Journal of Microbiological Methods 64 (2006) 250 – 265 www.elsevier.com/locate/jmicmeth
Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: Distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site Barbara Hendrickxa,b, Howard Juncac, Jolana Vosahlovad, Antje Lindnere, Irene Ru¨eggf, Margarete Bucheli-Witschelf, Folkert Faberg, Thomas Eglif, Margit Maue, Michael Schlo¨manne, Maria Brennerovad, VLadimir Brennerd, Dietmar H. Pieperc, Eva M. Topb,1, Winnie Dejonghea, Leen Bastiaensa, Dirk Springaela,h,* a
Environmental and Process Technology (Vito), Flemish Institute for Technological Research, B-2400 Mol, Belgium b Laboratory of Microbial Ecology and Technology, University of Ghent (UG), B-9000 Gent, Belgium c Biodegradation Group, German Research Centre for Biotechnology (GBF), 38124 Braunschweig, Germany d Department of Cell and Molecular Microbiology, Institute of Microbiology, 14200 Prague 4, Czech Republic e Interdisziplina¨res O¨kologisches Zentrum, TU Bergakademie Freiberg, D-09599 Freiberg, Germany f Department of Microbiology, Swiss Federal Institute for Environmental Science and Technology (EAWAG), 8600 Du¨bendorf, Switzerland g Department of Microbiology, Centre Ecological Evolutionary Studies, University of Groningen, 9750 AA Haren, The Netherlands h Laboratory of Soil and Water Management, Catholic University of Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium Received 1 August 2004; received in revised form 6 April 2005; accepted 11 May 2005 Available online 8 June 2005
Abstract Eight new primer sets were designed for PCR detection of (i) mono-oxygenase and dioxygenase gene sequences involved in initial attack of bacterial aerobic BTEX degradation and of (ii) catechol 2,3-dioxygenase gene sequences responsible for metacleavage of the aromatic ring. The new primer sets allowed detection of the corresponding genotypes in soil with a detection limit of 103–104 or 105–106 gene copies g 1 soil, assuming one copy of the gene per cell. The primer sets were used in PCR to assess the distribution of the catabolic genes in BTEX degrading bacterial strains and DNA extracts isolated from soils sampled from different locations and depths (vadose, capillary fringe and saturated zone) within a BTEX contaminated site. In both soil DNA and the isolates, tmoA-, xylM- and xylE1-like genes were the most frequently recovered BTEX catabolic genes. xylM and
* Corresponding author. Present address: Catholic University of Leuven (KUL), Laboratory for Soil and Water Management, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium. Tel.: +32 16 321604; fax: +32 16 321997. E-mail address: dirk.springael@agr.kuleuven.ac.be (D. Springael). 1 Present address: University of Idaho, Department of Biological Sciences, 83844-3051 Moscow, Idaho, USA. 0167-7012/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2005.04.018
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xylE1 were only recovered from material from the contaminated samples while tmoA was detected in material from both the contaminated and non-contaminated samples. The isolates, mainly obtained from the contaminated locations, belonged to the Actinobacteria or Proteobacteria (mainly Pseudomonas). The ability to degrade benzene was the most common BTEX degradation phenotype among them and its distribution was largely congruent with the distribution of the tmoA-like genotype. The presence of tmoA and xylM genes in phylogenetically distant strains indicated the occurrence of horizontal transfer of BTEX catabolic genes in the aquifer. Overall, these results show spatial variation in the composition of the BTEX degradation genes and hence in the type of BTEX degradation activity and pathway, at the examined site. They indicate that bacteria carrying specific pathways and primarily carrying tmoA/xylM/xylE1 genotypes, are being selected upon BTEX contamination. D 2005 Elsevier B.V. All rights reserved. Keywords: PCR detection; Aerobic BTEX biodegradation; Catabolic gene distribution; BTEX degrading isolates; BTEX contaminated site
1. Introduction BTEX (benzene, toluene, ethylbenzene and xylenes) are frequently occurring groundwater contaminants. BTEX can be biodegraded under both aerobic and anaerobic conditions and in situ bioremediation, either passive or active, is increasingly applied for the elimination of BTEX in groundwater (Lovley, 2001; Barker et al., 1987). Decisions as to whether a site should be contained and monitored or actively treated are largely made on an empirical basis. Basic knowledge about the distribution, population densities and activities of BTEX degrading organisms at the polluted sites can contribute to rational decision-making (Baldwin et al., 2003). The ability to rapidly and accurately detect BTEX biodegrading bacteria and their activity in the environment is therefore of major interest. This can be done by demonstrating the occurrence of catabolic genotypes involved in BTEX degradation or their corresponding mRNA in the aquifer by employing sensitive PCR and RT-PCR detection methods (Baldwin et al., 2003). Initial oxidative attack of BTEX converting the compound into a catechol structure and the cleavage of the catechol structure are key steps in aerobic BTEX degradation. As such, both activities are of direct interest as monitoring objects. Initial oxidative attack consists of direct oxidation of the aromatic ring via a mono-oxygenase (Kahng et al., 2001) or a dioxygenase attack (Zylstra and Gibson, 1989; Furukawa et al., 1993) or oxidation of the alkyl side chain which is catalyzed by mono-oxygenases (Burlage et al., 1989). Ring cleavage occurs by catechol 2,3dioxygenases (C23O) after which the structure is
further degraded into Krebs cycle intermediates (Harayama and Rekik, 1993). Phylogenetic studies of amino acid sequences of the proteins involved show that they can be divided into specific families and subfamilies showing significant sequence homology and indicating a common ancestry which allowed the design of group-specific primer sets for detection by PCR (Baldwin et al., 2003; Eltis and Bolin, 1996). In the past, several studies have reported PCR primers to detect and quantify the presence of specific genotypes encoding those key steps in BTEX biodegradation and their mRNA in environmental samples (Baldwin et al., 2003; Hallier-Soulier et al., 1996; Junca and Pieper, 2003; Mesarch et al., 2000; Meyer et al., 1999; Ogram et al., 1995; Okuta et al., 1998; Ringelberg et al., 2001). However, the recent availability of a lot of new sequence information on BTEX degradation genes indicates that previously published primer sets are not always suitable (Baldwin et al., 2003; Junca and Pieper, 2003). In addition, for some BTEX catabolic protein families there are no primers reported yet (Baldwin et al., 2003). Therefore, design and/or redesign of primers for PCR detection of BTEX catabolic genes is required. Previously, we reported the design of a degenerate primer set for the detection of tmoA-like genes, encoding a-subunit of subfamily 2 of the hydroxylase component of bacterial multi-component mono-oxygenases involved in BTEX degradation (Hendrickx et al., submitted). In this study, we report (i) an alternative primer set for detection of genes encoding subgroup I of subfamily 1 a-subunit of the hydroxylase component of bacterial multi-component monooxygenases, (ii) a new primer set for detecting genes
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involved in mono-oxygenase attack of the side chain of TEX, (iii) a new primer set for detecting genes encoding for iron–sulfur a-subunit of dioxygenase complexes and (iv) 4 alternative or novel primer sets for detection of the corresponding genes of the 4 major subfamilies of C23O proteins. The distribution of these genes in a set of BTEX degrading bacterial isolates and total community DNA from subsurface soil samples of a BTEX contaminated site was explored.
2. Materials and methods
and p-xylene which exceeded 10,000 times the regulatory criteria for groundwater concentrations of benzene in European countries. At A3, the concentration of benzene in the groundwater was 0.1 mg l 1 while A4 was not contaminated. The contents of the BTEX compounds in the groundwater were chemically determined by means of the US EPA 8260 modified method. Subsurface soil sample GP48 was a sandy aquifer sample obtained from a BTEX contaminated site situated in Belgium. It originated from a sampling point where the total BTEX concentration in the groundwater was around 50 mg l 1.
