Assessment of Microbial Diversity in Four Southwestern United States Soils by 16S rRNA Gene Terminal Restriction Fragment Analysis

Abstract

The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662-1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, HhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.Rapid analysis of diversity in complex microbial communities has remained an elusive but important goal in microbial ecology. Community diversity can be examined at several levels. The most simple analyses use DNA profiles (generated by PCR and sometimes followed by restriction digestion of amplification mixtures) to identify differences in the composition of communities. More refined approaches describe differences not only in community composition but also in community organization by measuring the number (richness) and relative abundance (structure or evenness) of species or phylotypes. The richness and evenness of biological communities reflect selective pressures that shape diversity within communities. Measuring these parameters is most useful when assessing treatment effects (e.g., physical disturbance, pollution, nutrient addition, predation, climate change, etc.) on community diversity. Diversity statistics can also indicate the ability of a community to recover from disturbance and utilize resources efficiently (4). An ideal method for analysis of diversity in complex microbial communities would enable the simultaneous measurement of composition, phylotype richness, and community structure. The method would be rapid and reproducible and would permit flexible sampling of the entire microbial community.Direct amplification of bacterial 16S rRNA genes from extracted soil DNA provides the most comprehensive and flexible means of sampling bacterial communities. Analysis of clone libraries of 16S rRNA genes amplified from different environments can pro...

The diversity and biogeography of soil bacterial communities

“…One limitation of the T-RFLP method is that it underestimates total bacterial diversity because the method resolves only a limited number of bands per gel (generally Ͻ100), and bacterial species can share phylotypes (37). However, the method does provide a robust index of bacterial diversity (35,36,38), and T-RFLP results are generally consistent with the results from clone libraries (39,40).…”

Changes in bacterial community of anthracene bioremediation in municipal solid waste composting soil

“…Some other profiling techniques, such as community-level physiological profiling, restriction fragment length polymorphism, denaturing gradient gel electrophoresis, and phospholipid fatty acid profiling, have been applied to investigate the composition of bacterial community in contaminated aquifers near MSW composting sites (Röling et al, 2000;Tian et al, 2005;Yu et al, 2010). Among available DNA profiling techniques, TRFLP has been used to monitor structural changes of various soil microbial communities (Dunbar et al, 2000;Singh et al, 2006;Grant et al, 2007). However, to the best of our knowledge, this is the first report to investigate the change of the bacterial community in the MSW composting soil in response to anthracene amendment and subsequent biodegradation.…”

Impacts of Mariculture on the Diversity of Bacterial Communities within Intertidal Sediments in the Northeast of China

“…Diversity indices, phylotype richness (S), ShannonWiener index (H), and evenness (E) were calculated to compare the relative complexities of microbial communities [45]. Statistically significant difference was calculated using Student's t-test of SPSS 17.0 (Chicago, IL, USA) software, and P values less than 0.05 were considered significant.…”