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Fig. S1. Evolutionary relationships of members of the enolase superfamily indicating misannotations present in public databases. According to the documented substrate specificity of representative members, cluster-1 comprises muconate cycloisomerases of Proteobacteria, cluster-2 chloromuconate cycloisomerases of Proteobacteria and cluster-3 muconate and chloromuconate cycloisomerases of Actinobacteria. TfdDII chloromuconate cycloisomerase of R. eutropha JMP134 (AAC44729) is related to other (chloro)muconate cycloisomerases. Enzymes outside of these clusters are highly unprobable to encode (chloro)muconate cycloisomerases. Based on the documented substrate specificity of proteins indicated by a black dot, the following clusters may be defined: cluster-4, enzymes related to L-Ala-D/L-Glu epimerase of Bacillus subtilis 168 (NP_389181); cluster-5, enzymes related to o-succinylbenzoate synthase of Bacillus subtilis 168 (CAB115056); cluster-6, enzymes related to mandelate racemase of Pseudomonas putida (AAA25887); cluster-7, enzymes related to L-Ala-D/L-Glu epimerase of Escherichia coli K-12 MG1655 (1JPD) and cluster-8, enzymes related o-succinylbenzoate synthase of Escherichia coli K-12 MG1655 (AAC75321). Enzymes previously assumed as indicating a catechol metabolic potential and annotated as (chloro)muconate cycloisomerases (Liang et al., 2009; 2011) are indicated by red squares.

Fig. S2. Normalized signal intensities of spots targeting the internal positive control plasmid carrying four distinct 50 mer oligonucleotide sequences in one (50 mer A), two (50 mer B), four (50 mer C) and eight copies (50 mer D) of the microarrays shown in Fig. 2. Experiments where a coefficient of determination R2 > 0.9 were obtained are labelled in light blue.

Fig. S3. (A) Correlation between the fluorescence intensity of spots on a microarray and the % of sequence identity with the target genome analysed for B. xenovorans LB400, P. putida F1, C. nercator JMP 134 and S. wittichii RW1 applying optimized experimental conditions. (B) The normalized signal intensities of spots targeting the internal positive control plasmid carrying four distinct 50 mer oligonucleotide sequences in one (50 mer A), two (50 mer B), four (50 mer C) and eight copies (50 mer D) on the IPC, which had been added in an amount of 3 pg to 100 ng genomic DNA are shown below each hybridization profile. The means of normalized intensities of three replicates are presented and the error bars represent SD.

Fig. S4. Normalized signal intensities of spots targeting the internal positive control plasmid carrying four distinct 50 mer oligonucleotide sequences in one (50 mer A), two (50 mer B), four (50 mer C) and eight copies (50 mer D). Experiments were performed in groundwater W14 and groundwater W16, in microcosms that did not receive additional carbon source (control) and in microcosms where naphthalene was added as a carbon source (naphthalene). The normalized intensity (NI) is given where an intensity of 1 corresponds to the mean fluorescence generated by a single internal control 50 mer when 3 pg of IPC had been added to 100 ng of DNA template or 6.7 pg of IPC had been added to cDNA generated from 2 μg of total RNA. The means of normalized intensities of three replicates are presented and the error bars represent SD.