2.1. Bacterial strains and growth conditions
2.3. Isolation of BTEX degraders
The bacterial BTEX degrading reference strains used in this study are described in Table 1 and were routinely grown at 30 8C on 869 medium or on Tris minimal medium (Mergeay et al., 1985) supplemented with BTEX as described (Hendrickx et al., in press). Phenanthrene and biphenyl were provided as a carbon source by placing crystals on the agar plates or adding them to the liquid cultures. BTEX, phenanthrene and biphenyl were purchased from Janssen Chemica (Beerse, Belgium).
Bacterial strains were isolated from the soil samples by either one of 5 different isolation procedures. Procedure 1 consisted of direct plating on R2A agar plates in the presence of BTEX vapors as described previously (Junca and Pieper, 2003). After incubation at 30 8C, colonies expressing meta-cleavage activity were retained. Procedure 2 consisted of enrichment in chloride free mineral medium MM5 (Hickey and Focht, 1990) supplemented with 50 mg l 1 BTEX 1:1:1:1 mixture as a sole source of carbon and energy. Growing cultures were plated on MM5 agar with BTEX and developing colonies were retained. In procedure 3, BTEX degraders were isolated from a micro-aerophylic chemostat inoculated with the soil samples (Faber, unpublished results). In procedure 4, BTEX degrading strains were isolated from an enrichment in Flavobacterium aquatile medium, containing 0.25 g l 1 yeast extract, 0.5 g l 1 peptone, 1 g l 1 caseinate, 0.25 g l 1 K2HPO4, cycloheximide (75 mg l 1) and different contents of soil (2.5%, 5%, and 10% wt/vol) at 14 8C. Procedure 5 consisted of enrichment of BTEX degraders at 12 8C in mineral medium modified from Evans et al. (1970), diluted to 25% of its original strength, adjusted to pH 7 by addition of a potassium phosphate buffer (50 mM) and further modified by adding 162 mg l 1 CaCl2 d 2H2O, by replacing citric acid with EDTA and a vitamin solution (Egli et al., 1988) with a BTEX mixture supplied via the vapour phase. After 6 days the culture was plated on a mineral medium plate and growing colonies retained after incubation in a BTEX atmosphere at 30 8C.
2.2. Subsurface soil samples used in this study Subsurface soil samples used for direct DNA extraction were sampled in March 2001 at an oil-refinery site, situated in Northern Bohemia (Czech Republic) containing a BTEX (mainly benzene) contaminated groundwater plume. Soil samples for isolation of BTEX degrading isolates were obtained from the same site in March and April 2001 and in April and May 2002. The subsurface soil samples were taken from different locations along the plume (A1, A2, A3 and A4) and from different depths designated as X, Y and Z, in which X represents the vadose zone (depth of 2.58 m), Y the capillary fringe (depth of 2.91 m) and Z the saturated zone (depth of 3.84 m). The tested soil samples were accordingly designated as A1X, A1Y, A1Z, A2X, A2Y, A2Z, A3X, A3Y, A3Z and A4Z. All were mixtures of three soil samples taken at the same place and depth within a distance of 1 m. The groundwater at sampling point A1 and A2 was strongly contaminated by BTEX containing 100 mg l 1 benzene, 10 mg l 1 toluene and l mg l 1 m-
Table 1 Description of used reference strains and results of PCR detection of BTEX degradation genes with the developed primer sets on reference strains Organism
Reference(s)
Initial attack
Lower pathway (meta-cleavage)
Mono-oxygenase
Dioxygenase
TBMD TMOA TOL XYLA TODC1 -F/-R -F/-R -F/-R -F/-R -F/-R B. sp. strain JS150
B, T, EB, tbmABCDEF, tbc2ABCDEF B. cepacia G4 B, T, tomA012345, tomB R. pickettii PKO1 B, T, EB, o-X, m-X, p-X, tbuA1UBVA2C (tbuT), tbuD (tbuR), tbuEFGKIHJ (tbuS), tbuX P. mendocina KR1 B, T, tmoXABCDEF (tmoST) P. aeruginosa JI104 B, bmoABCDEF P. stutzeri OX1 B, T, o-X, touABCDEF (touR), xylAM P. putida F1 B, T, EB, todFC1C2BADE, todGIH, (todST) P. putida mt-2 (PaW1) T, m-X, p-X, xylUWCMABN, xylXYZLTEGFJQKIH, (xylS, xylR) P. putida MT15 T, m-X, p-X, xylUWCMABN, xylXYZLTEGFJQKIH, cdo P. putida MT53 T, m-X, p-X, xylUWCMABN, xylXYZLTEGFJQKIH, cdo S. yanoikuyae B1 T, m-X, p-X, xylMAB, xylCXYFEGJQKIHT P. putida JHR Biphenyl, bphA1A2A3A4BCDEFG
(Johnson and Olsen, 1997; + Kahng et al., 2001) (Shields et al., 1995) + (Byrne et al., 1995) +
+
(Yen et al., 1991) (Kitayama et al., 1996a) (Bertoni et al., 1998)
+ + +
+ + +
+
+
+
+
+ +
+
(Zylstra and Gibson, 1989)
+ +
+
(Burlage et al., 1989)
+
(Keil et al., 1985a)
+
(Kok et al., 1999)
+
+
+ +
+
(Kim and Zylstra, 1999) Springael, unpublished
XYLE1 XYLE2 CDO TBUE TODE -F/-R -F/-R -F/-R -F/-R -F/-R
+
+
+
+ +
+
B. Hendrickx et al. / Journal of Microbiological Methods 64 (2006) 250–265
BTEX degrading capacity and relevant catabolic genotypesa
B, T, EB, o-X, m-X, p-X: growth on benzene, toluene, ethylbenzene, o-xylene, m-xylene, p-xylene, respectively; + = PCR signal; = no PCR signal. a The genes encoding the hydroxylase a-subunit of the aromatic ring mono-oxygenases, the terminal hydroxylase component and electron transfer component of the side chain mono-oxygenases, the iron–sulfur oxygenase a-subunit of the dioxygenase complex and the catechol 2,3-dioxygenases are underlined.
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2.4. Extraction of total DNA from bacterial cultures and from soil Total genomic DNA was extracted from bacterial strains grown in 5 ml Tris minimal medium exposed to BTEX vapors or in 5 ml 869 medium as described by Vanbroekhoven et al. (2004). Extraction of total DNA from aquifer material was performed as described previously (Hendrickx et al., in press).
