Supplementary Note, Tables and Figures
Isoeugenol Monooxygenase and Its Putative Regulatory Gene Are Located in the Eugenol Metabolic Gene Cluster in Pseudomonas nitroreducens Jin1 in “Archives of Microbiology”
Ji-Young Ryu1, Jiyoung Seo1, Tatsuya Unno1, Joong-Hoon Ahn3, Tao Yan4, Michael J. Sadowsky5, and Hor-Gil Hur1,2*
1Department of Environmental Science and Engineering; and 2International Environmental Research Center, Gwangju Institute of Science and Technology, Gwangju, 500-712, Korea
3Department of Bioscience and Biotechnology, Konkuk University, Seoul, 143-701, Korea
4Department of Civil and Environmental Engineering, University of Hawaii, Honolulu, Hawaii 96822, USA, and
5Department of Soil, Water, and Climate; and BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, 55108, USA
*Corresponding author: Hor-Gil Hur, Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology, Gwangju, Korea, 500-712, Tel: +82-970-2437, Fax: +82-970-2434, e-mail:
Supplementary Note
Description for ORFs found in P. nitroreducens Jin1
Based on comparison of the deduced amino acid sequence, there are one methyl-accepting chemotaxis protein (ORF 21), and 2 nucleoside-diphosphate-sugar epimerases (ORF 29 and ORF 38). The ORF 29 and ORF 38 have been assigned as nucleoside-diphosphate-sugar epimerses (Table. S2). The amino acid sequence of ORF 29 from P. nitroreducens Jin1 had 40.8% identity to the predicted nucleoside-diphosphate-sugar epimerase from Hahella chejuensis KCTC 2396 (Accession number ABC28150). The deduced amino acid sequence of the ORF 38 from Jin1 showed 69.0% identity and 64.4% identity to the predicted nucleoside-diphosphate-sugar epimerases from Y. mollaretii ATCC 43969 (Accession number ZP_00824666) and to the NmrA family protein from Polaromonas naphthalenivorans CJ2 Accession number ABM35941), respectively. The deduced amino acid sequences of ORF 29 and ORF38 exhibited significant identity to the predicted nucleoside-diphosphate-sugar epimerases from Y. mollaretii ATCC 43969, but the identity of amino acid sequences between ORF 29 and ORF 38 was only 30.1%. The ORFs 10, 17, 18 and 20 have been assigned as putative transcriptional regulators. The primary structure of ORF 10 had 40.8% to the transcriptional regulator from the Sphingobium herbicidovorans MH (Accession number CAF32814). ORF 17 and ORF 20 have been assigned as a putative transcriptional regulator and MarR family transcriptional regulator, respectively (Table. S2). The deduced amino acid sequence of ORF 18 of P. nitroreducens Jin1 revealed 26.9% identity (43.5% positives) and 26.0% identity (43.3% positives) to the TetR family transcriptional regulator from Delftia acidovorans SPH-1 (Accession number ABX35637), and to the probable transcriptional regulator, TetR family from Chromobacterium violaceum ATCC 12472 (Accession number AAQ59511), respectively.
We have also found ORF 8 and ORF 9, which showed 48.3% identity to the Rieske (2Fe-2S) protein from Novosphingobium aromaticivorans DSM 12444 (Accession number YP_496764) and 63.0% identity to the ferredoxin from P. putida GB-1 (Accession number YP_001668406), respectively. The ORF 8 and ORF 9 also exhibited 29.7% and 56.5% identity to the deduced amino acid sequences of ORF 36 and 35 encoding vanillin-O-demethylase, but they had opposite coding direction to ORF 35 and 36. Therefore, ORF 8 and ORF9 might be involved in the eugenol and isoeugenol metabolism as a demethylase which shows a similar function of vanillin-O-demethylase encoded by vanAB.
