bacteria (AAB) irreversibly spoil wines and represent a serious problem. Limited studies on the ecology of AAB during winemaking have been done due to the lack of rapid and precise techniques for their identification. RFLP analysis of PCR-amplified fragment of 16S rDNA was performed on AAB reference strains. The amplified rDNAs were approximately 870-bp long for all AAB species while no amplicons were detected for lactic acid bacteria and yeasts. Out of the four restriction enzymes tested, TaqI was the most efficient one and divided the studied AAB into six groups. However, complete differentiation among collection strains of Acetobacter pasteurianus and Gluconoacetobacter hansenii was not possible.     

Maturation of the murine cecal microbiota as revealed by terminal restriction fragment length polymorphism and 16S rRNA gene clone libraries

The maturation of murine cecal microbiota was determined by terminal restriction fragment polymorphism (T-RFLP) and 16S rRNA gene clone libraries. Cecal microbiota in specific pathogen free (SPF) mice aged four to 10 weeks were collected. The cluster of samples in 4-week-old mice was different from those of other ages based on T-RFLP profiles. The majority of clones obtained in this study belonged to the Clostridium coccoides (C. coccoides) group, the Bacteroides group or the Lactobacillus group. Phylogenetic analysis showed characteristic clusters composed of new operational taxonomic unit (OTU) of the C. coccoides and Bacteroides groups. The existence of a large number of yet unidentified bacteria inhabiting the murine cecum was demonstrated by 16S rRNA gene clone libraries. T-RFLP analysis data were more complex and more sensitive than the patterns generated by computer simulation of 16S rRNA gene clone library analysis data. T-RFLP revealed development with maturation of cecal microbiota including unidentified bacteria of SPF mice.     

Phylogenetic clustering of soil microbial communities by 16S rRNA but not 16S rRNA genes

We evaluated phylogenetic clustering of bacterial and archaeal communities from redox-dynamic subtropical forest soils that were defined by 16S rRNA and rRNA gene sequences. We observed significant clustering for the RNA-based communities but not the DNA-based communities, as well as increasing clustering over time of the highly active taxa detected by only rRNA.     

Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms

K.-B. JOUNG AND J.-C. CÔTÉ. 2001 .
Aims: To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them.
Methods and Results: Using 16S rRNA gene restriction fragment length polymorphisms generated by Hin dIII and Eco RI, 86 Bacillus thuringiensis strains were classified. This includes 80 B. thuringiensi s serovars and five more strains, kurstaki HD-1, subtoxicus , dendrolimus , tenebrionis and sandiego , to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis . All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia , at the 92·5% DNA relatedness rate.
Conclusions: 16S rRNA gene fingerprinting cannot only be used for the classification of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains.
Significance and Impact of the Study: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.     

Detection and identification of bacterial pathogens of fish in kidney tissue using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes

We report the application of a nucleic acid-based assay that enables direct detection and identification of bacterial pathogens in fish kidney tissue without the need for bacterial culture. The technique, known as terminal restriction fragment length polymorphism (T-RFLP), employs the polymerase chain reaction (PCR) using a primer pair that targets 2 highly conserved regions of the gene that encodes for the 16S small subunit of the bacterial ribosome. Each primer is 5' labeled with a different fluorescent dye, which results in each terminus of the resulting amplicon having a distinguishable fluorescent tag. The amplicon is then digested with a series of 6 restriction endonucleases, followed by size determination of the 2 labeled terminal fragments by capillary electrophoresis with laser-induced fluorescence detection. Comparison of the lengths of the full set of 12 terminal fragments with those predicted based on analyses of GenBank submissions of 16S sequences leads to presumptive identification of the pathogen to at least the genus, but more typically the species level. Results of T-RFLP analyses of genomic DNA from multiple strains of a number of fish bacterial pathogens are presented. The assay is further demonstrated on fish kidney tissue spiked with a known number of cells of Flavobacterium psychrophilum where a detection limit of ca. 30 CFU mg(-1) of tissue was estimated. A similar detection limit was observed for several other gram-negative pathogens. This procedure was also used to detect Aeromonas salmonicida and Renibacterium salmoninarum in the kidney tissue of 2 naturally infected salmonids.     

Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms

AIMS: To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them. METHODS AND RESULTS: Using 16S rRNA gene restriction fragment length polymorphisms generated by HindIII and EcoRI, 86 Bacillus thuringiensis strains were classified. This includes 80 B. thuringiensis serovars and five more strains, kurstaki HD-1, subtoxicus, dendrolimus, tenebrionis and sandiego, to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis. All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia, at the 92.5% DNA relatedness rate. CONCLUSION: 16S rRNA gene fingerprinting cannot only be used for the classification of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.     

Restriction fragment length polymorphisms of 16S rRNA genes in the differentiation of fast-growing mycobacterial species

Abstract DNA from several species of fast growing mycobacteria displayed a characteristic restriction fragment length polymorphism (RFLP) pattern when hybridizated to a Mycobacterium fortuitum 16S rRNA gene fragment. The resulting patterns were identical when comparing different strains belonging to the same species. The RFLP results were consistent with those obtained by DNA-DNA hybridization studies. Using this approach, we have been able to identify the number of copies for 16S rRNA genes in several fast-growing mycobacteria.     