TBUE-R and TODE-F/TODE-R were designated as tbmD-, tmoA-, xylM-, xylA-, todC1-, xylE1-, xylE2-, cdo-, tbuE- and todE-like genes, respectively. Novel generate and degenerate primers were designed based on Protein and DNA sequence alignments of the appropriate proteins/genes, constructed using the GCG Wisconsin protein and DNA analysis program (version 7.0) (Genetics Computer Group, Madison, Wisconsin). 2.7. PCR amplification of catabolic genes from pure strain and soil DNA
2.5. BOX-PCR fingerprint analysis PCR amplification of BOX repetitive sequences and pattern analysis was performed as described previously (Vanbroekhoven et al., 2004). For all strains, DNA concentrations were adjusted to 100 ng Al 1 and 1 Al was used in the PCR reaction. 2.6. Used primers and primer design Fig. 1 shows a schematic overview of the catabolic reactions of relevance in this paper and the involved target genotypes. The used primer sets, their target genes, locations in the gene and product lengths are described in Table 2. For convenience, genotypes amplified with primer sets TBMD-F/TBMD-R, TMOA-F/TMOA-R, TOL-F/TOL-R, XYLA-F/ XYLA-R, TODC1-F/TODC1-R, XYLE1-F/XYLE1R, XYLE2-F/XYLE2-R, CDO-F/CDO-R, TBUE-F/ tmoA tbmD
(CH3)
PCR amplification was carried out in a 50 Al reaction mixture containing 100 ng of pure strain DNA or soil DNA as templates. A 475-bp xylM gene fragment was amplified using primer set TOL-F/TOLR as described by Baldwin et al. (2003). Using the TBMD-F/TBMD-R, TMOA-F/TMOA-R, TOL-F/ TOL-R, XYLA-F/XYLA-R, TODC1-F/TODC1-R, TBUE-F/TBUE-R or TODE-F/TODE-R primer sets, the PCR mixture contained 1.25 U exTaq polymerase, 10 pmol of the forward primer, 10 pmol of the reverse primer, 200 AM of each dNTP and 5 Al of 10x exTaq reaction buffer (20 mM MgCl2). The Taq polymerase, dNTPs and PCR buffer were purchased from TaKaRa (TaKaRa Ex Taqk, TaKaRa Shuzo Co. Biomedical Group, Japan). Using the XYLE1-F/XYLE1-R, XYLE2-F/XYLE2-R or CDO-F/CDO-R primer set, tmoA tbmD
(CH3 )
(CH3 ) OH
OH OH CH 3
Initial attack
xylA xylM
CH 2 OH
OH
Mono-oxygenase OH
CH 3 CH 3
CH 3
CH 3
todC1
OH
OH
H H
OH
OH
OH
xylE1 xylE2
Ring cleavage OH
tbuE cdo todE
Dioxygenase
O COOH
Catechol 2,3-dioxygenase
OH
Fig. 1. Schematic presentation of BTEX catabolic enzyme reactions catalyzed by the proteins detected by the primer sets.
Table 2 Primer sets used in this study
640
65.5
This study
5V-CGAAACCGGCTT(C/T)ACCAA(C/T)ATG-3V 5V-ACCGGGATATTT(C/T)TCTTC(C/G)AGCCA-3V
505
61.2
Hendrickx et al., submitted
5V-TGAGGCTGAAACTTTACGTAGA-3V 5V-CTCACCTGGAGTTGCGTAC-3V
475
55
(Baldwin et al., 2003)
5V-CCAGGTGGAATTTTCAGTGGTTGG-3V 5V-AATTAACTCGAAGCGCCCACCCCA-3V
291
64
This study
5V-CAGTGCCGCCA(C/T)CGTGG(C/T)ATG-3V 5V-GCCACTTCCATG(C/T)CC(A/G)CCCCA-3V
510
66
This study
5V-CCGCCGACCTGATC(A/T)(C/G)CATG-3V 5V-TCAGGTCA(G/T)CACGGTCA(G/T)GA-3V 5V-GTAATTCGCCCTGGCTA(C/T)GTICA-3V 5V-GGTGTTCACCGTCATGAAGCG(C/G/T)TC-3V 5V-CATGTCAACATGCGCGTAATG-3V 5V-CATGTCTGTGTTGAAGCCGTA-3V
242
61.5
This study
906
64
This study
255
58
This study
5V-CTGGATCATGCCCTGTTGATG-3V 5V-CCACAGCTTGTCTTCACTCCA-3V
444
60
This study
5V-GGATTTCAAACTGGAGACCAG-3V 5V-GCCATTAGCTTGCAGCATGAA-3V
246
58
This study
1465
55
480
55
(Johnson, 1994) (Muyzer et al., 1993)
Sequence
TBMD-F/TBMD-R
5V-GCCTGACCATGGATGC(C/G)TACTGG-3V 5V-CGCCAGAACCACTTGTC(A/G)(A/G)TCCA-3V
27F/1492R
Subfamily 1 of a-subunits of hydroxylase component of multi-component mono-oxygenases Subfamily 2 of a-subunits of hydroxylase component of multi-component mono-oxygenases Subfamily 5 of hydroxylase component of two-component side chain mono-oxygenases Electron transfer component of two-component side chain mono-oxygenases Subfamilies D.1.B + D.1.C + D.2.A + D.2.B + D.2.C of a-subunits of Type D iron–sulfur multi-component aromatic dioxygenases Subfamily I.2.A of catechol extradiol dioxygenases Subfamily I.2.B of catechol extradiol dioxygenases cdo (U01826) of subfamily I.2.C of catechol extradiol dioxygenases tbuE (U20258) of subfamily I.2.C of catechol extradiol dioxygenases todE (Y18245), todE (Y18245), tobE (AF180147) of subfamily I.3.B of catechol extradiol dioxygenases Eubacterial 16S rRNA genes
38F/518R
Eubacterial 16S rRNA genes
TMOA-F/TMOA-R
TOL-F/TOL-R
XYLA-F/XYLA-R
TODC1-F/TODC1-R
XYLE1-F/XYLE1-R XYLE2-F/XYLE2-R CDO-F/CDO-R
TBUE-F/TBUE-R
TODE-F/TODE-R
5V-AGAGTTTGATCCTGGCTCAG-3V 5V-TACGGYTACCTTGTTACGACTT-3V 5V-GATCTTGGCTCAGGTTGAACGCTG-3V 5V-ATTACCGCGGCTGCTGG-3V
Amplicon size (bp)
255
Reference
Proteins targeted
B. Hendrickx et al. / Journal of Microbiological Methods 64 (2006) 250–265
PCR annealing temperature (8C)
Primer pair
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the PCR mixture contained 67 mM Tris/HCl (pH 8.8), 16.6 mM (NH4)2SO4, 0.45% Triton X-100, 0.2 mg gelatine mL 1, 120 AM of each dNTP, 2 mM MgCl2, 10 pmol primer-F, 10 pmol primer-R, 1.25 U Taq DNA polymerase (5 U Al 1). Primers were synthesized by Westburg (Westburg BV, Leusden, The Netherlands). The PCR temperature/time profile used for all primer sets, except for the XYLE2-F/XYLE2-R pair, was an initial denaturation of 5 min at 95 8C, followed by 35 cycles of denaturation for 1 min at 94 8C, annealing for 1 min at the temperature reported in Table 2 and elongation for 2 min at 72 8C. The last step included an extension for 10 min at 72 8C. The profile used with the XYLE2-F/XYLE2-R primer set consisted of an initial denaturation step of 5 min at 95 8C, followed by 35 cycles of denaturation for 15 s at 94 8C, annealing for 30 s at 64 8C and elongation for 45 s at 72 8C. The last step was an extension for 3 min at 72 8C. 1465-bp or 480-bp eubacterial 16S rRNA gene fragments were amplified using respectively primer set 27F/1492R and primer set 38F/518R as described (Johnson, 1994; Muyzer et al., 1993). In all cases, PCR was performed on Biometra (Biometra, Go¨ttingen, Germany) or Perkin Elmer (Perkin Elmer, Connecticut, USA) PCR machines. PCR products were analyzed by 2% agarose gel electrophoresis as described (Vanbroekhoven et al., 2004). 2.8. Sensitivity of PCR method To examine the limit of PCR detection for the different primer sets, a known amount of viable cells of different relevant BTEX degrading strains was added to two different sterilized soils, A2Z and GP48, at different final cell concentrations (i.e. approximately 108, 106, 104, 102 CFU g 1) prior to DNA-extraction. Inoculum cells were harvested from liquid cultures, washed twice and added in 100 Al suspension of appropriate cell densities to 1 g of sterilized soil. After extraction, total soil DNA was subsequently used as template for PCR with the different developed primer sets. 2.9. Sequence analysis of the PCR amplified catabolic gene and 16S rRNA gene fragments Amplicons resulting from PCR with the different primer sets were purified with the QIAquick PCR
Purification Kit from the Westburg company (Westburg, Leusden, The Netherlands). The purified PCR products of the BTEX catabolic gene sequences were sequenced double stranded by the Westburg company (Westburg, Leusden, The Netherlands). A similarity analysis of the DNA sequences was obtained by using the Advanced Blast Search program BLASTX for the BTEX catabolic gene sequences and BLASTN for the 16S rRNA gene sequences available from GenBank (GenBank, National Centre for Biotechnology Information, Rockville Pike Bethesda, USA). The BTEX catabolic gene sequences reported in this study are available from GenBank under accession numbers AY504971 to AY504995, while the 16S rRNA gene sequences are available under accession numbers AY510158 to AY510165, AY512600 to AY512644 and AY517534 to AY517541.