There are 7 hypothetical proteins (ORF 1, 3, 5, 13, 14, 15, and 31). Their closest similar proteins and identity are listed in Table 3. The deduced amino acid sequence of ORF 6 showed 79.4% identity to the helicase domain-containing protein from Pseudomonas aeruginosa PA7 (Accession number YP_001345476) and 76.2% identity to the type III restriction enzyme from P. aeruginosa PSE (Accession number ABR135150). The primary structure of ORF 7 exhibited 46.5% identity to the phospholipase A2 family protein from Aromatoleum aromaticum EbN1 (Accession number YP_159159). We have found two kinds of formaldehyde dehydrogenases, which are encoded by the ORF 11 and ORF 34 in the 55-kb region of P. nitroreducens Jin1 (Table. S2). The deduced amino acid sequence of ORF 11 showed very high identity (98.5%) to the glutathione-independent formaldehyde dehydrogenase from P. putida strain (BAA04743) (Ito et al. 1994), although DNA sequence identity was only 89.3%. On the other hands, the ORF 34 has been assigned as a putative glutathione-dependent formaldehyde dehydrogenase. An alignment of amino acid sequences between ORF 11 and ORF 34 revealed only about 20% identity, but amino acid residues in active and coenzyme binding sites were conserved in 2 putative formaldehyde dehydrogenases as revealed in the previous work (Ito et al. 1994).
Rest of ORFs, ORF 12, 28, and 33 have been assigned as a putative transport, alcohol dehydrogenase and putative gamma-glutamylcystein synthetase, respectively. The deduced amino acid sequence of the ORF 12 showed 68.6% identity to the putative transport from Polaromonas sp. JS666 (Accession number YP_546996). The amino acid sequences of ORF 5 located between ehyA and ehyB encoding the eugenol hydroxylase of Pseudomonas sp. HR199, which corresponds to ORF 31 of P. nitroreducens Jin1, has been assigned as the hypothetical protein (Priefert et al. 1999) (Table. S2). tap (CAB69494), trp (CAB69492) and trc1 and 2 of Pseudomonas sp. HR199 are considered being a putative transcriptional regulators, which have high amino acid sequence identity to the corresponding putative transcriptional regulators, ORF 17, 20, 18, and 37 of P. nitroreducens Jin1, respectively.
DNA blot analysis for iem
Genomic DNA of P. nitroreducens Jin1 was digested with restriction enzymes, HindIII, SalI, SmaI or XhoI and separated using an agarose gel. The gel was transferred into a Hybond-N+ membrane (Amersham, Piscataway, NJ, USA). For the synthesis of iem and ehyB probe, PCR DIG Probe synthesis kit (Roche, Penzberg, Germany) was used.
For iem primers, 5’-TGTTGTTCTTCGGTTCGGC-3’ and 5’-TTAAGG TCTGGGTACCCAGC-3’ were used, and for ehyB probe, 5’-GTGCCT TGACTCCTTCTTCG-3’ and 5’-TGCTCTTCTGTGCCGTAAAG-3’ were used. Hybridization was done with DIG Easy Hyb kit (Roche) according to the manufacture’s instruction.
References
Ito K, Takahashi M, Yoshimoto T, Tsuru D (1994) Cloning and high-level expression of the glutathione-independent formaldehyde dehydrogenase gene from Pseudomonas putida. J. Bacteriol. 176:2483-2491
Priefert H, Overhage J, Steinbuchel A (1999) Identification and molecular characterization of the eugenol hydroxylase genes (ehyA/ehyB) of Pseudomonas sp. strain HR199. Arch. Microbiol. 172:354-363
Supplementary Tables and Figures
Table S1. The list of primers for real-time PCR analysis
Primer name / Sequence (5’ à 3’) / PCR product size (bp)ehyA-F / TCT GCT TCA GAG AAT GCA GG / 135
ehyA-R / AGC CTA CTG TCA ACA ATG GC
calA-F / TCC TTA TGC AAC TGA AGG CG / 139
calA-R / CGT CGC AAA CGT GAA CTA TC
calB-F / AGA CCA GCA TTT CAT CCG AC / 138
calB-R / ATC GCT ACC TGA CTG ATG C
fcs-F / CAA ATC TTG CGA CAG CAG TG / 144
fcs-R / CTT ACG GAC TAT CGG CAG AG
ech-F / ATG TAG TAA AGG GAC TCG CG / 132
ech-R / CTG TGA TCT GGC CAT CTG TG
aat-F / GCA GTT CAA TTG CTC GAC TG / 140
aat-R / AGA AGC TGA TTC CTA TGC GG
vdh-F / TGA AGT TCA CTC GAC GTA CC / 145
vdh-R / TTT ACC CAT CGC CTG ATT GG
iemR-F / CAT TTT CCG TAG AGC CAC ACC / 122
iemR-R / CAT TGC GCA TCG ACG ATT TG
iem-F / TGT TGT TCT TCG GTT CGG C / 144
iem-R / TTC GGG TAA TGG CAA AGT CG
F and R indicate a forward primer and a reverse primer, respectively.
