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Junca Pieper2003 Jmm

  1. 1. Journal of Microbiological Methods 55 (2003) 697 – 708 www.elsevier.com/locate/jmicmeth Amplified functional DNA restriction analysis to determine catechol 2,3-dioxygenase gene diversity in soil bacteria Howard Junca, Dietmar H. Pieper * Department of Environmental Microbiology, GBF-German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Brunswick, Germany Received 31 March 2003; received in revised form 22 July 2003; accepted 22 July 2003 Abstract To determine phylogenetic diversity of a functional gene from strain collections or environmental DNA amplifications, new and fast methods are required. Catechol 2,3-dioxygenase (C23O) subfamily I.2.A genes, known to be of crucial importance for aromatic degradation, were used as a model to adapt the amplified ribosomal DNA restriction analysis to functional genes. Sequence data of C23O genes from 13 reference strains, representing the main branches of the C23O family I.2.A phylogeny, were used for simulation of theoretical restriction patterns. Among other restriction enzymes, Sau3A1 theoretically produce characteristic profiles from each subfamily I.2.A member and their similarities reassembled the main divergent branches of C23O gene phylogeny. This enzyme was used to perform an amplified functional DNA restriction analysis (AFDRA) on C23O genes of reference strains and 19 isolates. Cluster analyses of the restriction fragment profiles obtained from isolates showed patterns with distinct similarities to the reference strain profiles, allowing to distinguish four different groups. Sequences of PCR fragments from isolates were in close agreement with the phylogenetic correlations predicted with the AFDRA approach. AFDRA thus provided a quick assessment of C23O diversity in a strain collection and insights of its gene phylogeny affiliation among known family members. It cannot only be easily applied to a vast number of isolates but also to define the predominant polymorphism of a functional gene present in environmental DNA extracts. This approach may be useful to differentiate functional genes also for many other gene families. D 2003 Elsevier B.V. All rights reserved. Keywords: Amplified functional DNA restriction analysis; AFDRA; Catechol 2,3-dioxygenase catabolic gene diversity; Soil bacteria; BTEX biodegradation 1. Introduction mental microbial community composition, to detect new community members, activities, and functions, It is well documented that only a small fraction of have experienced a fast development in the last deca- environmental microorganisms can be cultured to date, des. Culture-independent approaches, usually focused and thus, culture-independent methods (Amann et al., on 16S rRNA phylogeny, have been applied for ana- 1995; Staley and Konopka, 1985) to describe environ- lysing populations in diverse environments (Torsvik and Ovreas, 2002), and various molecular fingerprint- * Corresponding author. Tel.: +49-531-6181-467; fax: +49-531- ing methods like denaturing gradient gel electrophore- 6181-411. sis (DGGE), temperature-gradient gel electrophoresis E-mail address: dpi@gbf.de (D.H. Pieper). (TGGE), ribosomal intergenic spacer analysis (RISA), 0167-7012/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0167-7012(03)00214-8
  2. 2. 698 H. Junca, D.H. Pieper / Journal of Microbiological Methods 55 (2003) 697–708 terminal restriction fragment length polymorphism aromatic ring fission activity, which play an essential (T-RLFP), upstream-independent ribosomal RNA am- role in the degradation of a wide range of aromatic plification analysis (URA), and polymerase chain pollutants. A large collection of genes coding for such reaction – single-strand conformation polymorphism an activity have been cloned and sequenced in the last (PCR-SSCP) have been optimised to obtain a fast years, being classified as a diverse gene family (Eltis and reliable overview on microbial community com- and Bolin, 1996). The gene phylogeny of these very positions and their shifts by changing environmental closely related sequences does not follow strictly a conditions (Kent and Triplett, 2002). Other fingerprint- taxonomical relation with the bacterial hosts, since ing methods like amplified ribosomal DNA restriction these genes are mainly found on plasmids, and their analysis (ARDRA) (Khetmalas et al., 2002) or PCR – evolution and conservation rates are heavily affected multiple enzyme restriction fragment length polymor- by traits like selection pressures, horizontal transfer, phism (PCR-MERFLP) (Porteous et al., 2002) have and mobile genetic elements (Williams et