3. Results and discussion 3.1. Design of novel PCR primer sets targeting BTEX initial attack mono- and dioxygenases The first novel primer set designed was primer set TMBMD-F/TBMDR allowing detection of all genes encoding a-subunits of subfamily 1 of the hydroxylase complex of aromatic mono-oxygenases (for the phylogenetic tree see Baldwin et al., 2003). The members in subfamily 1 are primarily phenol and cresol hydroxylase a-subunits, but include the toluene/benzene 2-hydroxylase a-subunit (Tb2m) encoded by the tbmD gene in strain JS150 (Johnson and Olsen, 1995) and the toluene 2-hydroxylase asubunit (TOM) encoded by the tomA3 gene in Burkholderia cepacia G4 (Shields et al., 1995). Because of the strong protein and DNA homology within the phenol/cresol hydroxylase a-subunits, it was not possible to design a primer set targeting only the tbmD and tomA3 genes. The novel TMBMD-F/ TBMDR primer set is an alternative for primer set PHE-F/PHE-R reported by Baldwin et al. (2003). However, primers TBMD-F and TBMD-R are less degenerate than PHE-F and PHE-R, which is of interest for diversity studies by DGGE (Hendrickx et al., submitted) and the TBMD-F/TBMD-R PCR product is 434-bp longer than the PHE-F/PHE-R fragment and yields more sequence information.
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Genes encoding more distantly related second, third and fourth subfamily a-subunits, should not be amplified, as they show 14 to 19 mismatches with the forward primer TBMD-F and 14 to 17 mismatches with the reverse primer TBMD-R. The new primers for detecting the BTEX side chain mono-oxygenases were based on the xylA gene. BTEX side chain mono-oxygenases are twocomponent enzyme systems consisting of a terminal hydroxylase component encoded by the xylM gene and an electron transfer component encoded by the xylA gene. More xylA gene sequences are available in GenBank than xylM gene sequences, and xylA and xylM are always found linked on TOL plasmids (Shaw and Harayama, 1992; Sentchilo et al., 2000). Moreover, a suitable primer set, i.e., TOL-F/TOL-R, was recently reported for PCR detection of xylM (Baldwin et al., 2003). The novel degenerate primer set, i.e., TODC1-F/ TODC1-R, for detecting genes involved in a direct dioxygenase attack of BTE, was designed to target simultaneously the genes of all five subfamilies of the iron–sulfur oxygenase a-subunits of the dioxygenase complex. The phylogenetic tree of the iron–sulfur oxygenase a-subunits of the dioxygenase complexes, reported by Baldwin et al. (2003), comprises two major types of a-subunits. The first type (N) consists primarily of naphthalene dioxygenase a-subunits. A second type (D) comprises two families, i.e., D.1 and D.2, which both consist of 3 subfamilies. Subfamilies D.1.A and D.2.C include BTEX dioxygenase a-subunits. The novel primer set is an alternative for the 5 dioxygenase primer sets recently described by Baldwin and co-workers (2003), which each allow the detection of the genes of a specific subfamily, and upgrades the primer set reported by Ogram et al. (1995) for detection of the todC1 gene of Pseudomonas putida F1. The ladder is a non-degenerate primer set and is not suitable for the detection of all other members of subfamily D.2.C and those of the other subfamilies. To test the theoretical assumptions concerning primer selectivity, PCR amplification with the 3 novel primer sets was performed with DNA from a variety of well-characterized BTEX degrading bacterial strains with and without the corresponding target gene as reported in Table 1. All strains were tested with each primer set. PCR amplification yielded pro-
257
ducts of the expected size and sequence (data not shown) for most of the strains it was expected for (data not shown). Nevertheless, some unexpected results were obtained with some strains. tbmD-like amplicons were also obtained with Pseudomonas mendocina KR1 and Pseudomonas aeruginosa JI104 DNA. The deduced protein sequences of the PCR products from KR1 and JI104 showed 95% identity with the phenol hydroxylase a-subunit of P. putida P-8 (Accession no. BAA74744) and 99% identity with the phenol hydroxylase a-subunit PhhN (Accession no. CAA55663), respectively. Similar results were reported by Baldwin et al. (2003) using primer pair PHE-F/PHE-R. Because both KR1 and JI104 produce methyl-substituted phenols from toluene, the existence of downstream phenol hydroxylases in that pathway and hence tbmD-related genes in those strains would be likely (Baldwin et al., 2003). Furthermore, no xylA PCR amplicon was obtained from P. putida MT15, although it carries the pWW15 TOL plasmid and although a xylM PCR amplicon was recovered using primer set TOL-F/ TOL-R (Table 1.). These results indicate that either xylA is not always linked with xylM or the xylA gene might show more sequence heterogeneity than deduced from reported sequences. Finally, PCR with primer set TODC1-F/TODC1-R yielded unexpectedly also PCR products with Burkholderia sp. strain JS150 and P. aeruginosa JI104 DNA. The deduced amino acid sequence of the PCR product of strain JS150 showed 98% amino acid identity with the terminal oxygenase large subunit McbAa of Ralstonia sp. JS705 involved in hydroxylation of chlorobenzene (Accession no. CAA06970) (van der Meer et al., 1998), whereas the PCR product of strain JI104 showed 100% identity with the biphenyl dioxygenase a subunit of biphenyl 2,3-dioxygenase BphA1 of Pseudomonas pseudoalcaligenes KF707 (Accession no. AAA25743) (Taira et al., 1992). Data by Robertson et al. (1992) indicate that catabolism of toluene in strain JS150 can also be initiated by a broad-substrate toluene/benzene dioxygenase similar to that of P. putida F1, whereas Kitayama et al. (1996b) reported growth of strain JI104 on biphenyl suggesting that it contains a bph operon including todC1-like genes. The presence and detection of todC1-like genes in JS150 and JI104 agree with those observations and can be explained by them.