13
Table S2. Homology analyses of amino acid sequences for 38 open reading frames (ORFs) found in cluster containing eugenol and isoeugenol metabolism of P. nitroreducens Jin1
# of ORF / Number of amino acid / Closest similar protein (gene name) * / Amino acid identity / Accession number§%† / Number of aa‡
ORF 1 / 781 / Hypothetical protein PSPTO_5632 / 30.6 / 772 / YP_001621410
ORF 2 / 694 / Site-specific recombinase phage integrase family protein / 37.7 / 698 / NP_794482
ORF 3 / 204 / Hypothetical protein Mmwyl1_4221 / 27.1 / 202 / YP_001343051
ORF 4 / 488 / Phage integrase family protein / 37.3 / 475 / YP_001343050
ORF 5 / 192 / Hypothetical protein / 68.9 / 192 / ABR13514
ORF 6 / 463 / Helicase domain-containing protein / 79.4 / 466 / YP_001345476
ORF 7 / 645 / Phospholipase A2 family protein / 46.5 / 626 / YP_159159
ORF 8 / 353 / Rieske (2Fe-2S) protein / 48.3 / 351 / YP_496764
ORF 9 / 317 / Ferredoxin / 63.0 / 316 / YP_001668406
ORF 10 / 325 / Transcriptional regulator / 40.8 / 324 / CAF32814
ORF 11 / 399 / Glutathione-independent formaldehyde dehydrogenase / 98.5 / 399 / BAA04743
ORF 12 / 806 / Putative transport / 68.6 / 808 / YP_546996
ORF 13 / 368 / Hypothetical protein / 48.0 / 360 / BAD11010
ORF 14 / 453 / Hypothetical protein Bpro_0131 / 51.6 / 458 / YP_546994
ORF 15 / 548 / Hypothetical protein / 66.3 / 552 / BAD11008
ORF 16 / 255 / Coniferyl alcohol dehydrogenase (calA) / 100 / 255 / CAB69495¶
ORF 17 / 609 / Transcriptions-activator-protein (tap) / 95.1 / 609 / CAB69494
ORF 18 / 223 / TetR family transcriptional regulator (trc1) / 100 / 192 / ABX35637
ORF 19 / 480 / Coniferyl aldehyde dehydrogenase (calB) / 99.0 / 480 / CAB69493
ORF 20 / 177 / Transcriptions-regulator-protein (trp) / 96.3 / 136 / CAB69492
ORF 21 / 532 / Methyl-accepting chemotaxis protein (mcp) / 94.4 / 532 / CAB69491
ORF 22 / 422 / Beta-ketothiolase (aat) / 94.1 / 431 / CAB69490
ORF 23 / 630 / Feruloyl-CoA-synthetase (fcs) / 99.2 / 589 / CAB69489
ORF 24 / 482 / Vanillin dehydrogenase (vdh) / 98.8 / 481 / CAB69488
ORF 25 / 276 / Enoyl-CoA-hydratase/aldolase (ech) / 99.6 / 244 / CAB69487
ORF 26 / 316 / Transcriptional regulator (iemR) / 97.5 / 316 / CAB69486
ORF 27 / 478 / Isoeugenol monooxygenase (iem) / 97.9 / 505 / CAB69485
ORF 28 / 336 / Alcohol dehydrogenase, zinc-binding domain protein (adh) / 99.4 / 336 / CAB69484
ORF 29 / 286 / Predicted nucleoside-diphosphate-sugar epimerase (ndse) / 97.2 / 286 / CAB69483
ORF 30 / 517 / Eugenol hydroxylase flavoprotein subunit (ehyB) / 99.8 / 517 / CAB69482
ORF 31 / 228 / Hypothetical protein / 98.2 / 228 / CAB69481
ORF 32 / 117 / Eugenol hydroxylase cytochrome C subunit precursor (ehyA) / 98.3 / 117 / CAB69480
ORF 33 / 545 / Putative gamma-glutamylcysteine synthetase (gcs) / 95.2 / 545 / CAB69479
ORF 34 / 372 / Putative formaldehyde dehydrogenase, glutathione-dependent (fdh) / 98.9 / 372 / CAB69478