al., 2002). been developed to distinguish single 16S rDNA PCR Conserved regions of the C23O genes have been fragments from isolates or gene libraries to get a rapid selected in several studies as suitable PCR targets in overview on taxonomical diversity in cultivable or culture-independent approaches, showing a correlation noncultivable microbial fractions. in relative C23O abundances dependent of pollutants However, a number of bacterial genes such as those levels (Erb and Wagner-Dobler, 1993; Mesarch et al., involved in antibiotic resistance, antimicrobial produc- 2000; Meyer et al., 1999; Ringelberg et al., 2001; tion or pollutant degradation are selected or present Wikstrom et al., 1996). independently of the rate of evolution and taxonomy of In this study, we developed and evaluated an the bacterial host (Davison, 1999). Thus, assessment amplified DNA restriction analysis targeting a func- of community functions needs reliable tools to analyse tional gene (amplified functional DNA restriction those functions rather than taxonomical composition. analysis, AFDRA) in a collection of strains sharing PCR-based techniques have been used to detect func- a phenotypic character, meta-cleavage activity. We tional/catabolic genes in environmental isolates or examined the suitability of this method to directly environmental DNA, and diversity is usually assessed analyse soil DNA by a combination of PCR serial by sequencing of genes from isolates or PCR clone dilution assays and AFDRA analyses. This ap- libraries (Buchan et al., 2001; Duarte et al., 2001; proach allowed us to determine C23O gene copy Hamelin et al., 2002; Yeates et al., 2000). Recent numbers and predominant C23O gene variants in reports showed that PCR-amplified fragments of cat- soil samples. abolic genes from environmental DNA can be sepa- rated by DGGE (Henckel et al., 1999; Nicolaisen and Ramsing, 2002), and RFLP analyses were used to 2. Materials and methods select distinctive restriction patterns of single ampli- cons for further sequence determinations (Bakermans 2.1. Microorganisms, samples, isolation, and culture and Madsen, 2002; Braker et al., 2000; Yan et al., conditions 2003). A more detailed knowledge on catabolic genes, retrieved by culture-independent methods and from Reference strains P. stutzeri AN10 (Bosch et al., isolates, can significantly improve our understanding 2000), P. putida CF600 (Bartilson and Shingler, 1989), of microbial functioning and degradation processes in P. putida G7 (Ghosal et al., 1987), P. putida H the environment, which would help to design new (Herrmann et al., 1995), Pseudomonas sp. IC (Car- bioremediation strategies (Widada et al., 2002). How- rington et al., 1994), P. aeruginosa JI104 (Kitayama et ever, no fast and reliable tools are available to rapidly al., 1996), Sphingomonas sp. KF711 (Moon et al., analyse gene phylogeny in respective culture collec- 1996), P. putida mt-2 (Nakai et al., 1983), P. putida tions or to determine abundant gene variants and their mt53 (Keil et al., 1985), P. stutzeri OM1 (Ouchiyama phylogeny in the environment. et al., 1998), P. stutzeri OX1 (Arenghi et al., 2001), P. Catechol 2,3-dioxygenases (C23O) comprise a fam- putida HS1 (Benjamin et al., 1991), P. putida 3,5X ily of genes coding for a group of enzymes with (Hopper and Kemp, 1980), and isolated bacterial
  3. 3. H. Junca, D.H. Pieper / Journal of Microbiological Methods 55 (2003) 697–708 699 strains were cultured on R2A agar (DIFCO) at 30 jC 0.25 AM of each primer (synthesized by Invitrogen) for 3 days. and 0.3 U/Al Taq DNA polymerase (Promega). Bacterial strains were isolated by direct plating on For amplification of soil DNA, PCR was per- R2A agar of appropriate soil dilutions from samples formed in a PCRExpress gradient thermocycler obtained from four sampling points of a BTEX- (Hybaid) as follows: an initial step at 95 jC for 3 contaminated area in Czech Republic. After 3 days min, followed by 35 cycles of denaturation at 94 jC of incubation at 30 jC, colonies were tested by for 45 s, annealing at 55 jC for 45 s, and elongation at spraying with a 100 mM aqueous catechol solution, 72 jC for 90 s. For amplification of DNA from and those exhibiting meta-cleavage activity, indicated isolates, the PCR program comprised an initial step by yellow coloration of the medium, were selected, at 95 jC for 3 min, 10 cycles of denaturation at 94 jC streaked, and purified. for 45 s, annealing for 45 s at a starting temperature of For culture-independent analyses, soil samples 60jC with a decrease in annealing temperature of 1 were collected from the capillary fringe zone (zone jC per cycle, elongation for 90 s