Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomi

Estuarine systems are the major conduits for the transfer of nitrate from agricultural and other terrestrial-anthropogenic sources into marine ecosystems. Within estuarine sediments some microbially driven processes (denitrification and anammox) result in the net removal of nitrogen from the environment, while others (dissimilatory nitrate reduction to ammonium) do not. In this study, molecular approaches have been used to investigate the diversity, abundance, and activity of the nitrate-reducing communities in sediments from the hypernutrified Colne estuary, United Kingdom, via analysis of nitrate and nitrite reductase genes and transcripts. Sequence analysis of cloned PCR-amplified narG , napA , and nrfA gene sequences showed the indigenous nitrate-reducing communities to be both phylogenetically diverse and also divergent from previously characterized nitrate reduction sequences in soils and offshore marine sediments and from cultured nitrate reducers. In both the narG and nrfA libraries, the majority of clones (48% and 50%, respectively) were related to corresponding sequences from delta-proteobacteria. A suite of quantitative PCR primers and TaqMan probes was then developed to quantify phylotype-specific nitrate ( narG and napA ) and nitrite reductase ( nirS and nrfA ) gene and transcript numbers in sediments from three sites along the estuarine nitrate gradient. In general, both nitrate and nitrite reductase gene copy numbers were found to decline significantly ( P < 0.05) from the estuary head towards the estuary mouth. The development and application, for the first time, of quantitative reverse transcription-PCR assays to quantify mRNA sequences in sediments revealed that transcript numbers for three of the five phylotypes quantified were greatest at the estuary head.

Estuarine sediments are the location for significant bacterial removal of anthropogenically derived inorganic nitrogen, in particular nitrate, from the aquatic environment. In this study, rates of benthic denitrification (DN), dissimilatory nitrate reduction to ammonium (DNRA), and anammox (AN) at three sites along a nitrate concentration gradient in the Colne estuary, United Kingdom, were determined, and the numbers of functional genes ( narG , napA , nirS , and nrfA ) and corresponding transcripts encoding enzymes mediating nitrate reduction were determined by reverse transcription-quantitative PCR. In situ rates of DN and DNRA decreased toward the estuary mouth, with the findings from slurry experiments suggesting that the potential for DNRA increased while the DN potential decreased as nitrate concentrations declined. AN was detected only at the estuary head, accounting for ∼30% of N ₂ formation, with 16S rRNA genes from anammox-related bacteria also detected only at this site. Numbers of narG genes declined along the estuary, while napA gene numbers were stable, suggesting that NAP-mediated nitrate reduction remained important at low nitrate concentrations. nirS gene numbers (as indicators of DN) also decreased along the estuary, whereas nrfA (an indicator for DNRA) was detected only at the two uppermost sites. Similarly, nitrate and nitrite reductase gene transcripts were detected only at the top two sites. A regression analysis of log( n + 1) process rate data and log( n + 1) mean gene abundances showed significant relationships between DN and nirS and between DNRA and nrfA . Although these log-log relationships indicate an underlying relationship between the genetic potential for nitrate reduction and the corresponding process activity, fine-scale environmentally induced changes in rates of nitrate reduction are likely to be controlled at cellular and protein levels.

Terminal restriction fragment length polymorphism (tRFLP) is a potentially high-throughput method for the analysis of complex microbial communities. Comparison of multiple tRFLP profiles to identify shared and unique components of microbial communities however, is done manually, which is both time consuming and error prone. This paper describes a freely accessible web-based program, T-Align (http://inismor.ucd.ie/~talign/), which addresses this problem. Initially replicate profiles are compared and used to generate a single consensus profile containing only terminal restriction fragments that occur in all replicate profiles. Subsequently consensus profiles representing different communities are compared to produce a list showing whether a terminal restriction fragment (TRF) is present in a particular sample and its relative fluorescence intensity. The use of T-Align thus allows rapid comparison of numerous tRFLP profiles. T-Align is demonstrated by alignment of tRFLP profiles generated from bacterioplankton communities collected from the Irish and Celtic Seas in November 2000. Ubiquitous TRFs and site-specific TRFs were identified using T-Align.

Summary: Quantitative polymerase chain reaction (Q‐PCR) amplification is widely applied for determining gene and transcript numbers within environmental samples. This research evaluated Q‐PCR reproducibility via Taq Man assays quantifying 16S rRNA gene and transcript numbers in sediments, within and between replicate Q‐PCR assays. Intra‐assay variation in 16S rRNA gene numbers in replicate DNA samples was low (coefficients of variation; CV from 3.2 to 5.2%). However, variability increased using replicated standard curves within separate Q‐PCR assays (CV from 11.2% to 26%), indicating absolute comparison of gene numbers between Q‐PCR assays was less reliable. 16S rRNA transcript quantification was evaluated using standard curves of diluted RNA or cDNA (before, or following, reverse transcription). These standard curves were statistically different with cDNA‐derived curves giving higher r ² values and Q‐PCR efficiencies. Template concentrations used in Q‐PCR also affected 16S rRNA gene and transcript numbers. For DNA, 10 ⁻³ dilutions yielded higher gene numbers than 10 ⁻¹ and 10 ⁻² dilutions. Conversely, RNA template dilution reduced numbers of transcripts detected. Finally, different nucleic acid isolation methods also resulted in gene and transcript number variability. This research demonstrates Q‐PCR determination of absolute numbers of genes and transcripts using environmental nucleic acids should be treated cautiously.