Cultivation‐independent in situ molecular analysis of bacteria involved in degradation of pentachlorophenol in soil

Abstract

The central aim of this study was to determine which components of an indigenous bacterial community in pristine grassland soil were capable of degrading pentachlorophenol (PCP) using two cultivation-independent, in situ, molecular techniques. The first involved polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) amplification of 16S rRNA genes from DNA and RNA, respectively, extracted from PCP-amended soil. The second involved stable isotope probing (SIP), with incubation of soil with 13C-PCP and molecular analysis of 13C-labelled RNA, derived from cells incorporating PCP or its breakdown products, after separation from 12C-RNA by ultracentrifugation. Bacterial communities were characterized by denaturing gradient gel electrophoresis (DGGE) analysis of amplification products. PCP was degraded at an approximate rate of 1.18+/-0.25 (SEM) mg kg-1 day-1 and 39% of the measurable PCP fraction was degraded after incubation for 63 days. PCP degradation was associated with significant changes in bacterial community structure, leading to the appearance of seven bands in both DNA- and RNA-based DGGE profiles, the latter providing clearer evidence of qualitative shifts in community structure. The majority of novel bands increased in relative intensity during the first 35 days and subsequently decreased in relative intensity as incubation continued. Sequence and phylogenetic analysis of six of these bands indicated most to have closest database relatives that were uncultured bacteria with sequence homologies to reported hydrocarbon degraders. No band could be detected in RNA-SIP-DGGE profiles derived from 13C-RNA fractions at day 0 but several faint bands appeared in these fractions after incubation of soil for 4 days, indicating assimilation of PCP or its degradation products. These bands increased in intensity during subsequent incubation for 21 days and decreased with further incubation. With one exception, RNA-SIP-DGGE and RNA-DGGE profiles were similar, indicating that RNA-targeted DGGE, in this case, provided a good indication of the metabolically active microbial community.

Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots

“…DNA was eluted in sterile water at 65 1C for 30 min and used as a template for PCR amplification under the conditions mentioned above. In most cases as reported by Mahmood et al (2005), further rounds of band excision, PCR amplification and DGGE analysis were necessary before sequencing the PCR products (Genome express, Meylan, France). A particular attention was paid on 13 Clabelled populations and those impacted by glucosinolates, specifically in case of minor DGGE bands.…”

Analysis of bacterial core communities in the central Baltic by comparative RNA–DNA-based fingerprinting provides links to structure–function relationships

“…Currently, DNA-based fingerprints are assumed to reflect the taxa present in a community, whereas RNA-based fingerprints reflect the active members of the community (Logue and Lindström, 2010;Prosser et al, 2010). A seminal experimental study combining nucleic acid-based fingerprints with stable isotope probing by Mahmood et al (2005) showed the validity of this concept by finding that six out of seven major RNA-based phylotypes in soil microcosms were actively degrading pentachlorophenol. This led us to the assumption that a comparison of RNA-based community fingerprints of bacterioplankton with DNA-based fingerprints from the same samples could provide criteria to identify the active members of the core community.…”

The use of stable isotope probing to identify key iron-reducing microorganisms involved in anaerobic benzene degradation

“…SIP involves the incorporation of 13 C-label into cellular biomarkers such as nucleic acids, followed by density gradient separation of labeled nucleic acids and molecular identification of the involved microbes (Radajewski et al, 2000). Today, the SIP technique has greatly increased our understanding of the role of specific microbial community members in diverse environmental settings (Madsen, 2006) and identified key microbes utilizing various aromatic and chlorinated hydrocarbons (Manefield et al, 2002;Jeon et al, 2003;Padmanabhan et al, 2003;Mahmood et al, 2005;Yu and Chu, 2005;Kasai et al, 2006). The goal of the present study was to identify the microorganisms in our enrichment specifically responsible for anaerobic benzene degradation.…”