at 72 jC, followed of essentially water-saturated soil just above the water by 25 PCR cycles at a constant annealing temperature table) and the saturated zone (zone below the water of 55 jC, final elongation step at 72 jC for 8 min, and table) of a highly contaminated area (BTEX concen- further storage of the reactions at À 20 jC. trations in the groundwater of 320, 97, 6, and 13 mg/ To determine the minimum number of C23O l of benzene, toluene, ethylbenzene, and xylenes, copies detected by PCR with this set of primers, respectively, as determined by Aquatest Prague). Total an equimolar mixture of 15 C23O PCR amplicons DNA was extracted in triplicates from 500 mg of each (from 13 reference strains and from isolate numbers soil sample and purified with the Fast Prep Soil DNA 9 and 19), was serially diluted and aliquots of these Extraction Kit (BIO101). Concentration of DNA was dilutions applied in triplicates to PCR reactions. The determined by using PicoGreen dsDNA Quantitation maximum dilution at which a PCR signal of the Kit (Molecular Probes) and a microtiter plate reader as expected size was detected in one of the triplicates described previously (Weinbauer and Hofle, 2001). ¨ was determined analysing 3 Al of the PCR reactions by gel electrophoresis (1.5% agarose, 10 cm length, 2.2. Primer design and PCR conditions 1 Â TAE running buffer, 1 h at 95 V and visualized by ethidium bromide staining) (Sambrook et al., The primers C23O-ORF-F 5VAGG TGW CGT 1989). As the average molecular mass of the 934- SAT GAA MAA AGG 3Vand C23O-ORF-R 5VTYA bp C23O fragments is 570 kDa, it can be calculated GGT SAK MAC GGT CAK GAA 3Vwere designed that a single PCR amplicon molecule corresponds to to amplify 934 bp comprising the complete open approximately 9.5 Â 10À 10 ng of DNA. The mini- reading frames of the subfamily I.2.A C23O genes. mum number of C23O copies necessary to produce a Degenerations are placed according to all the possible detectable PCR amplification with the primers variable positions in the alignment of 20 C23O assayed was used to extrapolate the number of reference sequences (EMBL/GenBank/DBBJ acces- copies per milligram of soil (Ringelberg et al., sion numbers M33263, AY112717, S77084, 2001) by determination of the maximum dilution X80765, D83057, X77856, M65205, X60740, of soil DNA at which a PCR signal is detected in J C 4 8 8 5 , A F 0 3 9 5 3 4 , A B 0 0 1 7 2 2 , V 0 11 6 1 , six independent experiments. The inhibitory effect of AF226279, AF102891, AJ496739, D83042, JC5654, soil DNA extracts on C23O PCR amplification U01825, AY228547, X06412). As template DNA, 4- efficiency was determined by spiking soil DNA Al aliquots of a total of 50-Al supernatant from (5 Al containing a total of 10 ng of DNA) extracted colonies boiled for 10 min (Kanakaraj et al., 1998) from a control sample of the saturated zone of the or 5 Al of serial dilutions of soil DNA (prepared from study area with no previous BTEX contamination 50 Al of a total DNA extraction of 500 mg of soil) and devoid of amplifiable C23O genes, with 1 ng of were applied in a PCR mixture containing 1 Â PCR an equimolar mixture of 15 C23O PCR amplicons Buffer (Promega) supplemented with 1.5 mM MgCl2, (equivalent to 107 C23O gene copies). By serial 200 AM of each deoxyribonucleotidetriphosphate, dilutions in triplicates, the maximum dilution at
  4. 4. 700 H. Junca, D.H. Pieper / Journal of Microbiological Methods 55 (2003) 697–708 which a C23O PCR signal of the expected size is 2.5. DNA sequencing and phylogenetic analyses detected was determined. Nucleotide sequencing of PCR fragments or plas- 2.3. In silico PCR-RFLP analyses mids with cloned inserts was carried out on both strands using Taq dye –deoxy terminator in an ABI Predictions and simulations of the restriction frag- 373A automatic DNA sequencer (Perkin-Elmer Ap- ment length of 13 C23O DNA sequences from refer- plied Biosystems) following the protocols provided by ence strains was performed by a restriction mapping the manufacturer. Primers used for sequence reactions software (Heiman, 1997) with around 300 different were the same as for PCR. Alignments were per- endonuclease recognition sites. Enzymes that poten- formed with CLUSTAL X 1.8 windows interface of tially produce phylogenetic informative fragments the CLUSTALW program using default values were selected after detailed inspection and comparison (Thompson et al., 1997). DNA alignments were edited of the predicted restriction positions and calculation of and translated with the GeneDoc program (Nicolas, the produced fragment lengths. Matrices to represent 1997). Phylogenetic trees were obtained with the discrete values