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A qualitative procedure of purified DNA/RNA co-extraction from complex organic matter, …

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Results and Discussion
- Detection of aromatic catabolic gene expression in heterogeneous organic matter used for reduction of volatile organic compounds (VOC) by biofiltration

Bacterial strains

The application of sensitive methods requires efficient negative and positive controls to determine both the reaction efficiency and the cross contaminations in all analytical procedures.

The ability to degrade and/or utilize methylbenzenes such as toluene, ortho-, meta-, para-xylene, pseudocumene (1,2,4-trimethylbenzene), hemellitol (1,2,3-trimethylbenzene), mesitylene (1,3,5-trimethylbenzene) or 1,2,3,4-tetramethylbenzene is exhibited by a wide variety of general bacteria (Table 1) (Lang 1996, Fredrickson et al. 1995). Many strains showed multiple sources of carbon requirement but only two bacteria, strains F199 (Fredrickson et al. 1991) and a mutant of strain OXI (Di Lecce et al. 1997), utilized toluene and all isomers of xylene as a sole carbon and energy source.

Several different pathways have been proposed in a variety of different microorganisms and, during the past three decades, much research has been devoted to elucidating the aromatic hydrocarbons metabolisms (Smith 1990) and genetic relationship (Singer and Finnerty 1984). Based on studies of these properties, interesting conclusions were drawn by Van der Meer et al. (1992). Regarding mechanisms of genetic adaptation, the assumptions that “common ancestral genes have spread among different microorganisms”, “the divergence of descendants does not necessarily correspond to the evolutionary distances determined from those organisms” and “the organization of catabolic gene suggest that several different gene clusters may be combined as modules, to which other peripheral genes may be added producing expansion of the existing degradative pathways” have been particularly illuminating.

Considering the convergent metabolic pathways, our purpose was to evaluate specific activities of single compounds within the mono aromatic degradation pathway. The specificity of the inductive mechanism was evinced by the use of similar molecules such as xylenes and toluene. For this purpose, we chose two strains with the characteristics described in Table 2. Microbial growth in the presence of VOCs as sole carbon source was evaluated by optical density and confirmed with respirometric assay. Because of the high VOC volatility, it was not possible to determine the exact C/CO2 conversion rate with the latter method (data not shown).

Strain 14 was used as positive control for xylene enriched matrices and strain C18 was used as a positive control for growing on toluene enriched matrices. In addition, two E. coli strains (BL21 and TOP10), grown in the same matrices, were used as xylene and toluene gene negative controls (see below).

Design and test of primers for amplification of specific degradative enzymes of xylene and toluene pathways

In order to test the expression of genes coding for specific degradative enzymes of xylene and toluene pathways, different primers were synthesized to amplify conserved portions of xylene monooxygenase hydrolase component (xylM, AF019635) and the alpha subunit-terminal oxygenase component of toluene 2 monoxygenase (tmbD, L40033).

Two pairs of primers (see Table 3 for details) resulted as highly specific for the chosen genes. In fact, by using genomic DNA from C18 (Tol+) and 14 (Xyl+) isolated strains as template, these primers resulted in specific amplifications of xylM and tmbD genes by PCR. In the case of the bacterial DNA of the strain isolated for growing on toluene (C18), PCR amplification gave rise to a specific amplified product corresponding to 311 bp of toluene 2 monooxygenase partial gene (primers tol2fwd and tol2rev). In the case of bacterial DNA deriving from the strain isolated for growing on xylene (strain 14), PCR amplification with primers xylfwd and xylrev gave a specific amplification product corresponding to 269 bp of xylene monooxygenase partial gene (not shown). The specificity of the designed primers was demonstrated by performing PCR reactions with the two pairs of primers with DNA extracted from E. coli cells (strain BL21 and TOP10), which gave no amplification products. The correctness of the amplification products was confirmed by DNA sequencing of each one.

