PCR-denaturing gradient gel electrophoresis (PCR-DGGE) is widely used in microbial ecology for the analysis of comparative community structure. However, artifacts generated during PCR-DGGE of mixed template communities impede the application of this technique to quantitative analysis of community diversity. The objective of the current study was to employ an artificial bacterial community to document and analyze artifacts associated with multiband signatures and preferential template amplification and to highlight their impacts on the use of this technique for quantitative diversity analysis. Six bacterial species (three Betaproteobacteria, two Alphaproteobacteria, and one Firmicutes) were amplified individually and in combinations with primers targeting the V7/V8 region of the 16S rRNA gene. Two of the six isolates produced multiband profiles demonstrating that band number does not correlate directly with alpha-diversity. Analysis of the multiple bands from one of these isolates confirmed that both bands had identical sequences which lead to the hypothesis that the multiband pattern resulted from two distinct structural conformations of the same amplicon. In addition, consistent preferential amplification was demonstrated following pairwise amplifications of the six isolates. DGGE and real rime PCR analysis identified primer mismatch and PCR inhibition due to 16S rDNA secondary structure as the most probable causes of preferential amplification patterns. Reproducible DGGE community profiles generated in this study confirm that PCR-DGGE provides an excellent high-throughput tool for comparative community structure analysis, but that method-specific artifacts preclude its use for accurate comparative diversity analysis. (C) 2013 Elsevier B.V. All rights reserved.
Plant establishment during phytostabilization of legacy mine tailings in semiarid regions is challenging due to low pH, low organic carbon, low nutrients, and high toxic metal(loid) concentrations. Plant-associated bacterial communities are particularly important under these harsh conditions because of their beneficial services to plants. We hypothesize that bacterial colonization profiles on rhizoplane surfaces reflect deterministic processes that are governed by plant health and the root environment. The aim of this study was to identify associations between bacterial colonization patterns on buffalo grass (Buchloe dactyloides) rhizoplanes and both plant status (leaf chlorophyll and plant cover) and substrate biogeochemistry (pH, electrical conductivity, total organic carbon, total nitrogen, and rhizosphere microbial community). Buffalo grass plants from mesocosm- and field-scale phytostabilization trials conducted with tailings from the Iron King Mine and Humboldt Smelter Superfund Site in Dewey-Humboldt, Arizona, were analyzed. These tailings are extremely acidic and have arsenic and lead concentrations of 2-4 g kg-1 substrate. Bacterial communities on rhizoplanes and in rhizosphere-associated substrate were characterized using fluorescence in situ hybridization and 16S rRNA gene amplicon sequencing, respectively. The results indicated that the metabolic status of rhizoplane bacterial colonizers is significantly related to plant health. Principal component analysis revealed that root-surface Alphaproteobacteria relative abundance was associated most strongly with substrate pH and Gammaproteobacteria relative abundance associated strongly with substrate pH and plant cover. These factors also affected the phylogenetic profiles of the associated rhizosphere communities. In summary, rhizoplane bacterial colonization patterns are plant specific and influenced by plant status and rhizosphere biogeochemical conditions.
PMID: 18424534;PMCID: PMC2446573;Abstract:
Bacterial diversity in mine tailing microbial communities has not been thoroughly investigated despite the correlations that have been observed between the relative microbial diversity and the success of revegetation efforts at tailing sites. This study employed phylogenetic analyses of 16S rRNA genes to compare the bacterial communities present in highly disturbed, extremely (pH 2.7) and moderately (pH 5.7) acidic lead-zinc mine tailing samples from a semiarid environment with those from a vegetated off-site (OS) control sample (pH 8). Phylotype richness in these communities decreased from 42 in the OS control to 24 in the moderately acidic samples and 8 in the extremely acidic tailing samples. The clones in the extremely acidic tailing sample were most closely related to acidophiles, none of which were detected in the OS control sample. The comparison generated by this study between the bacteria present in extremely acidic tailing and that in moderately acidic tailing communities with those in an OS control soil provides a reference point from which to evaluate the successful restoration of mine tailing disposal sites by phytostabilization. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
To explore the bacteria involved in the oxidation of arsenite (As(III)) under denitrifying conditions, three enrichment cultures (ECs) and one mixed culture (MC) were characterized that originated from anaerobic environmental samples. The oxidation of As(III) (0.5 mM) was dependent on NO(3) (-) addition and N(2) formation was dependent on As(III) addition. The ratio of N(2)-N formed to As(III) fed approximated the expected stoichiometry of 2.5. A 16S rRNA gene clone library analysis revealed three predominant phylotypes. The first, related to the genus Azoarcus from the division Betaproteobacteria, was found in the three ECs. The other two predominant phylotypes were closely related to the genera Acidovorax and Diaphorobacter within the Comamonadaceae family of Betaproteobacteria, and one of these was present in all of the cultures examined. FISH confirmed that Azoarcus accounted for a large fraction of bacteria present in the ECs. The Azoarcus clones had 96% sequence homology with Azoarcus sp. strain DAO1, an isolate previously reported to oxidize As(III) with nitrate. FISH analysis also confirmed that Comamonadaceae were present in all cultures. Pure cultures of Azoarcus and Diaphorobacter were isolated and shown to be responsible for nitrate-dependent As(III) oxidation. These results, taken as a whole, suggest that bacteria within the genus Azoarcus and the family Comamonadaceae are involved in the observed anoxic oxidation of As(III).