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B. Hendrickx et al. / Journal of Microbiological Methods 64 (2006) 250–265
3.2. Design of PCR primer sets targeting (alkyl) catechol 2,3-dioxygenases (C23O) Proteins involved in BTEX degradation can be found in subfamilies I.2.A, I.2.B and I.2.C within family I.2 and in subfamily I.3.B within family I.3 of the phylogenetic tree of C23O amino acid sequences reported by Eltis and Bolin (1996). Subfamily I.2.A contains the C23O sequences of mainly fluorescent Pseudomonas bacteria, whereas subfamily I.2.B includes the C23O sequences of mainly Sphingomonas bacteria. Primer sets for the detection of C23O genes of subfamilies I.2.A and I.2.B were previously reported by Hallier-Soulier et al. (1996), Okuta et al. (1998), Meyer et al. (1999) and Mesarch et al. (2000). Since then, many new C23O sequences became available. As those reported primers did not always matched with all presently available sequences, we adapted and/or designed new degenerate primer sets, which would allow the detection of genes encoding for subfamily I.2.A and subfamily I.2.B. C23O proteı¨ns, i.e., primer set XYLE1-F/ XYLE1-R and primer set XYLE2-F/XYLE2-R. Subfamily I.2.C comprises mainly C23O sequences involved in phenol degradation derived from a wide variety of bacterial genera (Pseudomonas, Comamonas, Burkholderia and Ralstonia). In addition, it contains two C23O proteı¨ns involved in BTEX degradation for which no primers for PCR detection have been reported, i.e., the cdo gene coding for the C23OII Cdo in P. putida MT15 (Keil et al., 1985a) and the tbuE gene coding for the C23O TbuE in Ralstonia pickettii PKO1 (Kukor and Olsen, 1996). Since the members of subfamily I.2.C C23O protein sequences showed a low degree of similarity between each other, no subfamily I.2.C specific primer set could be designed. Instead, non-degenerate Cdo and TbuE C23O gene specific primer sets, named CDO-F/ CDO-R and TBUE-F/TBUE-R respectively, were designed. Family I.3 consist of subfamilies I.3.A and I.3.B, and includes C23O sequences from different Pseudomonas and Rhodococcus strains, which are mainly involved in biphenyl degradation. However, subfamily I.3.B also contains the 3-methylcatechol 2,3-dioxygenases TodE found in P. putida F1 (Zylstra and Gibson, 1989) and P. putida DOT-T1 (Mosqueda et al., 1999) and TobE of P. putida PB4071 (W. Li and
H. M. Tan, unpublished results), all involved in toluene degradation. As no primer sets to detect the corresponding gene sequences exist, a suitable primer set TODE-F/TODE-R was designed. PCR amplification with the primer sets targeting the different C23O DNA sequences was performed with DNA from all BTEX degrading bacterial strains reported in Table 1 including strains with and without the target gene (Table 1). All strains were tested with all primer sets. PCR products of expected size were obtained for strains carrying the corresponding target gene and the fragments showed the corresponding gene sequences. However, use of the TODE-F/ TODE-R primer set resulted into some aspecific fragments in addition to the expected fragment. The translated xylE1 amplicon sequences from strains MT15 and MT53 showed 100% identity with other Pseudomonas C23O protein sequences including XylE of the pWWO TOL plasmid (Greated et al., 2002; Keil et al., 1985b), demonstrating the presence of TOL-like xyl genes in those strains. Unexpectedly, for Burkholdeia sp. strain JS150, a todE-like gene PCR amplicon was obtained. The deduced protein showed 90% amino acid sequence identity with TodE of P. putida F1 (Zylstra and Gibson, 1989) and P. putida DOT-T1 (Mosqueda et al., 1999). Previously, we detected a todC1-like gene in strain JS150. Those data strongly suggest the presence of a tod-like pathway in JS150. 3.3. Sensitivity of detection of the PCR with the different primer sets in soil To examine the sensitivity of the PCR to detect the target catabolic genes in soil, decreasing concentrations of different bacterial strains, carrying relevant BTEX degradation genes (P. putida mt-2 (xylA, xylE1), S. yanoikuyae B1 (xylE2), P. putida MT15 (xylE1, cdo), Ralstonia pickettii PKO1 (tbuA1, tbuE) and P. putida F1 (todC1, todE)) were simultaneously added to both sterilized soils A2Z and GP48. DNA was extracted and subjected to PCR amplification. Except for primer sets TODC1-F/TODC1-R and TODE-F/TODE-R with detection limits of 105–106 gene copies g 1 soil, all primer sets detected the corresponding catabolic gene sequences in both soils with a detection limit of ca. 103–104 gene copies g 1 soil, assuming one copy of the gene per cell. The experiment was performed twice with both soils and
B. Hendrickx et al. / Journal of Microbiological Methods 64 (2006) 250–265
gave identical results (data not shown). No signals were obtained with DNA from sterilized uninoculated soils. The observed detection limits are commonly found for PCR detection of functional genes in environmental samples such as soil (Kowalchuk et al., 1999), compost (Kowalchuk et al., 1999) or seawater (Sinigalliano et al., 1995). 3.4. Distribution of BTEX degradation genes in BTEX degrading bacteria isolated from a BTEX polluted site A total of 81 BTEX degrading isolates was obtained from the site. BOX-PCR was used to discriminate between potential clonal isolates, recognizing 52 different BOX-PCR groups (Fig. 2). From each BOX-PCR group, the 16S rRNA gene of at least one isolate was partially sequenced for identification and all isolates were examined for the presence of the BTEX degradation genotypes and growth on BTEX compounds. For each BTEX degradation gene, PCR products obtained from the isolates were randomly selected for sequencing to examine if the recovered gene fragments were indeed the corresponding target genes. For all contaminated locations, Actinobacteria and Proteobacteria comprised the two main groups of resident culturable BTEX degrading bacteria, while from the uncontaminated location only Actinobacteria were isolated. The Actinobacteria included mainly Rhodococcus and Arthrobacter strains. Proteobacteria included mainly g-Proteobacteria, and more specifically Pseudomonas. Similarly, Cavalca et al. (2003) isolated from a BTEX-polluted aquifer, treated by air-sparging, bacteria belonging to both the classes of the Proteobacteria (Pseudomonas, Azoarcus and Bradyrhizobium) and the Actinobacteria (Microbacterium and Mycobacterium). On the other hand, Stapleton and Sayler (2000), mostly isolated Proteobacteria and no Actinobacteria. The proliferation of BTEX degrading Proteobacteria therefore seems to be a major characteristic of adaptation to BTEX in BTEX contaminated sites. Recently, at least at the site examined in this study, this was confirmed by means of an in situ microcosm study in which aquifer material from an uncontaminated location was placed into the contaminated groundwater plume. In the microcosms, the aquifer community was clearly developing into a community dominated by fluorescent Pseudomonads. In contrast, at the non-contami-
259
nated area both Actinobacteria and Proteobacteria were found (Hendrickx et al., in press). With a few exceptions, bacterial isolates of the same BOX-PCR group utilized the same BTEX substrates. Most of the obtained isolates utilized benzene as C-source, while only two isolates grew on o-xylene. The ability to utilize TEX compounds as a carbon source is not always accompanied by the ability to utilize benzene and vice versa (Stapleton and Sayler, 2000). In our study, benzene was the main contaminant which probably resulted into a major distribution of the ability to utilize benzene in the bacterial community of that site. In contrast, o-xylene was not detected in the groundwater at the site and the ability to degrade o-xylene was the least detected BTEX degradative phenotype among the isolates. The tmoA-like gene was the most represented BTEX initial attack genotype among the isolates, i.e., 59 and 39 of the isolates and BOX-PCR groups, respectively, carried the tmoA-genotype, followed by the xylM gene which was present in 39 of the isolates and 20 of the BOX-PCR groups. The other initial attack genotypes tbmD, todC and xylA were present in 28, 8 and 3 isolates and 12, 4 and 2 BOX-PCR groups, respectively. For all primer sets targeting genotypes encoding initial attack proteins, no aspecific signals were obtained and all PCR products showed the expected sizes. Moreover, all sequences obtained from randomly selected PCR products matched in BlastX similarity analysis with the BTEX initial attack genes expected to be detected by the respective primer sets showing the specificity of the primer sets. The recovered and sequenced tbmD-like initial attack genes showed a very high similarity with mono-oxygenases involved in phenol degradation instead of BTEX degradation indicating that at least the sequenced tbmD-like genes are rather involved in mono-oxygenase attack of a phenol compound than of BTEX itself. The phenol compound might have been derived from initial attack of the BTEX by a true BTEX mono-oxygenase such as those encoded by tmoA-like genes. From 3 of the 8 isolates showing todC1 genes, the todC1 PCR products were sequenced. The deduced protein sequences were most closely related to the BedC1 sequence. bedC1 is the todC1-like gene of a benzene degrading isolate showing the specialized adaptation to benzene degradation on the examined site.
