Detection and enumeration of aromatic oxygenase genes by multiplex and real-time PCR

Abstract

Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5 degrees C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 x 10(2) copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.

FIG. 1.

(A) Phylogenetic analysis of the large subunits of aromatic dioxygenase genes. The tree was constructed by the neighbor-joining method (54) and bootstrapping analysis. Designations at branch points, e.g., D.1.A, indicate type (D), family (2), and subfamily (A). Subfamilies of genes were used to perform alignments leading to the identification of PCR primer sets. (B) Phylogenetic analysis of the large subunits of ring-hydroxylating monooxygenase genes. Isolates are described in Table 1.

FIG. 2.

NAH primer specificity confirmation by agarose gel electrophoresis. The specificity of each primer set was confirmed by PCR with positive- and negative-control templates (A), followed by hybridization experiments with gene probes created from positive-control strains (B). Lanes: M, 100- to 3,000-bp markers; 1, P. putida G7; 2, Burkholderia sp. strain DNT; 3, P. putida F1; 4, P. putida HS1; 5, P. putida mt-2.

FIG. 3.

Multiplex PCR amplification. Multiplex PCR amplification performed with mixtures of positive-control DNAs. Lanes: M, 100- to 3,000-bp markers; 1, NAH/PHE primers with P. putida G7 and Pseudomonas sp. strain CF600 DNAs; 2, TOL/TOD primers with P. putida HS1 and P. putida F1 DNAs; 3, BPH2/BPH4 primers with P. pseudoalcaligenes KF707 and Rhodococcus sp. strain RHA1 DNAs.

FIG. 4.

(A) Melting curve of amplification product with primer BPH1. No-template control (○) and 1 ng of Rhodococcus sp. strain RHA1 (×) were used. The melting point of the PCR product can be identified by the peak in the plot of −dF/dT as a function of temperature during a 0.2°C/s temperature ramp at the end of the run. For the BPH4 product shown, the observed melting temperature is approximately 92°C. Subsequent real-time PCR with the BPH4 primers used a polymerization temperature of 87°C. (B) Amplification plot with primer BPH4. Different numbers of copies were used as follows: 10⁷ (▪), 10⁶ (□), 10⁵ (⧫), 10⁴ (◊), and 10³ (•) copies per reaction mixture. ○, no-template control.