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.

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

Plant species is considered to be one of the most important factors in shaping rhizobacterial communities, but specific plant-microbe interactions in the rhizosphere are still not fully understood. Arabidopsis thaliana, for which a large number of naturally occurring ecotype accessions exist, lacks mycorrhizal associations and is hence an ideal model for rhizobacterial studies. Eight Arabidopsis accessions were found to exert a marked selective influence on bacteria associated with their roots, as determined by terminal-restriction fragment length polymorphism (T-RFLP) and ribosomal intergenic spacer analysis (RISA). Community differences in species composition and relative abundance were both significant (P <0.001). The eight distinct and reproducible accession-dependent community profiles also differed from control bulk soil. Root exudates of these variants were analysed by high performance liquid chromatography (HPLC) to try to establish whether the unique rhizobacterial assemblages among accessions could be attributed to plant-regulated chemical changes in the rhizosphere. Natural variation in root exudation patterns was clearly exhibited, suggesting that differences in exudation patterns among accessions could be influencing bacterial assemblages. Other factors such as root system architecture are also probably involved. Finally, to investigate the Arabidopsis rhizosphere further, the phylogenetic diversity of rhizobacteria from accession Cvi-0 is described.

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Influence of Arabidopsis thaliana accessions on rhizobacterial communities and natural variation in root exudates

Effects of selected root exudate components on soil bacterial communities.

In highly urbanized areas, wastewater treatment plant (WWTP) effluent can represent a significant component of freshwater ecosystems. As it is impossible for the composition of WWTP effluent to match the composition of the receiving system, the potential exists for effluent to significantly impact the chemical and biological characteristics of the receiving ecosystem. We assessed the impacts of WWTP effluent on the size, activity, and composition of benthic microbial communities by comparing two distinct field sites in the Chicago metropolitan region: a highly urbanized river receiving effluent from a large WWTP and a suburban river receiving effluent from a much smaller WWTP. At sites upstream of effluent input, the urban and suburban rivers differed significantly in chemical characteristics and in the composition of their sediment bacterial communities. Although effluent resulted in significant increases in inorganic nutrients in both rivers, surprisingly, it also resulted in significant decreases in the population size and diversity of sediment bacterial communities. Tag pyrosequencing of bacterial 16S rRNA genes revealed significant effects of effluent on sediment bacterial community composition in both rivers, including decreases in abundances of Deltaproteobacteria, Desulfococcus, Dechloromonas, and Chloroflexi sequences and increases in abundances of Nitrospirae and Sphingobacteriales sequences. The overall effect of the WWTP inputs was that the two rivers, which were distinct in chemical and biological properties upstream of the WWTPs, were almost indistinguishable downstream. These results suggest that WWTP effluent has the potential to reduce the natural variability that exists among river ecosystems and indicate that WWTP effluent may contribute to biotic homogenization.

Wastewater Treatment Effluent Reduces the Abundance and Diversity of Benthic Bacterial Communities in Urban and Suburban Rivers

Biodegradation of ciprofloxacin in water and soil and its effects on the microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) is increasingly being used to examine microbial community structure and accordingly, a range of approaches have been used to analyze data sets. A number of published reports have included data and results that were statistically flawed or lacked rigorous statistical testing. A range of simple, yet powerful techniques are available to examine community data, however their use is seldom, if ever, discussed in microbial literature. We describe an approach that overcomes some of the problems associated with analyzing community datasets and offer an approach that makes data interpretation simple and effective. The Bray-Curtis coefficient is suggested as an ideal coefficient to be used for the construction of similarity matrices. Its strengths include its ability to deal with data sets containing multiple blocks of zeros in a meaningful manner. Non-metric multi-dimensional scaling is described as a powerful, yet easily interpreted method to examine community patterns based on T-RFLP data. Importantly, we describe the use of significance testing of data sets to allow quantitative assessment of similarity, removing subjectivity in comparing complex data sets. Finally, we introduce a quantitative measure of sample dispersion and suggest its usefulness in describing site heterogeneity.