2
ND
B
tb mD, tmoA
A2w8
2
Pseudomonas putida
B
tb mD, tmoA, todC1
March 2001
A2w4
2
ND
B
tb mD, tmoA
1
A2Y
March 2001
A2Y25
2
Stenotrophomonas sp.
m-X, p-X
2 3 4 5 6 7 8
tb mD, tmoA
2
A1Y
March 2001
A1Y11
2
Pseudomonas putida
T, EB, m-X,p-X
xylE1
3
A1 groundwater
March 2001
A1w4'
Pseudomonas putida
B
tb mD, tmoA, xylM, todC1, xylE1
3
A1 groundwater
March 2001
A1w3'
2 2
ND
B
tb mD, tmoA, xylM, todC1, xylE1
4
A1 mix of all zones
May 2 002
F 27
3
Agrobacterium sp.
BTEX-mix
tm oA
5
A1Y
March 2001
Amico5
4
Arthrobacter sp.
(B)
tm oA
6
A1Y
March 2001
Amico7
4
Arthrobacter sp.
(B)
tm oA
7
A1 mix of all zones
May 2 002
E31
BTEX-mix
tm oA
A1 mix of all zones A3X
May 2 002 April 2002
C2 0 A3X2
3 3
Georgenia sp.
8 9
2
Hydrogenophaga sp. ND
BTEX-mix B
9
tb mD, tmoA tm oA, xylM, todC1, xylE1
9
A3X
April 2002
A3X4
2
Pseudomonas jessenii
B
10 11 12 13 14 15 16 17 18 19 20 21 22
tm oA, xylM, todC1, xylE1
9
A3Z
April 2002
A3Z2
2
ND
B
tm oA, xylM, todC1,xylE1
10
A2Y
March 2001
Ami co27
Arthrobacter sp.
B, BTEX-mix
tm oA
11
A2Y
March 2001
Ami co51
4 4
Rhodococcus sp.
B, BTEX-mix
tm oA
12
A1Y
March 2001
Ami co42
4
Rhodococcus sp.
B, BTEX-mix
tm oA
13
A1X
May 2 002
A1X B1-5
1
Rhodococcus sp.
Catechol
tm oA, xylM, xylE1, todE
14 15
A1Y
A1Y13 A1- 1
2
A1 mix of all zones
March 2001 April 2001
3
Pseudomonas sp. Arthrobacter sp.
(BTEX-mix) BTEX-mix
tm oA tm oA, xylE1
16
A1 mix of all zones
April 2001
A3- 104
3
Pseudomonas sp.
BTEX-mix
tm oA
17
A1 mix of all zones
April 2001
A1- 13
3
Sphingomonas sp.
BTEX-mix
tm oA, xylE2
18
A1 mix of all zones
April 2001
A1- 69
3
Arthrobacter polychromogenes
BTEX-mix
tm oA
19
A1 mix of all zones
April 2001
A1- 10
3
ND
BTEX-mix
tm oA
20
A1X
May 2 002
A1X dBTEX1-4
1
Pseudomonas veronii
Catechol
xylM, xylE 1
21
A1Y
May 2 002
A1Y dBTEX1-5
1
Pseudomonas sp.
Catechol
xylM, xylE 1
22 23
A1X A1Y
May 2 002 May 2 002
A1X B1-4 A1Y B2-4
1 1
Pseudomonas fluorescens Pseudomonas veronii
B B
xylM, xylE 1 tb mD, tmoA, xylM , xylE1
23
A1Y
May 2 002
A1Y B1-4
1
ND
B
23
tb mD, tmoA, xylM , xylE1
23
A1Y
May 2 002
A1Y B3-4
1
Pseudomonas veronii
B
tb mD, tmoA, xylM , xylE1
23
A1Y
May 2 002
A1Y C2-5
1
Pseudomonas veronii
B
tb mD, xylM,xylE1
23
A1Y
May 2 002
A1Y C3-5
1
ND
B
24 25
tb mD, xylM,xylE1
24
A1X
May 2 002
A1X B2-5
1
Pseudomonas veronii
B, T, EB
xylM, xylE 1
25
A2Y
April 2001
IA2YCDA
1
Pseudomonas putida
B
tb mD, xylM
26
A3Y
May 2 002
A3Y dBTEX1-5
1
Pseudomonas sp.
B
xylM, xylE 1
26
A3Y
May 2 002
A3Y Xyl3 -5
1
ND
B
26
xylM, xylE 1
26
A3Y
May 2 002
A3Y Xyl2 -4
1
Pseudomonas sp.
B
xylM, xylE 1
26
A3Y
May 2 002
A3Y Xyl1 -4
1
ND
B, T
xylM, xylE 1
26
A3Y
May 2 002
A3Y dBTEX2-4
1
Pseudomonas sp.
B, T, EB
27 28 29
xylM, xylE 1
27
A1X
May 2 002
A1X Xyl1 -5
1
Sphingomonas sp.
B, T, EB
tm oA, xylM, xylE2
28
A1X
April 2001
IA1XBOX
1
Rhodococcus sp.
B
tm oA, xylM, xylE1
29
A2Y
April 2001
IA2YCDO
1
Pseudomonas sp.
B
xylM, xylE 1
30
A1Y
April 2001
IA1YICDB
1
Pseudomonas sp.
T, m-X, p-X
30 31 32
xylM, xylE 1
30
A1Y
April 2001
IA1YICDA
1
ND
T, m-X, p-X
xylM, xylE 1, cdo
31
A2X
April 2001
IA2XCDB
1
Pseudomonas putida
T, m-X, p-X
xylA, xylM, xylE1
32
A1 mix of all zones
April 2001
A1- 8
3
Sphingomonas sp.
BTEX-mix
tm oA, xylE2
33
A1X
April 2002
A1X/2A
2
Pseudomonas veronii
B, T, EB
tb mD, tmoA, xylE1
33
A1X
April 2002
A1X/2B
2
ND
B, T, EB
33
tb mD, tmoA, xylE1
33
A1X
April 2002
A1X/3B
2
ND
(B), (m-X), (p-X)
tb mD, tmoA, xylE1
33
A1X
April 2002
A1X/4A
2
ND
(X-mix), (m-X)
tb mD, tmoA, xylE1
33 33
A1X A1X
April 2002 April 2002
A1X/3A A1X/4B
2 2
Pseudomonas veronii Pseudomonas veronii
B, T, EB B, T
tb mD, tmoA, xylE1 tb mD, tmoA, xylE1
34
A1Y
May 2 002
A1Y C1-5
1
ND
B, T, EB
34
tb mD, xylM,xylE1
34
A1Y
May 2 002
A1Y Xyl3-5
1
ND
B, T, EB
tb mD, xylM,xylE1
34
A1Y
May 2 002
A1Y dBTEX2-5
1
Pseudomonas veronii
B, T, EB
tb mD, xylM,xylE1
35
A3Y
April 2002
A3Y3
2
Pseudomonas gingerii
B, T,m-X,p-X
35 36 37 38
tm oA, xylA,xylM,xylE1
35
A3X
April 2002
A3X3
2
ND
B, T,m-X,p-X
tm oA, xylA,xylM,xylE1
36 37
A2Y EAWAG
March 2001
4
March 2001
Amico3 KZ1
5
Arthrobacter sp. ND
(X-mix) B,T, EB, (p-X),m-X, o-X
tm oA tm oA, todE
38
A1 groundwater
March 2001
A1w2
2
Stenotrophomonas maltophilia
m-X, p-X
39
tm oA
38
A1 groundwater
March 2001
A1w3
2
ND
m-X, p-X
tm oA
39
A4Z
March 2001
A4Z24
2
Arthrobacter sp.