ORF 35 / 317 / Vanillate-O-demethylase oxidoreductase, reductase subunit (vanB) / 98.7 / 317 / CAB69477
ORF 36 / 354 / Vanillate-O-demethylase oxidoreductase a-subunit (vanA) / 98.9 / 354 / CAB69476
ORF 37 / 236 / Transcriptional regulator (vanR) / 82.2 / 191 / CAB64602
ORF 38 / 284 / Predicted nucleoside-diphosphate-sugar epimerase (ndse) / 97.5 / 284 / CAB69475
*Closest similar proteins of each ORF were obtained using the PSI- BLAST algorithm or the protein sequence from Pseudomonas sp. HR199 in the patent EP0845532.
† The percentages of identity were obtained from comparison of aligned regions of amino acid sequences of proteins. Deduced amino acid sequences of ORF 1- 15 were compared to closest similar proteins collected from GenBank. Deduced amino acid sequences of ORF 16 - 38 were compared to corresponding amino acid sequences of Pseudomonas sp. HR199 in the patent EP0845532.
‡ Number indicates amino acid sequence size of closest similar protein
§Number indicates accession number of closest similar proteins collected from the GenBank
¶Accession numbers starting with CAB694 indicate amino acid sequences of Pseudomonas sp. HR199 in the patent EP0845532.
13
FIG. S1.
Fig. S1. Image of Southern blotting for iem and ehyB genes to know copy number.
FIG. S2.
Fig. S2 Nucleotide and deduced amino acid sequences of iemR and iem of P. nitroreducens Jin1. The putative Shine-Dalgarno (S/D) sequences of iemR (lower strand) and iem (upper strand) are boxed. The translational termination sites are indicated by three star marks. The positions of putative hairpin-like structures downstream of iemR and iem are indicated by inverted arrows.
Fig. S3
FIG. S3 Homologies of the transcriptional regulator IemR (a) and isoeugenol monooxygenase (Iem) (b) of P. nitroreducens Jin1 with other bacterial transcriptional regulators, and bacterial oxygenases, respectively. (a); The amino acid sequences of ORF 3 from Pseudomonas sp. HR199 (Accession number CAB69486), the probable transcriptional regulator from P. putida IE27(Accession number BAF62887), were aligned to the IemR of P. nitroreducens Jin1 deduced from iemR, and (b); The amino acid sequences of lignostilbene-dioxygenase from Pseudomonas sp. HR199 (HR199_LSD) (Accession number CAB69485), isoeugenol monooxygenase from P. putida IE27 (IE27_Iso), lignostilbene-α,β-dioxygenase isozymes I from Sphingomonas paucimobilis TMY1009 (TMY1009_LSD-I) (Accession number AAC60447), were aligned to the isoeugenol monooxygenase of P. nitroreducens Jin1 (Jin1_Iem) deduced from iem. Gaps (-) are introduced into the sequences to improve an alignment. White color letters on black background indicate identical amino acid residues. Conservative amino acid residues are specified by white color letters on dark gray background. Black color letters on gray background indicate block of similar residues. Weakly similar residues are specified by black color letters on light gray background.
13