for presence or absence of a predict- option available on the CLUSTAL program through able restriction fragment size in the sequence data set the Neighbour Joining (N-J) algorithm method. Dis- were generated for each selected enzyme. Distance tances were generated using Kimura Matrix, and tree estimations on these matrices were performed with stability was supported through Bootstrap analysis. NJ Treecon software (Van de Peer and De Wachter, 1993) trees were visualized with the NJplot program (Per- by Simple Matching or by Nei and Liu methods. Tree riere and Gouy, 1996). Values of more than 50% of topology was inferred by the UPGMA clustering 1000 replications (seed value 111) are shown on method. Those results were compared with C23O appropriate branches. A similarity matrix to compare protein phylogeny. PCR-RFLP gel patterns was calculated using Bio1D v.99.02 Software (Vilber Lourmat), with Jaccard coef- 2.4. Amplified functional DNA restriction analysis ficients with 3% confidence applied to both compared bands. A dendrogram of similarity values in the Selected restriction enzymes (NEB) were used in matrix was calculated using the UPGMA algorithm. reactions in a final volume of 20 Al containing the 1 Â The sequences of gene fragments reported in this buffer recommended by the manufacturer, 3 U of the study are available under the EMBL/ GenBank/ DBBJ enzyme, and approximately 200 ng of PCR product, accession numbers AJ544921 to AJ544938. incubated at optimal temperature for 4 h. The restriction fragments patterns were resolved by gel electrophoresis in a 5.5% Nusieve 3:1 (FMC 3. Results and discussion Bioproducts) agarose matrix (14 Â 11 cm) in 1 Â TBE buffer (Sambrook et al., 1989), at 120 V (80 3.1. Targeting the functional gene by PCR mA), until the bromophenol blue dye in the loading buffer was reaching the front edge (approximately From soil samples contaminated with BTEX, a 5 h). The loading buffer comprised glycerol (30%, v/ broad set of microorganisms exhibiting catechol 2,3- v) in water, xylene cyanol, and bromophenol blue at dioxygenase activity, identified based on a simple test final concentrations of 0.025%. To correctly detect all for induction of this enzyme activity during growth on bands and intensities onto the gel, the loading buffer agar plates, was isolated. All isolates referred here contained dyes at 1/10 strength of the standard con- could use benzene and/or toluene as sole source of centrations (Sambrook et al., 1989), avoiding their carbon and energy. collateral effect of dark background. For staining, the We hypothesized that these isolates could carry gel was placed in ethidium bromide solution (10 mg/l) C23O genes members of the I.2.A subfamily (Eltis for 30 min. The gel was visualised, images acquired and Bolin, 1996), because this group of enzymes is and stored on a gel documentation system (Vilber known to be involved in the degradation of aromatic Lourmat). compounds in several environmental strains. A prim-
  5. 5. H. Junca, D.H. Pieper / Journal of Microbiological Methods 55 (2003) 697–708 701 er set was designed to amplify the complete C23O acid sequencing of the genes. The theoretical simula- gene of members in the I.2.A subfamily, annealing at tion was performed through the generation of discrete the start and stop codon positions. For the forward matrices of restriction fragment sizes produced in primer, annealing was optimised by placing its 5V C23O sequences deposited on EMBL/GenBank/ side 10 bases upstream of the C23O starting codon. DDBJ databases. These matrices were further analysed This primer set was tested on 13 reference strains to find restriction enzymes producing specific frag- (see Materials and methods) known to carry C23O ment profiles for different genes, and a restriction subfamily I.2.A gene variants and on a soil bacterial pattern related to gene phylogeny. In this search, out strain collection. From all the reference strains and of approximately 300 enzymes tested on each of the 13 from 30 out of 37 bacterial isolates tested, a signal of selected sequences, some enzymes, among them AluI, the expected size was successfully amplified, where- BsiHKAI, BstUI, HhaI, HinP1I, MspR9I, PalI, as 7 isolates did not give any amplification product, Sau3AI, and TaqI, were found as theoretically produc- indicating that they probably harbour a C23O of ing characteristic profiles from each subfamily I.2.A another subfamily. Nineteen of those isolates, show- member. ing an amplification product of 934-bp in size, were randomly selected for further analysis. 