B, T
40
tm oA
41 42 43 44 45 46 47 48 49 50 51 52
BTEX catabolic genes
A2w7
March 2001
A2 groundwater
BTEX substrate
March 2001
A2 groundwater
1
16S rRNA gene Identification
A2 groundwater
1
Identification
1
Sampling date
Isolation procedure
1
Soil sample
BOX-PCR
100
90
70
80
60
50
Pearson correlation (Opt:1.86%) [0.0%-100.0%] BOX-PCR
Isolate
B. Hendrickx et al. / Journal of Microbiological Methods 64 (2006) 250–265
BOX-PCR group
260
40
A3Z
March 2001
A3Z19
2
Rhodococcus sp.
T, EB, X-mix
tm oA
40
A2Y
March 2001
A2Y26
2
Rhodococcus sp.
T, EB, X-mix
tm oA
41
A2X
March 2001
A2X9
2
ND
T, EB, X-mix
tm oA, xylM
41 41
A2Y A2Y
March 2001
2
March 2001
A2Y7' A2Y6
2
Rhodococcus sp. Rhodococcus sp.
T, EB, X-mix T, EB, X-mix
tm oA, xylM tm oA, xylM
41
A2Y
March 2001
A2Y5'
2
ND
T, EB, X-mix
tm oA, xylM
42
A3Y
April 2002
A3Y2
2
Rhodococcus sp.
B,T, EB, 0-X
tm oA
43
A1Y
March 2001
A1Y15
2
Ralstonia eutropha
B, T, EB
tb mD, tmoA
44
A2Y
March 2001
Amico6
4
Variovorax sp.
B,T, EB, BTEX-mix
tb mD, cdo
45
A1 mix of all zones
May 2 002
H8
3
Pseudomonas stutzeri ssp.
BTEX-mix
tm oA, xylM, xylE1
46
A1Y
April 2002
A1Y/3A
2
Pseudomonas veronii
(EB), (m-X)
tb mD, xylE1
47
A3Z
March 2001
A3Z17
B, EB, X-mix
tm oA
48
A3Z
March 2001
A3Z18
2 2
Arthrobacter sp. Arthrobacter sp.
B, EB, X-mix
tm oA
49
A1 mix of all zones
May 2 002
B33
3
Aquaspirillum sp.
BTEX-mix
tm oA
50
A1 mix of all zones
May 2 002
G1 4
3
Agrobacterium sp.
BTEX-mix
tm oA
51
A3Z
April 2002
A3Z4
2
Pseudomonas marginalis
B, T
tb mD, tmoA, xylM , todC1 , xylE1
51
A3Z
April 2002
A3Z1
2
Pseudomonas marginalis
B, p-X
tb mD, tmoA, xylM , todC1 , xylE1
52
A1 mix of all zones
May 2 002
A23
3
Acidovorax sp.
BTEX-mix
tb mD, tmoA
Fig. 2. UPGMA clustering of BOX-PCR fingerprints of the 81 bacterial strains isolated from BTEX contaminated soil samples.
B. Hendrickx et al. / Journal of Microbiological Methods 64 (2006) 250–265
tmoA-like genes have been shown to be implicated in benzene, toluene and o-xylene degradation (Bertoni et al., 1998; Byrne et al., 1995; Kitayama et al., 1996a; Yen et al., 1991), todC1 in (ethyl)benzene and toluene degradation (Zylstra and Gibson, 1989) and xylM/xylA in toluene, m- and pxylene degradation (Burlage et al., 1989; Keil et al., 1985a; Kok et al., 1999). In this study, isolates carrying tmoA-like genes and todC1-like genes showed a BTEX degradation range as expected from previous observations. However, many isolates containing xylM-like genes did not grow on either toluene, m- and p-xylene or ethylbenzene (BOXPCR groups: 3, 9, 22, 23, 25, 26, 28 and 29), but instead only utilized benzene. Benzene cannot be attacked by a XylM/XylA enzyme system since it does not carry an alkyl side chain. The bacteria belonging to those BOX-PCR groups carry in addition also tmoA- and/or todC1-like genes, which might be implicated in benzene degradation in those strains. The recovered xylM/xylA genes might be truncated versions of the TOL plasmid pathway which would nevertheless allow them to initiate degradation of toluene and xylene for consumption of the metabolites by synergistic communities. Many bacteria carry different initial attack genotypes. The presence of multiple catabolic genotypes with a similar catabolic activity in a single bacterial strain is not that remarkable and has been shown for Burkholderia sp. strain JS150 (Kahng et al., 2001) and in different. P. putida strains (Cavalca et al., 2000). About the respective role and activity of multiple initial attack systems in BTEX degrading bacteria in the presence of single or mixed BTEX compounds is not much known. Interestingly, as found for some TOL plasmid carrying reference strains, many of xylM carrying BTEX degraders did not contain a xylA-analogue. The xylE1-like genotype was the most recovered C23O gene among the isolates, being present in 42 and 19 of the isolates and BOX-PCR groups, respectively, followed in order by the xylE2-, cdo-/todEand tbuE-genotype. As for the initial attack genotypes, no aspecific signals were obtained with the primers targeting C23O genes, except for primer pair TODEF/TOER. All PCR products showed the expected sizes and all sequences from randomly selected PCR products matched in BlastX similarity analysis with the C23O genes expected to be
261
detected by the respective primer sets showing the specificity of the primer sets. As expected, xylE1 was often found in combination with xylM. However, some unexpected combinations of initial attack enzymes and C23O enzymes were observed. todE methylcatechol 2,3-dioxygenase-like genes which were only detected in two BOX-PCR groups were never found in combination with a todC1-like initial attack gene. Moreover, cdo-like genes which up to now were only detected in TOL plasmid containing bacteria in combination with xylM and xylE1 (Keil et al., 1985a,b), were in this study only combined with xylM-, xylE1-like genes in one Pseudomonas strain belonging to BOX-PCR group 30. The cdo C23OIIlike gene was also once combined with a tbmD-like initial attack gene in a Variovorax strain (BOX-PCR group: 44), indicating the combination of cdo and xylM as originally found in P. putida strains MT15 and MT53 is not always consistent. Interestingly, xylM-like genes were also detected in one Sphingomonas strain (BOX-PCR group: 27) and in some Rhodococcus strains (BOX-PCR groups: 13, 28, and 41), whereas the tmoA-like gene was also detected in different a-Proteobacteria like Agrobacterium (BOX-PCR groups: 4 and 50) and Sphingomonas (BOX-PCR groups: 17, 27 and 32) and in Gram-positives like Arthrobacter (BOX-PCR groups: 5, 6, 10, 15, 18, 36, 39, 47 and 48). This result might be an indication for the occurrence of horizontal transfer of BTEX catabolic genes in the aquifer community and moreover, that lateral gene transfer in the aquifer occurred across phylogenetic boundaries. A similar observation was done by Cavalca et al. (2003) who suggested interspecies transfer of the tmoA genes in a BTEX contaminated aquifer based on the occurrence of tmoA-analogues in different BTEX degrading isolates (Pseudomonas, Mycobacterium and Bradyrhizobium) from that aquifer. 3.5. PCR detection of BTEX degradation genes in contaminated subsurface soil samples from a BTEX polluted site tbmD-, tmoA-, xylM-, xylE1- and cdo-like genes were recovered from DNA extracts from all contaminated locations and this for al three zones X, Y, Z, though the cdo- and tbmD-like gene signals were weak for samples derived from sampling point A3
= no PCR signal; (+)
(+) (+) (+)
(+) (+) (+) 1
3 + 3 +
3 6
3 (+) + 6 1 2 ++ + (+) b0.0002 b0.0002 b0.0002 b0.0002 b0.0002 b0.0002 0.01–0.1 b0.0002
N100 NA NA
N10 NA NA
N1 NA NA
N10 NA NA
+ (+) (+)
3 ++ + +
3 3 2
+ + +
(+) 3 (+) 6 (+)
1 1
(+) (+)
1 2
+ + +
2 1 1 + + + 2 1 2 2 (+) 5 (+) + + + 5 1 8 2 ++ + 3 + + + + N10 NA NA N1 NA NA N10 NA NA N100 NA NA A1 A2 A2
A2 A3 A3
A3 A4
A1Z A2X A2Y
A2Z A3X A3Y
A3Z A4Z
S = PCR results soil; I = number of bacterial isolates which give a PCR signal, for these the Z-zone and groundwater were seen as equal; NA=not applicable; (+) = presence of a weak PCR signal; + = presence of a strong PCR signal; ++ = presence of a very strong PCR signal.