Fig. S5. (A) Taxonomic distribution of the 16S rRNA gene sequences where probes have been printed on the microarray. 16S rRNA gene sequences representing probes against phyla where no hybridization was observed under the experimental conditions (Aquifex pyrophilus, Aquificae; Fibrobacter succinogenes, Fibrobacteres; Thermotoga maritima, Thermotogae; Thermodesulfobacterium thermophilum, Thermodesulfobacteria; Fusobacterium nucleatum, Fusobacteria; Gemmatimonas aurantiaca, Gemmatimonadetes; Chlorobium luteolum, Chlorobi; Deferribacter desulfuricans,Deferribacteres; Chrysiogenes arsenatis, Chrysiogenes; Verrumicrobium spinosum, Verrumicrobiota; Mycoplasma genitalum, Tenericutes; Dictyoglomas turgidum, Dictyoglomi; Planctomyces brasiliensis, Planctomycetes; Nitrospira moscoviensis, Nitrospira; Chloroflexus aurantiacus, Chloroflexi; Leptospira borgpetersenii, Borrelia afzelii, Treponema socranskii, Spirochaeta americana, Spirochaetes) are not shown. Cluster-1, γ-Proteobacteria; cluster-2, β-Proteobacteria; cluster-3, α-Proteobacteria; cluster-4, δ-Proteobacteria; cluster-5, e-Proteobacteria; cluster-6, Bacteroidetes; cluster-7, Cyanobacteria; cluster-8, Clostridia; cluster-9, Bacilli; cluster-10, Deinococcus-Thermus; cluster-11, Acidobacteria; cluster-12, Actinobacteria. Abbreviations: Enb, Enterobacter; Kle, Klebsiella; Ser, Serratia; Sal, Salmonella; Shi, Shigella; Esc, Escherichia; Erw, Erwinia; Yer, Yersinia; Pro, Proteus; Vib, Vibrio; Ali, Alilvibrio; Pas, Pasteurella; Acb, Actinobacillus; Aer, Aeromonas; She, Shewanella; Alt, Alteromonas; Thi, Thiomicrospira; Leu, Leucothrix; Hal, Halomonas; Alv, Alcanivorax; Aci, Acinetobacter; Oce, Oceanospirillum; Mar, Marinomonas; Ole, Oleispira; Pse, Pseudomonas; Car, Cardiobacterium; Mec, Methylococcus; Chr, Chromatium; Rsi, Rickettsiella; Xan, Xanthomonas; Ste, Stenotrophomonas; Hyd, Hydrogenophilus; Nei, Neisseria; Tha, Thauera; Nim, Nitrosomonas; Nis, Nitrosospira; Mep, Methylophilus; Alc, Alcaligenes; Bor, Bordetella; Bur, Burkholderia; Ral, Ralstonia; Cup, Cupriavidus; Lep, Leptothrix; Del, Delftia; Com, Comamonas; Acv, Acidovorax; Rhs, Rhodospirillum; Pic, Rickettsia; Sph, Sphingomonas; Pal, Parvularcula; Cau, Caulobacter; Rhi, Rhizobium; Pac, Paracoccus; Ant, Antarctobacter; Ros, Roseobacter; Deb, Desulfobacter; Dev, Desulfovibrio; Myx, Myxococcus; Syn, Syntrophobacter; Geo, Geobacter; Peb, Pelobacter; Dem, Desulfuromonas; Der, Dermocarpella; Hel., Helicobacter; Cam, Campylobacter; Fla, Flavobacterium; Cel, Cellulophaga; Bao, Bacteroides; Por, Porphyromonas; Spb, Sphingobacterium; Chi, Chitinophaga; Flt, Flexithrix; Flb, Flexibacter; Osc, Oscillatoria; Ana, Anabaena; Der, Desulfurella; Prc, Prochlorococcus; Pem, Pelotomaculum; Des, Desulfosporosinus; Spo, Sporomusa; Clo, Clostridium; Pae, Paenibacillus; Bac, Bacillus; Str, Streptococcus; Lac, Lactococcus; Lab, Lactobacillus; Enc, Enterococcus; Sta, Staphylococcus; Dei, Deinococcus; The, Thermus; Abm, Acidobacterium; Prp, Propionicella; Stm, Streptomyces; Mic, Micrococcus; Art, Arthrobacter; Acm, Actinomyces; Cor, Corynebacterium; Die, Dietzia; Myc, Mycobacterium; Noc, Nocardia; Rho, Rhodococcus. (B) Abundance of respective 16S rDNA or 16S rRNA is given as normalized intensity. The abundance in control microcosms which did not receive additional carbon source are given in darker shades of colour, whereas abundance in microcosms where naphthalene was added is given in lighter shades of colour. The normalized intensity (NI) is given where an intensity of 1 corresponds to the mean fluorescence generated by a single internal control 50 mer when 3 pg of IPC had been added to 100 ng of DNA template or 6.7 pg of IPC had been added to cDNA generated from 2 μg of total RNA. The means of normalized intensities of three replicates are presented and the error bars represent SD.

Fig. S6. (A) Evolutionary relationships of intradiol dioxygenases. All genes encoding those proteins were targeted by probes printed on the microarray. Cluster-1, catechol 1,2-dioxygenases of Proteobacteria; cluster-2, catechol 1,2-dioxygenases of Actinobacteria; cluster-3, chlorocatechol 1,2-dioxygenases; cluster-4, hydroxyhydroquinol 1,2-dioxygenases; cluster-5 and -6, α- and β-subunits of protocatechuate 3,4-dioxygenases respectively. (B) Abundance of genes or transcripts is given as normalized intensity. The abundance in control microcosms, which did not receive additional carbon are given in darker shades of colour, whereas abundance in microcosms where naphthalene was added as a carbon source are given in lighter shades of colour. The normalized intensity (NI) is given where an intensity of 1 corresponds to the mean fluorescence generated by a single internal control 50 mer when 3 pg of IPC had been added to 100 ng of DNA template or 6.7 pg of IPC had been added to cDNA generated from 2 μg of total RNA. The means of normalized intensities of three replicates are presented and the error bars represent SD.