PCR amplification analysis on nucleic acid (DNA and RNA) extracted from solid biofilter matrices

Several different organic samples were withdrawn from a laboratory scale biofilter fed with single VOCs for several months. A methodology was set up to co-extract and purify the nucleic acids from these solid matrices to avoid contamination from organic and composed aromatic substances (data not shown). The amount of DNA extracted, as judged from serial dilutions of the samples and comparative electrophoresis gel analysis with a known amount of same size DNA and RNA standards, was about 100–500 ng DNA (10 kb) and 0.5–2 μg rRNA 16S from 100 mg matrix (data not shown).

To evaluate the extraction method efficiency and monitor the expression of xylM and tmbD genes, nucleic acids extracts originated from xylenes, methyl ethyl ketone, butyl acetate, ethyl acetate adapted matrices, were split into two aliquots to prepare pure template of DNA or RNA. In order to obtain pure DNA, half the sample was incubated at 37°C with RNAase (Sigma) at 100 μg/ml for 10 min. After further extraction with phenol/chloroform, the sample was used for the PCR reactions.

The presence of bacterial strains selected for their ability to form degradative pathways of xylene and toluene was then assayed by PCR on extracted DNA. As shown in Fig. 1, panels (a) and (b), efficient amplification of the 16S rDNA partial gene, for all the extracted samples harvested from the various matrices, confirmed the standardization of the extraction and purification methods. The possible presence of the two isolated bacterial strains was confirmed by PCR amplification of xylene monooxygenase (Panel a) and toluene 2 monoxygenase (Panel b) partial genes. PCR analysis confirmed the presence of xylene monooxygenase gene only in nucleic acids purified from the xylene pre-adapted matrix (Lane 1), while it was absent in the other samples. This evidence confirmed the high VOC selective pressure operated on the microorganisms subsequently grown on several matrices. Evaluation of the possible presence of the gene for toluene 2 monoxygenase was used as a control for method specificity because toluene is an aromatic compound similar to xylenes. The complete absence of this gene, in the nucleic acids purified material from xylene pre-adapted matrices (Panel b), resulted as a useful negative control regarding process selection within the matrix.

Reverse transcription and amplification of a specific fragment of cDNA starting from previously purified mRNA.

In order to estimate the expression of the xylene monooxygenase gene at mRNA level, the RNA purified from the xylene adapted matrix was converted to cDNA and used as a template for the PCR reactions. The RNAs for reverse-transcription were obtained by treating one out of two aliquots of all samples, extracted and purified from different matrices, with 5 U DNase RNase-free (Sigma, Milan, Italy), at 37°C for 30 min. Subsequently, the reverse-transcription reactions and PCR on the obtained cDNA were performed as described in Materials and methods.

Figure 2 shows the results of RT-PCR reactions. Efficient amplification of xylene monooxygenase was obtained only from samples originating from xylene adapted matrix (panel a, lane 1), confirming the sensitivity and specificity of the procedure to evaluate the expression of this gene. On the contrary, toluene 2 monooxygenase expression was, as expected, not revealed (data not shown). In any case, as a control of the RT-PCR reaction, the expression of rRNA 16S was always evaluated to confirm the integrity of the material and microorganisms viability. In each analysis, particular attention was paid to ensuring that there was no DNA cross-contamination in the RNA sample, by carrying out the RT-PCR reactions on RNA samples in the absence of the reverse transcriptase enzyme (Fig. 2, panel b). The lack of the transcript for thmD gene, as demonstrated by the absence of amplification product, might therefore suggest the absence of this enzyme. Further analysis at protein level will address this issue.

The main difficulty in applying nucleic acids technology to microbial traceability in environmental solid matrices is due to the fact that the common protocols for nucleic acids extraction in denaturing conditions involve a significant contamination of the samples with organic and composed aromatic substances released by organic matter. These compounds may significantly alter further PCR analysis by inhibiting the reverse transcriptase activity and DNA polymerase, thus affecting performance. Moreover, the standard procedures have been shown to be not particularly efficient in the extraction and purification of good quality nucleic acids, possibly because of the co-extraction of active DNAse and RNAse present in the matrices (Griffiths et al. 2000). 


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