1
I
1
S I S I
(+) (+) 1
S I
1 (+)
S I
11 13 + +
S I S I S
6 11
I S
+ + 9 8
I S
6 + 10 +
I S
+ + NA NA
Xylenes Ethyl-benzene
NA NA NA NA
Toluene Benzene
NA NA
vadose capillary fringe saturated vadose capillary fringe saturated vadose capillary fringe saturated saturated A1 A1 A1X A1Y
TBUE -F/-R CDO -F/-R XYLE2 -F/-R XYLE1 -F/-R TODC1 -F/-R XYLA -F/-R TOL -F/-R TMOA -F/-R TBMD -F/-R ) 1
Concentration in the groundwater (mg L Soil Location Zone samples
(Table 3 and Fig. 3). For the non-contaminated samples, only tmoA-like sequences were found. No aspecific fragments were recovered from soil DNA (Fig. 3). In addition, as for the isolates, signals always matched the expected size fragment (Fig. 3). Since all sequenced fragments obtained with the isolates showed the right gene sequence, we did not clone and sequence PCR products obtained with the direct soil DNA extracts. Nevertheless, in other more recent work, we showed that gene fragments amplified with the reported primer sets from soil DNA extracts indeed corresponded to the target catabolic gene (Hendrickx et al., in press; Hendrickx et al., unpublished results). Strong signals were especially found for the tmoA, xylM and xylE genes which were congruent with the data obtained for the isolates in which those genes were also the most frequently encountered. In a similar study, Guo et al. (1997) showed by hybridization that DNA from contaminated subsurface soil samples collected along a gradient of BTEX concentrations at a fuel oil-contaminated site, was significantly enriched in tmo and especially xylE1, relative to DNA from the non-contaminated site. In their study, no hybridization was observed with todC1C2BA. Also in our study, recovery of todC1-like genes was rather low and was only observed for contaminated samples with low BTEX concentrations. As previously noticed for the isolates, xylM and xylA genes were not necessarily detected together as weak xylA-like gene signals were only found for sampling points A2 and A3 while xylM was detected in most samples. Similar to the isolates, no tbuE-like genes were amplified from the samples and from the uncontaminated sampling point A4, only tmoA-like genes were obtained. Although the overall picture was the same, some incongruency between the PCR results obtained with the direct DNA extracts and with the isolates exists. todE-like genes were detected in the isolates but not in the soil DNA. This might be due to the rather poor detection limit with soil DNA (105–106 copies g 1 soil) observed when using this primer set. Vice versa, some BTEX degrading genotypes detected in specific soil samples were not recovered in the corresponding BTEX degrading bacteria isolated from the same samples, indicating that not all BTEX degraders were isolated. For example, todC1-like genes were only amplified
TODE -F/-R
B. Hendrickx et al. / Journal of Microbiological Methods 64 (2006) 250–265
Table 3 Characteristics of the tested soil samples and results of the PCR with the different primer sets on soil template DNA and DNA from the isolates
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+ -
+
X
-
A1
+
Z
-
A4
Z A1
X A1
Z
- +
todE Z
cdo
A1
X A3 Y A1 Z
-
A1
X
- +
+
tbuE A1
Z A1
A2 A1 X Z
Z A4
- +
xylE2
-
todC1 A3
A2 X A1 Z
xylA
xylE1
+
A1
+
A1 Z A2 X
-
tbmD Z A2 X
tmoA
263
Fig. 3. Examples of PCR product analogues of the indicated catabolic genotypes obtained with direct DNA extracts from the contaminated site using primer set TMOA-F/TMOA-R and the primer sets developed in this study. The soil sample from which the PCR result was obtained is indicated above each lane. Lanes indicated with and + show results of negative (no DNA added) and positive controls, respectively. Positive controls for PCR with tmoA, tbmD, xylA, xylM, todC1, xylE1, xylE2, tbuE, cdo and todE were genomic DNAs from R. picketii PKO1, B. cepacia G4, P. putida mt-2, P. putida mt-2, P. putida F1, P. putida mt-2, S. yanoikuyae B1, R. picketii PKO1, P. putida MT15 and P. putida F1, respectively. The arrows indicate expected size fragments (see Table 2).
from DNA from samples from location A3 while todC1-like genes were detected in bacteria isolated from the groundwater at sampling points A1 and A2. The degraders carrying these genes might have been missed in our isolation process or might be not culturable using the procedures we applied for isolation (Amann et al., 1995). In conclusion, in this study, we report the development of eight novel primer sets for PCR detection of gene sequences encoding for mono-oxygenases and dioxygenase enzyme proteins involved in the initial attack of BTEX and encoding for catechol 2,3-dioxy-
genase involved in aromatic ring meta-cleavage. Both the results obtained for the soil DNA extracts as for the isolates indicate that strains with specific BTEX catabolic genotypes and hence, corresponding catabolic pathways are being selected upon BTEX contamination. These genotypes are especially related to the canonical xylM/xylE sequences found on the TOL plasmid and to tmoA, which both could be recovered from all contaminated areas of the site. In addition, xylE1 and xylM were not detected neither in DNA nor isolates from the uncontaminated area. We found similar results at another uncontaminated location at
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that site (Hendrickx et al., in press). tmoA-like genes were recovered from both contaminated and non-contaminated samples, but in another study, we showed that by DGGE fingerprinting, tmoA-like genes recovered from the contaminated and uncontaminated areas show significant sequence diversity (Hendrickx et al., in preparation). These results indicate a primary role of these sequences in adaptation of the community to the BTEX contamination and in in situ degradation of BTEX at the examined site.
Acknowledgements This work was supported by grant QLK3-CT200000731 of the European Commission. We thank M. Cernik, T. Lederer and D. Dubin for providing soil samples, T. Vallaeys for help in primer design, and P.A. Williams, J.J. Kukor, M.S. Shields, G.J. Zylstra, P. Barbieri and D.T. Gibson for kindly providing the reference strains used in this study.
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