3.3. Characterization and comparison of C23O diversity in bacterial isolates by AFDRA and DNA 3.2. Selection of restriction enzymes producing sequencing patterns clustering as the divergent branches of C23O protein phylogeny by using predictions and After a meticulous comparison of the predictions, simulations on reported DNA sequences we selected the restriction enzyme Sau3A1 for the experiments. This enzyme could cleave the C23O To assess the diversity of the C23O genes, a fast genes in such a way that the resulting clustering of screening method to identify potential redundant or the matrix (37 Â 14), representing discrete values for highly similar genes based on a restriction fragment presence or absence of a predictable restriction frag- simulation, was developed. This method was com- ment size (37 theoretical restriction fragments from 11 pared with results obtained by a conventional nucleic reference strains; Fig. 1), reassembled the main diver- Fig. 1. Cluster dendrogram of theoretical Sau3AI restriction fragments produced from C23O genes of P. stutzeri AN10, P. putida CF600, P. putida G7, P. putida H, Pseudomonas sp. IC, P. aeruginosa JI104, P. putida mt-2, P. putida mt53, P. stutzeri OM1, P. putida HS1, P. putida 3,5X. The corresponding discrete matrix of presence (1) or absence (0) of a specific restriction fragment size is given to the right. Columns 1 – 37 represent, respectively, the following sizes in base pairs: 10, 14, 18, 19, 21, 23, 30, 34, 35, 39, 44, 55, 58, 88, 92, 104, 111, 112, 126, 133, 138, 141, 152, 153, 156, 157, 163, 174, 177, 232, 239, 269, 285, 306, 321, 443, and 447.
  6. 6. 702 H. Junca, D.H. Pieper / Journal of Microbiological Methods 55 (2003) 697–708 gent branches of the C23O phylogeny (Eltis and Bolin, tion patterns present in the isolates. A selection of 1996). Furthermore, the predicted fragment size range those representative patterns found with distinct sim- produced by this enzyme (10 – 447 bp) could be ilarities to the reference strain profiles are shown in separated by high-resolution agarose gel electrophore- Fig. 2. The most dominant pattern, observed in 13 of sis. Sau3A1 restriction assays were performed using 19 isolates of C23O PCR-RFLP, was equal to that PCR C23O gene amplifications from seven reference obtained from P. stutzeri AN10. Another common strains (Fig. 2) selected to cover the main divergent pattern, which shared some restriction fragment sizes branches of the protein gene phylogeny. Comparisons with P. putida G7, was present in four isolates. The of the restriction patterns under optimised electropho- PCR fragment from a single isolate produced a retic conditions resolved the informative restriction restriction pattern closely related to that from P. putida fragment patterns of reference strain amplifications mt-2. Another PCR fragment from a single isolate in the >35- to < 600-bp range, effectively discriminat- showed a restriction pattern sharing, to a lesser extent, ing down to 5-bp size differences, as evidenced by the band sizes with the P. putida 3,5X C23O gene. It can comparison of 133-bp and 138-bp fragments. The thus be hypothesized that the isolates are dominated same procedure was performed on 19 PCR-amplified by catechol 2,3-dioxygenase genes closely related to products of bacterial isolates, which were producing the P. stutzeri AN10 gene, whereas other isolates distinctive restriction fragment patterns. Digestions harbour far divergent evolutionary variants of catechol were usually complete under the conditions used, 2,3-dioxygenases. and only in case of high concentrations of amplifica- To confirm the reliability of this assay, the frag- tion product subjected to digestions, faint bands due to ments from all the isolates mentioned above were incomplete digests were observed. completely sequenced, and the resulting sequences Cluster analyses of the restriction fragment profiles were aligned against C23O DNA sequences of refer- obtained allowed to distinguish four groups of restric- ence strains. In the phylogenetic tree of the deduced Fig. 2. Agarose gel-generated cluster dendrogram illustrating the relationship of C23O genes based on similarity of restriction fragment patterns produced by Sau3AI digestions. Digestions of the 934-bp C23O PCR fragments amplified from the reference strains P. stutzeri AN10, P. putida CF600, P. putida G7, Pseudomonas sp. IC, P. putida mt-2, P. putida HS1, P. putida 3,5X, and isolated bacterial strains of group A: isolates number 2, 9, 4, 12, and 13; group C: isolate number 14; group B: isolate number 18; and group D: isolate number 1. Marker lane (MWM) shows DNA marker V (Roche), in the upper side are written the corresponding base pair length values of each DNA fragment.