Fig. S7. (A) Evolutionary relationships of muconate/chloromuconate cycloisomerases. All genes encoding those proteins were targeted by probes printed on the microarray. Cluster-1, muconate cycloisomerases; cluster-2, chloromuconate cycloisomerases. (B) Abundance of genes or transcripts is given as normalized intensity. The abundance in control microcosms, which did not receive additional carbon source, are given in darker shades of colour, whereas abundance in microcosms where naphthalene was added as a carbon source are given in lighter shades of colour. The normalized intensity (NI) is given where an intensity of 1 corresponds to the mean fluorescence generated by a single internal control 50 mer when 3 pg of IPC had been added to 100 ng of DNA template or 6.7 pg of IPC had been added to cDNA generated from 2 μg of total RNA. The means of normalized intensities of the three replicates are presented and the error bars represent SD.

Fig. S8. (A) Evolutionary relationships of maleylacetate reductases. All genes encoding those proteins were targeted by probes printed on the microarray. Cluster-1, maleylacetate reductases involved in hydroxyhydroquinol degradation by β-Proteobacteria and related enzymes; clusters 2, 5 and 7, maleylacetate reductases involved in chlorocatechol degradation by Proteobacteria; cluster-3, maleylacetate reductases involved in pentachlorophenol or lindane degradation; cluster-4, maleylacetate reductase involved in chlorocatechol degradation by Rhodococcus; cluster-6, maleylacetate reductases involved in hydroxyhydroquinol or sulfocatechol degradation by α-Proteobacteria and related enzymes; cluster-8, maleylacetate reductases involved in hydroxyhydroquinol degradation by Actinobacteria. (B) Abundance of genes or transcripts is given as normalized intensity. The abundance in control microcosms, which did not receive additional carbon source, are given in darker shades of colour, whereas abundance in microcosms where naphthalene was added as a carbon source are given in lighter shades of colour. The normalized intensity (NI) is given where an intensity of 1 corresponds to the mean fluorescence generated by a single internal control 50 mer when 3 pg of IPC had been added to 100 ng of DNA template or 6.7 pg of IPC had been added to cDNA generated from 2 μg of total RNA. The means of normalized intensities of three replicates are presented and the error bars represent SD.

Fig. S9. (A) Evolutionary relationships of integral-membrane alkane hydroxylases. All genes encoding those proteins were targeted by probes printed on the microarray. Cluster-1, alkane hydroxylases of Actinobacteria related to AlkB1 and AlkB2 of Rhodococcus sp. Q15 (Whyte et al., 2002); cluster-2, alkane hydroxylases of Proteobacteria related to AlkB of Pseudomonas fluorescens Pf-5, cluster-3, alkane hydroxylases related to Mvan_3100 of Mycobacterium vanbaalenii PYR-1, cluster-4, alkane hydroxylases of Actinobacteria related to AlkB3 of Rhodococcus sp. Q15; cluster-5, alkane hydroxylases of Acinetobacter spp., cluster-6, alkane hydroxylases related to AlkB of Alkanivorax borkumensis SK2; cluster-7, alkane hydroxylases of Proteobacteria related to AlkB1 and AlkB2 of Pseudomonas aeruginosa PAO1 and cluster-8, p-cymene and xylene monooxygenases. (B) Abundance of genes or transcripts is given as normalized intensity. The abundance in control microcosms, which did not receive additional carbon source, are given in darker shades of colour, whereas abundance in microcosms where naphthalene was added as a carbon source are given in lighter shades of colour. The normalized intensity (NI) is given where an intensity of 1 corresponds to the mean fluorescence generated by a single internal control 50 mer when 3 pg of IPC had been added to 100 ng of DNA template or 6.7 pg of IPC had been added to cDNA generated from 2 μg of total RNA. The means of normalized intensities of three replicates are presented and the error bars represent SD.

Fig. S10. (A) Evolutionary relationships of cytochrome P450 alkane hydroxylases (CYP153). All genes encoding those proteins were targeted by probes printed on the microarray. Cluster-1, CYP153 alkane hydroxylases related to AphG of Mycobacterium sp. HXN1500; cluster-2, CYP153 alkane hydroxylases related to ABO_0201 of Alcanivorax borkumensis SK2; cluster-3, CYP153 alkane hydroxylases related to AhpG4 of Sphingopyxis macrogoltabida (CAH61456). (B) Abundance of genes or transcripts is given as normalized intensity. The abundance in control microcosms which did not receive additional carbon source are given in darker shades of colour, whereas abundance in microcosms where naphthalene was added as a carbon source is given in lighter shades of colour. The normalized intensity (NI) is given where an intensity of 1 corresponds to the mean fluorescence generated by a single internal control 50 mer when 3 pg of IPC had been added to 100 ng of DNA template or 6.7 pg of IPC had been added to cDNA generated from 2 μg of total RNA. The means of normalized intensities of three replicates are presented and the error bars represent SD.

