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