Application of reverse transcriptase PCR for monitoring expression of the catabolic dmpN gene in a phenol-degrading sequencing batch reactor

Abstract

A modified freeze-thaw method in combination with reverse transcriptase PCR was developed for monitoring gene expression in activated sludge. The sensitivity of the methodology was determined by inoculating nonsterile activated sludge samples with 3-chlorobenzoate-degrading Pseudomonas putida PPO301(pRO103), which contains the catabolic tfdB gene. tfdB mRNA was detected in 10 mg of activated sludge inoculated with 10 4 CFU of the target organism. This technique was subsequently utilized to analyze the in situ expression of the catabolic dmpN gene in a sequencing batch reactor (SBR) bioaugmented with phenol-degrading P. putida ATCC 11172. Greatest dmpN expression was observed 15 min after maximum phenol concentration was reached in the reactor and 15 min after the start of aeration. Decreased phenol concentrations in the reactor corresponded to reduced levels of dmpN expression, although low levels of dmpN mRNA were observed throughout the SBR cycle. These results indicate that concentration of phenol in the reactor and the onset of aeration stimulated transcriptional activity of the dmpN gene. The information obtained from this study can be used to alter SBR operational strategies so as to lead to more effective bioaugmentation practices.

Expression of lip genes during growth in soil and oxidation of anthracene by Phanerochaete chrysosporium

“…Monitoring the physiological state of microbes in situ provides a useful tool to help ensure bioremediation performance. RT-PCR-based approaches have been applied to Pseudomonas putida in bioreactors (47) and during remediation of naphthalene-contaminated soils (16). Methods which employ this type of mRNA analysis have distinct advantages over other approaches used with white-rot fungi, such as quantitation of ergosterol (11) or PCR-based quantitation of fungal DNA (28).…”

RNA fingerprinting of microbial community in the rhizosphere soil of grain legumes

“…Direct extraction of mRNA from soil [10] and quantification of mRNA by an RNase protection assay [11] have been used for naphthalene dioxygenase in soil and for soluble methane monooxygenase in aquifer sediments [12]. Reverse transcriptase-PCR (RT-PCR) amplification of mRNA for soluble methane monooxygenase in aquifer sediments [13] and for lignin peroxidase in soils [14] has also been performed. One of the major prerequisites for such methods is knowledge of the sequence flanking the target region for the design of amplification primers for PCR.…”

Detection of mRNA by Reverse Transcription-PCR as an Indicator of Viability in Escherichia coli Cells

“…Detection of mRNA might therefore be a good indicator of living cells or those only recently dead at the time of sampling. Detection of mRNA by Northern blot hybridization has been used as an indicator of microbial metabolic activity in aquatic and soil samples (17,28,35), and detection of mRNA by reverse transcription-PCR (RT-PCR) was used to monitor gene expression in activated sludge (33). While these studies undoubtedly reflected the activities of viable cells in natural environments, their main purpose was not to distinguish between living and dead cells.…”