  7. 7. H. Junca, D.H. Pieper / Journal of Microbiological Methods 55 (2003) 697–708 703 C23O protein sequences from reference strains and to P. putida mt-2 C23O protein numbering) to tyro- isolates (Fig. 3), the sequences obtained from isolates sine (11 isolates, subgroup A1) or histidine (2 isolates, are clustering in five different branches. This closely subgroup A2) (Fig. 3). This difference does not affect corresponds to the results obtained by the restriction a Sau3AI recognition site and therefore could not be analysis. In addition, all the sequences that were discriminated by PCR-RFLP. In such cases where supposed to carry the same or a closely related gene small differences like point mutations should be polymorphism are, in fact, at sequence level sharing detected, a screening technique not retrieving gene this feature (Figs. 2 and 3). In the case of group A, phylogeny information but with higher sensitivity for comprising 13 isolates, a single point mutation dif- single nucleotide substitutions like SSCP (Orita et al., ference is present, producing a difference in the 1989) or DGGE (Muyzer et al., 1993) could be then coding of the amino acid at position 218 (referred applied. Fig. 3. Phylogenetic tree of deduced amino acid sequences of C23O gene fragments from isolates (shaded) and from reference strains (indicated by their EMBL/GenBank/DDBJ accession number, abbreviated organism name, and strain designation). Alignment of a 268-amino-acid length block was performed with CLUSTAL W using default values. N-J phylogenetic tree was generated using the option available in CLUSTAL W program. Bootstrap values above 50% from 1000 neighbor-joining trees are indicated to the left of the nodes. Bar represents 2 amino acid changes per 100 amino acids.
  8. 8. 704 H. Junca, D.H. Pieper / Journal of Microbiological Methods 55 (2003) 697–708 3.4. AFDRA of new C23O sequences follow the struction of gene phylogeny. AFDRA proved thus to expected relation with gene phylogeny be a valuable tool to screen PCR fragments of func- tional genes from isolates, avoiding the redundant work The PCR-RFLP discrete matrix (40 Â 18) of C23O of repeated sequencing of the same gene from different members, including the four new sequences observed isolates or colonies, saving time and resources, and in isolates and comprising three new PCR-RF sizes giving a fast and reliable overview of the predominance only present in these isolates is shown in Fig. 4. The and phylogenetic affiliation of the genes in a group of thereby generated dendrogram reassemble the main environmental bacterial isolates. It can be particularly divergent clustering topology of the C23O protein useful in studies determining functional gene diversity phylogeny (Fig. 3). The application of PCR-RFLP in in collections of numerous environmental isolates, as a closely related group of sequences diminishes the well as to screen PCR fragments of functional genes potential limitations of the RFLP technique (Hillis et amplified from environmental DNA. al., 1996), as neither larger insertion/deletions or rear- rangement events nor the convergent fragments were 3.5. Quantitative and qualitative applications of the observed in the sequences analysed. However, the AFDRA on soil DNA cluster analysis of the bands resolved on the gel showed an outgrouping restriction pattern of the P. putida The efficiency and the minimum amount of C23O CF600 C23O gene, and its 141-bp fragment was copies detected with our primers designed to target the incorrectly assigned as clustering with fragments C23O subfamily I.2.A were determined, as a prereq- corresponding to 138 bp. That is an expected limitation uisite for a quantitative and qualitative application of of the technique due to the experimental error in AFDRA to DNA extracted from soil. PCR products calculation of very similar fragment sizes. Neverthe- were observed in all triplicates when the PCR mixture less, the generation and detection of a high number of contained 10À 8 ng of DNA (corresponding to approx- discrete characters allowed an approximated recon- imately 10 template molecules) and usually in one of Fig. 4. Cluster dendrogram and corresponding discrete matrix of presence – absence of a restriction fragment size of theoretical restrictions of C23O genes with Sau3AI from reference strains and isolates. Reference strains are P. stutzeri AN10, P. putida CF600, P. putida G7, P. putida H, Pseudomonas sp. IC, P. aeruginosa JI104, P. putida mt-2, P. putida mt53, P. stutzeri OM1, P. putida HS1, P. putida 3,5X. Group A includes sequences from isolates number 2, 9, 4, 12, and 13, group C includes isolate number 14, group B includes isolate number 18, and group D includes isolate number 1. The resultant 40 columns represent the following sizes in base pairs (1 – 40, respectively): 10, 14, 18, 19, 21, 23, 27, 30, 34, 35, 39, 44, 55, 58, 88, 92, 104, 111, 112, 126, 133, 138, 141, 147, 152, 153, 156, 157, 163, 174, 177, 232, 239, 269, 285, 306, 321, 353, 443, and 447.