Ordination and significance testing of microbial community composition derived from terminal restriction fragment length polymorphisms: application of multivariate statistics

The export of nitrogen from urban catchments is a global problem, and denitrifying bacteria in stream ecosystems are critical for reducing in-stream N. However, the environmental factors that control the composition of denitrifying communities in streams are not well understood. We determined whether denitrifying community composition in sediments of nine streams on the eastern fringe of Melbourne, Australia was correlated with two measures of catchment urban impact: effective imperviousness (EI, the proportion of a catchment covered by impervious surfaces with direct connection to streams) or septic tank density (which affects stream water chemistry, particularly stream N concentrations). Denitrifying community structure was examined by comparing terminal restriction fragment length polymorphisms of nosZ genes in the sediments, as the nosZ gene codes for nitrous oxide reductase, the last step in the denitrification pathway. We also determined the chemical and physical characteristics of the streams that were best correlated with denitrifying community composition. EI was strongly correlated with community composition and sediment physical and chemical properties, while septic tank density was not. Sites with high EI were sandier, with less fine sediment and lower organic carbon content, higher sediment cations (calcium, sodium and magnesium) and water filterable reactive phosphorus concentrations. These were also the best small-scale environmental variables that explained denitrifying community composition. Among our study streams, which differed in the degree of urban stormwater impact, sediment grain size and carbon content are the most likely drivers of change in community composition. Denitrifying community composition is another in a long list of ecological indicators that suggest the profound degradation of streams is caused by urban stormwater runoff. While the relationships between denitrifying community composition and denitrification rates are yet to be unequivocally established, landscape-scale indices of environmental impact such as EI may prove to be useful indicators of change in microbial communities.

Urban Stormwater Runoff Drives Denitrifying Community Composition Through Changes in Sediment Texture and Carbon Content

Urbanization leads to degradation in water quality and has a major effect on the biota of streams, but its effect on microbial communities is not as well understood. DNA-techniques that target functional genes are being used to examine microbial communities, but less frequently applied to freshwater aquatic systems. Our aim was to determine whether terminal restriction fragment length polymorphism and sequence analysis of polymerase chain reaction-amplified (PCR) nosZ gene sequences could be used to show if there were measurable differences in the denitrifying community in two urban streams in catchments with contrasting degrees of catchment urbanization. Community structure in the sediments and associated riparian zones were studied at the contrasting sites. We showed that the denitrifying community in the sediments and riparian soils of the two streams were significantly different. There were also significant differences between the sediment and riparian zone communities within each of the sites. Terminal restriction fragment length polymorphism analysis proved to be a valuable technique that could resolve patterns of the denitrifying community in streams of contrasting degrees of urbanization, but sequence analysis was required to confirm the identity of the amplified products.

Analysis of denitrifying communities in streams from an urban and non-urban catchment

Urbanization is an important ecological modifier of stream catchments and significantly alters C type and concentration and heavy metal input to aquatic systems. C and heavy metals are important determinants of microbial community structure (population dynamics) and function (physiological processes). Understanding how changes at the landscape scale affect key nutrient-transformation processes (e.g., denitrification) and C metabolism (e.g., methanogenesis) at the micro scale requires simultaneous determination of the structural and functional responses of bacterial communities. We compared total bacterial (16S ribosomal ribonucleic acid [rRNA] gene) and denitrifying bacterial (nosZ gene) community structures in sediments taken from an urban and a nonurban stream before and after C and Zn additions. Microcosms containing stream sediment were supplemented with either proteinaceous or leaf-leachate C, increasing the C content by ∼36 and 130 mg/L, respectively. C was added in the presence and absence ...

https://www.jstor.org/stable/10.1899/11-009.1

Shane E. Perryman

Papers

Ordination and significance testing of microbial community composition derived from terminal restriction fragment length polymorphisms: application of multivariate statistics

Urban Stormwater Runoff Drives Denitrifying Community Composition Through Changes in Sediment Texture and Carbon Content

Analysis of denitrifying communities in streams from an urban and non-urban catchment

Sediment bacterial community structure and function in response to C and Zn amendments: urban and nonurban streams

The lower Broken Creek : aspects of water quality and growth of Azolla species