Fig. S11. (A) Evolutionary relationships of benzylsuccinate synthases. All genes encoding those proteins were targeted by probes printed on the microarray. Cluster-1, benzylsuccinate synthases from β-Proteobacteria; cluster-2, benzylsuccinate synthases from δ-Proteobacteria; cluster-3, benzylsuccinate synthases from methanogenic consortia; cluster-4, unidentified BSSA-homologues. (B) Abundance of genes or transcripts is given as normalized intensity. The abundance in control microcosms, which did not receive additional carbon source, is given in darker shades of colour, whereas abundance in microcosms where naphthalene was added as a carbon source is given in lighter shades of colour. The normalized intensity (NI) is given where an intensity of 1 corresponds to the mean fluorescence generated by a single internal control 50 mer when 3 pg of IPC had been added to 100 ng of DNA template or 6.7 pg of IPC had been added to cDNA generated from 2 μg of total RNA. The means of normalized intensities of three replicates are presented and the error bars represent SD.

Fig. S12. (A) Evolutionary relationships of benzoyl coenzyme A reductases. All genes encoding those proteins were targeted by probes printed on the microarray. Cluster-1, BcrA α-subunits of benzoyl coenzyme A reductases related to that of Thauera aromatica K172, cluster-2, BzdQ α-subunits of benzoyl coenzyme A reductases related to that of Azoarcus sp. CIB; cluster-3, putative activators of 2-hydroxyglutaryl-CoA dehydratases; cluster-4, BzdP δ-subunits of benzoyl coenzyme A reductases related to that of Azoarcus sp. CIB and cluster-5, BcrD δ-subunits of benzoyl coenzyme A reductases related to that of T. aromatica K172. (B) Abundance of genes or transcripts is given as normalized intensity. The abundance in control microcosms, which did not receive additional carbon source, are given in darker shades of colour, whereas abundance in microcosms where naphthalene was added as a carbon source are given in lighter shades of colour. The normalized intensity (NI) is given where an intensity of 1 corresponds to the mean fluorescence generated by a single internal control 50 mer when 3 pg of IPC had been added to 100 ng of DNA template or 6.7 pg of IPC had been added to cDNA generated from 2 μg of total RNA. The means of normalized intensities of three replicates are presented and the error bars represent SD.

Fig. S13. (A) Evolutionary relationships of ferredoxins. All genes encoding those proteins were targeted by probes printed on the microarray. Cluster-1, ferredoxins mediating electron transport in Rieske non-haem iron oxygenase enzyme systems and cluster-2, ferredoxins mediating electron transport in soluble diiron monooxygenase enzyme systems. (B) Abundance of genes or transcripts is given as normalized intensity. The abundance in control microcosms, which did not receive additional carbon source is given in darker shades of colour, whereas abundance in microcosms where naphthalene was added as a carbon source are given in lighter shades of colour. The normalized intensity (NI) is given where an intensity of 1 corresponds to the mean fluorescence generated by a single internal control 50 mer when 3 pg of IPC had been added to 100 ng of DNA template or 6.7 pg of IPC had been added to cDNA generated from 2 μg of total RNA. The means of normalized intensities of three replicates are presented and the error bars represent SD.

Fig. S14. (A) Evolutionary relationships of α-subunits of soluble diiron monooxygenases. All genes encoding those proteins were targeted by probes printed on the microarray. Cluster-1, α-subunits of phenol/methylphenol monooxygenases; cluster-2, α-subunits of alkene monooxygenases; cluster-3, α-subunits of benzene/toluene monooxygenases. (B) Abundance of genes or transcripts is given as normalized intensity. The abundance in control microcosms, which did not receive additional carbon source, is given in darker shades of colour, whereas abundance in microcosms where naphthalene was added as a carbon source are given in lighter shades of colour. The normalized intensity (NI) is given where an intensity of 1 corresponds to the mean fluorescence generated by a single internal control 50 mer when 3 pg of IPC had been added to 100 ng of DNA template or 6.7 pg of IPC had been added to cDNA generated from 2 μg of total RNA. The means of normalized intensities of three replicates are presented and the error bars represent SD.

Fig. S15. Schematic presentation of the features of the pICDNA control plasmid. Specifically, the organization of the 50 mer A, B, C and D are shown.

Fig. S16. Array hybridization image using labelled amplified DNA obtained from the W14 control microcosm. Controls (positives and negatives) were arranged as frames around each of the 16 subgrids forming together the entire microarray.

Fig. S17. Array hybridization image using labelled amplified cDNA obtained from the W16 control microcosm. Controls (positives and negatives) were arranged as frames around each of the 16 subgrids forming together the entire microarray.

Table S1. Proteins which had previously served as targets for microarray studies and which were discussed as being indicative for a catechol or pyrene metabolic potential (Liang et al., 2009; 2011).

Table S2. Detailed information on all probes printed on the array.

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