Species of Borrelia distinguished by restriction site polymorphisms in 16S rRNA genes

Abstract Three phyletic groups of Borrelia associated with Lyme disease, B. burgdorferi, B. garinii and group VS461 can be distinguished from each other and other species of Borrelia by Bfa I restriction site polymorphisms in PCR amplified 16S rRNA genes. One strain isolated from an Ixodes pacificus tick in California that was previously unclassifiable was distinguishable from B. burgdorferi by an Mnl I restriction site polymorphism.     

Differentiation of Bartonella species using restriction endonuclease analysis of PCR-amplified 16S rRNA genes

Abstract Differentiation of the four Bartonella species which were formerly classified as Rochalimaea using restriction endonuclease analysis of PCR-amplified citrate synthase gene fragments has previously been described. However, attempts to extend this method to include all members of Bartonella were confounded when amplification of the gene fragment from strains of B. bacilliformis each yielded two products of differing sizes. An alternative differentiation scheme for Bartonella species was developed based on restriction endonuclease analysis of their 16S rRNA genes. As the complete 16S rRNA gene sequences of all extant Bartonella species are available, the usefulness of specific endonucleases could be theoretically predetermined rather than discovered empirically. The potential usefulness of the restriction enzymes Ddel and Mnll was established using this approach, and this potential was confirmed in practice as all eight species could be distinguished from each other.     

Rapid identification of Listeria species by using restriction fragment length polymorphism of PCR-amplified 23S rRNA gene fragments

A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua. Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.     

Polymerase chain reaction amplification and restriction fragment length polymorphism analysis of 16S rRNA genes from methanogens

For restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, the rDNA fragments of 1.5 kb were amplified by polymerase chain reaction (PCR) from crude cell lysates of various methanogenic species which were prepared by a combined technique of ultrasonic treatment and protease digestion. The PCR products were purified by the polyethylene glycol precipitation method and treated with various restriction enzymes. The 16S rDNA fragments digested with HaeIII or HhaI gave species-specific RFLP profiles on simplified agarose gel electrophoresis. 16S rDNA gragments of 0.4 kb from the bulk DNA extracted from mixed populations of anaerobic sludge were also amplified by PCR with a pair of methanogen-specific primers and cloned directly by the T-A cloning technique. The cloned 16S rDNAs from recombinants were reamplified by PCR, and RFLP pattern analysis was performed following digestion with HhaI. The PCR-RFLP analysis of 16S rDNA with the present protocol can be completed within one day, provided that sufficient amounts of test cells are available, and has great promise as a simple and rapid technique for identification of methanogens. A combined method consisting of PCR amplification, direc cloning with T vectors, and RFLP analysis of 16S rDNA is also useful for rapid estimation of the mixed population structure of methanogens without the need for cultivation and isolation.     

Horizontal heterogeneity of denitrifying bacterial communities in marine sediments by terminal restriction fragment length polymorphism analysis

Although it is widely believed that horizontal patchiness exists in microbial sediment communities, determining the extent of variability or the particular members of the bacterial community which account for the observed differences among sites at various scales has not been routinely demonstrated. In this study, horizontal heterogeneity was examined in time and space for denitrifying bacteria in continental shelf sediments off Tuckerton, N.J., at the Rutgers University Long-Term Ecosystem Observatory (LEO-15). Characterization of the denitrifying community was done using PCR amplification of the nitrous oxide reductase (nosZ) gene combined with terminal restriction fragment length polymorphism analysis. Spatial scales from centimeters to kilometers were examined, while temporal variation was assayed over the course of 1995 to 1996. Sorenson's indices (pairwise similarity values) were calculated to permit comparison between samples. The similarities of benthic denitrifiers ranged from 0.80 to 0.85 for centimeter scale comparisons, from 0.52 to 0.79 for meter level comparisons, and from 0.23 to 0.53 for kilometer scale comparisons. Sorenson's indices for temporal comparisons varied from 0.12 to 0.74. A cluster analysis of the similarity values indicated that the composition of the denitrifier assemblages varied most significantly at the kilometer scale and between seasons at individual stations. Specific nosZ genes were identified which varied at centimeter, meter, or kilometer scales and may be associated with variability in meio- or macrofaunal abundance (centimeter scale), bottom topography (meter scale), or sediment characteristics (kilometer scale).     

Bacterial community profiles on feathers during composting as determined by terminal restriction fragment length polymorphism analysis of 16S rDNA genes

Composting is one of the more economical and environmentally safe methods of recycling feather waste generated by the poultry industry, since 90% of the feather weight consists of crude keratin protein, and feathers contain 15% N. However, the keratin in waste feathers is resistant to biodegradation and may require the addition of bacterial inocula to enhance the degradation process during composting. Two keratin-degrading bacteria isolated from plumage of wild songbirds and identified as Bacillus licheneformis (OWU 1411T) and Streptomyces sp. (OWU 1441) were inoculated into poultry feather composts (1.13×10⁸ cfu g^–1 feathers) and co-composted with poultry litter and straw in 200-l compost vessels. Composting temperatures, as well as CO₂ and NH₃ evolution, were measured in these vessels to determine the effects of inoculation on the rate and extent of poultry feather decomposition during composting. Terminal restriction fragment length polymorphisms of 16S rRNA genes were used to follow changes in microbial community structure during composting. The results indicated that extensive carbon conversion occurred in both treatments (55.5 and 56.1%). The addition of the bacterial inocula did not enhance the rate of waste feather composting. The microbial community structure over time was very similar in inoculated and uninoculated waste feather composts.