Materials and Methods

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

Genomic DNA of isolated strains grown in LB broth (Triptone 10 g l⁻¹, yeast extract 5 g l⁻¹, NaCl 10 g l⁻¹) was extracted as described previously (Versalovic et al. 1991).

Extractions were performed essentially as described by Griffiths et al. (2000). Briefly, 100 mg powdered organic matrix, obtained from each bioreactor after freezing, lyophilization and further homogenization, were dissolved in 0.1 ml CTAB extraction buffer and 0.1 ml of phenol/chloroform/isoamyl alcohol (25:24:1 by vol.) (pH 8.0).

After extraction-purification steps, pellets containing nucleic acids were washed with 70% (v/v) ethanol, air dried and resuspended in 30 μl Tris/EDTA buffer (pH 7.4 and RNase free).

The extracted DNA and RNA quality was further verified by analysis on agarose gel stained with ethidium bromide (0.5 μg/ml).

Extracted nucleic acids, 30 μl, were treated with (3 U) DNase (Sigma and RNase-free) for 1 h at 37°C. The reaction mixture was then mixed with guanidine thiocianate (GTC) up to 200 μl and 200 μl sodium acetate, 2 M pH 4.0, followed by 40 μl chloroform/isoamyl alcohol (24:1) and 200 μl phenol acid. The suspension was then vigorously stirred and placed on ice for 15 min. Subsequently, it was centrifuged at 13,00 wg for 10 min (4°C). To the collected supernatant, 1 vol. ethanol was added and then frozen for 30 min at −20°C, followed by centrifugation at 13,000 wg for 20 min (4°C). The pellet of RNA was further washed in 70% (v/v) ethanol and again centrifuged at 13,000 g for 5 min (4°C). At the end, the pellet was resuspended in 30 μl diethylpyrocarbonate (DEPC) treated water.

The RNA presence and quality was estimated on agarose gel (0.5–1% w/v) in 15% (v/v) formaldehyde, ethidium bromide, 0.5 μg/ml, run in MOPS buffer (20 mM, pH 7.0, 10 mM sodium acetate; 1 mM EDTA, pH 8.0).

The 16S rDNAs were selectively amplified (Gene Amp PCR System 2400, Perkin Elmer thermal cycler) from purified genomic DNA or from boiled cells routinely cultured on LB broth. Oligonucleotide primers were designed to anneal to conserved positions in the 3′ and 5′ regions of bacterial 16S rRNA genes (Al-Robaiy et al. 2001). The forward primer prbfo corresponding to position 509–525 of E. coli 16S rRNA whose upper sequence was 5′-ACTACGTGCCAGCAGCC-3′. The reverse primer was prbre 5′-GGACTACCAGGGTATCTAATCC-3′ corresponding to the complement of position 784–805 of E. coli 16S rRNA. The amplification product thus resulted as 297 bp.

The reaction mixture, in a final volume of 100 μl, was placed in 0.2 ml thin wall Eppendorf test-tubes. The reaction mixture, contained Taq Polimerase (Perkin-Elmer) 2.5 U, template DNA solution (20–200 ng genomic DNA), 0.5 μM of each primer, 4% (v/v) DMSO, MgCl₂ 1.5 μM and 10 μl buffer 10× (Perkin-Elmer). Amplification conditions were as follows: a preliminary denaturation of 10 min at 94°C followed by 40 cycles of 45 s at 94°C (denaturation), 30 sec at 66°C (annealing), 30 s at 72°C (extension) and a final cycle of 8 min at 72°C. PCR products were electrophoresed at 10 V cm⁻¹ in 1.3% agarose in TAE buffer containing ethidium bromide (0.5 μg/ml).

The primers to amplify catabolic enzyme genes have been chosen to identify the key enzymes involved on the xylene and toluene aerobic degradation pathways. Due to the presence of various isoforms of the key enzymes in different bacterial species, the choice of primers has been limited to extremely conserved DNA regions using the BLAST program analysis of the National Center for Biotechnology Information of the National Institutes of Health (http://www.ncbi.nlm.nih.gov/blast).

For xylene pathways, the sequence of PCR primer, xylfwd, was 5′-CATTCTTTCTTTGGCTTGGCTTAGTGG-3′, corresponding to nucleotide 4988–5014 of xylene monooxygenase hydroxylase component (xylM) of AF019635 sequence, and the primer, xylrev, was 5′-CCTCAATCTTTATCGCATCTTTGACGG-3′, corresponding to nucleotide 5256–5230 of the same sequence. For the toluene degradation pathway, the alpha subunit-terminal oxygenase component, tbmD gene, of toluene/benzene-2-monooxygenase (Johnson and Olsen 1995) was chosen to design the following primers: tol2fwd primer sequence 5′-CGCTGGACTGACAAGTGGTTCTGG-3′, corresponding to 2838–2861 of L40033 Gene Bank sequence, and tol2rev reverse primer 5′-TTCTCCGAGAGCCATTGCATCTCTT-3′, corresponding to 3148–3124 position of the same sequence.

The reaction mixture components were the same as those described above, with amplification conditions as follows: a preliminary denaturation of 120 s at 94°C followed by 35 cycles of 45 s at 94°C, 50 s at 58°C, 60 s at 72°C and 8 min at 72°C (extension). PCR products were electrophoresed at 10 V cm⁻¹ in 1.3% agarose in TAE buffer containing ethidium bromide (0.5 μg/ml).

The RT-PCR were performed on the extracted RNA, after a preliminary phase of denaturation at 70°C for 5 min followed by rapid cooling on ice. The reverse-transcription reactions were performed in 0.2 ml thin wall Eppendorf tubes in 50 μl final volume, using a mixture of exaprimers (Invitrogen, Milan, Italy) at 10 ng/μl, 200 μM of every dNTP, 200 U of M-MLV-RT (Invitrogen, Milan, Italy), 4 mM DTT, 40 U RNase inhibitor (Invitrogen, Milan, Italy), and 10 μl 5× buffer (Invitrogen, Milan, Italy). The reverse-transcription reaction was performed on 5 μl of the extracted RNA at 37°C for 1 h. The PCR was performed on 5 μl of produced cDNA as described above. Reaction tests where the sample had been omitted and replaced with an equal volume of water were used as negative control, and the presence of the rRNA 16S bacterial gene was tested as positive control.