  9. 9. H. Junca, D.H. Pieper / Journal of Microbiological Methods 55 (2003) 697–708 705 the triplicates when it contained 10À 9 ng of DNA can be assumed that the soil extract decreased the (corresponding to approximately one template mole- C23O amplification efficiency by a factor of 100. This, cule), independent of the complexity of the applied in turn, indicates that the contaminated soils analysed experimental C23O gene composition (Fig. 5A). Soil would contain approximately 106 C23O copies/g of DNA extractions yielded 100 – 300 ng of DNA/g of soil. To determine if the amplification affects the soil for the samples analysed. C23O PCR products diversity recovered, AFDRA was applied on the PCR were observed from as low as 20 pg of soil DNA as products obtained from artificial C23O gene mixtures template (Fig. 5B), indicating that 1 g of soil of the containing 10– 104 gene copies. The primers did not sampling point analysed would contain at least 104 show evidence for preferential amplification of any C23O gene copies, independent if the soil was collect- specific polymorphism under the conditions tested ed from the capillary fringe or the saturated zone. (Fig. 5C; lanes 17 and 18). The pattern obtained from However, a control soil DNA extract devoid of C23O an equimolar mixture of 15 C23O gene variants was genes and spiked with 107 C23O gene copies showed very complex and did not reassemble clearly any of the an amplification signal only, when the serial dilution single C23O gene profiles, even when the applied contained at least 100 copies of C23O genes. Thus, it template mixture contains only approximately 10 C23O copies. On the other hand, the primers amplified preferentially the most abundant polymorphism out of Fig. 5. Detection limit, diversity comprised, and performance in artificial mixtures or environmental DNA of the primers targeting C23O subfamily I.2.A and assessment of predominant gene variants in soil DNA by AFDRA approach. (A) Detection limit of primers designed to target C23O subfamily I.2.A. Lanes 1 – 5 show PCR products obtained from 10À 6, 10À 8, 10À 10, 10À 11, and 10À 12 ng, respectively, of an equimolar mixture of 15 C23O genes. Lanes 6 – 10 show PCR products obtained from 10À 5, 10À 7, 10À 9, 10À 10, and 10À 11 ng, respectively, of an equimolar mixture of 14 C23O gene variants supplemented with a 10-fold excess of the P. putida CF600. Lanes M were loaded with 200 ng of DNA Molecular Weight Marker VIII (Roche). (B) Determination of the C23O copy number in contaminated soils. Total DNA extracted from 500 mg of soil (100 ng) was resuspended in 50 Al of H2O with a DNA concentration of 2 ng/Al and appropriate dilutions subjected to PCR amplification. PCR products obtained from DNA extracted from a soil sample of the capillary fringe zone (lanes 11 – 13) or from the saturated zone (lanes 14 – 16) were analysed by agarose gel electrophoresis. PCR products correspond to those obtained from 200, 20 or 2 pg of soil DNA, respectively. Lanes 1kb were loaded with 100 ng of GeneRuler 1 kb DNA ladder (MBI Fermentas). (C) AFDRA patterns obtained from PCR-amplified artificial C23O mixtures or direct amplifications of soil DNA. Lanes 17 and 18 show AFDRA of the PCR amplification products obtained from 10À 6 or 10À 8 ng of an equimolar mixture of 15 C23O subfamily I.2.A gene variants. Lanes 19 and 20 show AFDRA of the PCR amplification product obtained from 10À 5 or 10À 7 ng of an equimolar mixture of 14 C23O gene variants supplemented with a 10-fold excess of the P. putida CF600 gene. Lanes 21 and 22 show AFDRA of the PCR amplification product obtained from 200 or 20 pg of soil DNA of the capillary fringe zone, lanes 23 and 24 AFDRA of the PCR amplification product obtained from 200 or 20 pg of soil DNA of the saturated zone, lanes 25 – 28 AFDRA of the C23O genes from P. stutzeri AN10, P. putida CF600, P. putida G7, and P. putida mt-2, respectively. Lanes M were loaded with 120 ng of DNA Molecular Weight Marker V (Roche).
  10. 10. 706 H. Junca, D.H. Pieper / Journal of Microbiological Methods 55 (2003) 697–708 a complex template mixture containing a 10-fold Gallegos, Prof. Dr. M. Tsuda, Dr. A. Kitayama, Dr. R. excess of the P. putida CF600 C23O gene, (Fig. 5C; Bosch, Prof. Y. Kim, Prof. V. Shingler, Dr. D. Young, lanes 19, 20, and 26). These results show the potential and Prof. D. Kunz for kindly providing the reference use of AFDRA to rapidly determine the predominant strains used in this work. We also thank Dr. Andreas gene polymorphism in complex environmental DNA Felske for critical reading of the manuscript and mixtures. When AFDRA was applied to PCR products Robert Witzig for soil DNA quantification. This work obtained from 200 pg of soil DNA, characteristic was supported by grant QLK3-CT-2000-00731 from patterns were observed. When PCR-amplified DNA the European Community. extracted from the capillary fringe zone was analysed (lane 21), a characteristic pattern identical to that obtained from P. putida AN10 (lane 25) or group A References isolates was observed. This result perfectly matches with the information obtained by screening of a PCR Amann, R.I., Ludwig, W., Schleifer, K.H., 1995. Phylogenetic iden- clone library constructed with a 527-bp C23O frag- tification and in situ detection of individual microbial cells with- out cultivation. Microbiol. Rev. 59, 143 – 169. ment amplified from this soil sample (Junca and Arenghi, F.L., Berlanda, D., Galli, E., Sello, G., Barbieri, P., 2001. Pieper, unpublished data). The C23O gene composi- Organization and regulation of meta cleavage pathway genes for tion in the saturated zone differed qualitatively from toluene and o-xylene derivative degradation in Pseudomonas that of the capillary fringe horizon (Fig. 5C; lane 23), stutzeri OX1. Appl. Environ. Microbiol. 67, 3304 – 3308. evidencing the coexistence of two most abundant Bakermans, C., Madsen, E.L., 2002. Diversity of 16S rDNA and C23O polymorphisms: one closely related to group naphthalene dioxygenase genes from coal – tar – waste – contami- nated aquifer waters. Microb. Ecol. 44, 95 – 106. C isolates and another equal to that obtained from P. Bartilson, M., Shingler, V., 1989. Nucleotide sequence and expres- putida AN10 –group A isolates. sion of the catechol 2,3-dioxygenase-encoding gene of phenol- catabolizing Pseudomonas CF600. Gene 85, 233 – 238. 3.6. Concluding remarks Benjamin, R.C., Voss, J.A., Kunz, D.A., 1991. Nucleotide sequence of xylE from the TOL pDK1 plasmid and structural comparison with isofunctional catechol-2,3-dioxygenase genes from TOL, In this report, we demonstrated that the ARDRA pWW0 and NAH7. J. Bacteriol. 173, 2724 – 2728. approach can be transferred to assess functional di- Bosch, R., Garcia-Valdes, E., Moore, E.R., 2000. Complete nucleo- versity of environmental isolates in a very convenient tide sequence and evolutionary significance of a chromosomally first selection providing preliminary phylogenetic in- encoded naphthalene-degradation lower pathway from Pseudo- monas stutzeri AN10. Gene 245, 65 – 74. formation, for further focused and dedicated biochem- Braker, G., Zhou, J., Wu, L., Devol, A.H., Tiedje, J.M., 2000. ical characterization, point mutation determinations or Nitrite reductase genes (nirK and nirS) as functional markers sequencing work, instead of random or massive to investigate diversity of denitrifying bacteria in Pacific north- screenings. The method proved to be also appropriate, west marine sediment communities. Appl. Environ. Microbiol. coupled to a quantitative survey, to assess predomi- 66, 2096 – 2104. nance of gene variant(s) in DNA extracted directly Buchan, A., Neidle, E.L., Moran, M.A., 2001. Diversity of the ring- cleaving dioxygenase gene pcaH in a salt marsh bacterial com- from environmental samples. Future works will de- munity. Appl. Environ. Microbiol. 67, 5801 – 5809. termine if the technique can be optimised to refine its Carrington, B., Lowe, A., Shaw, L.E., Williams, P.A., 1994. The phylogenetic resolution power, by additional testing lower pathway operon for benzoate catabolism in biphenyl-uti- of multiple restrictions of a single PCR fragment lizing Pseudomonas sp. strain IC and the nucleotide sequence of (Porteous et al., 2002) or by integrated comparisons the bphE gene for catechol 2,3-dioxygenase. Microbiology 140, 499 – 508. of profiles obtained in independent restrictions with Davison, J., 1999. Genetic exchange between bacteria in the envi- different enzymes (Sikorski et al., 2002). ronment. Plasmid 42, 73 – 91. Duarte, G.F., Rosado, A.S., Seldin, L., de Araujo, W., van Elsas, J.D., 2001. 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