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Molecular genetic investigation of the abietane diterpenoid degradation pathway of pseudomonas abietaniphila… Martin, Vincent J.J. 1999

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MOLECULAR GENETIC INVESTIGATION OF THE ABIETANE DITERPENOID DEGRADATION PATHWAY OF PSEUDOMONAS ABIETANIPHILA BKME-9 by VINCENT J.J. MARTIN B.Sc, McGill University, 1989 M.Sc, University of Guelph, 1993 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1999 © Vincent J.J. Martin » 1999 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of fa\Q](k)'Q>)QjjO&j { ^yyiUKlpLoO-y The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date ABSTRACT Pseudomonas abietaniphila BKME-9 is able to degrade abietane diterpenoids, via a dioxygenolytic pathway. Tn5 transposon mutagenesis and inverse PCR were used to clone and sequence the dit gene cluster which encodes enzymes and a transcription regulator of the catabolic pathway for abietane diterpenoid degradation in P. abietaniphila BKME-9. The gene cluster is located on a 16.7 kb EcoRI-EcoRI DNA fragment containing 13 complete and 1 partial open reading frames (ORFs). The genes ditAl ditA2 and ditA3 encode the a and p subunits and the ferredoxin of a new class of ring-hydroxylating dioxygenases. Sequence analysis of the ferredoxin indicated that it is likely to be a [4Fe-4S]- or [3Fe-4S]-type ferredoxin, and not a [2Fe-2S]-type ferredoxin, as found in all previously described ring-hydroxylating dioxygenases. Expression in E. coli of ditAl A2 and dit A3, resulted in a functional enzyme. The diterpenoid dioxygenase attacks 7-oxodehydroabietic acid but not dehydroabietic acid (DhA), at C-l 1 and C-12, producing 7-oxo-ll,12-dihydroxy-8,13-abietadien acid. The dioxygenase mutant strain BKME-941 {ditA l:\ln5) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxoDhA in cell suspensions assays, demonstrating that the dioxygenase is essential and central to the abietane catabolic pathway. Using xylE reporter gene transcriptional fusions, it was shown that abietic, dehydroabietic, 7-oxodehydroabietic, isopimaric and 12,14-dichlorodehydroabietic acids induce the expression of ditAl and ditA3. In addition to the aromatic ring-hydroxylating dioxygenase genes, the dit cluster encodes the extradiol ring cleavage dioxygenase ditC and the IclR-type transcriptional regulator ditR. The ditC gene is required for the growth of P. abietaniphila on abietanes. Cell suspensions of the ditC mutant strain produced a yellow colored supernatant and accumulated several DhA metabolites. Although ditR is not required for the growth of strain BKME-9 on abietanes, catechol-2,3-dioxygenase activity of xylE ii reporter strains with a dilR.Km* mutation demonstrated that it encodes a transcriptional activator of ditA3 and possibly a repressor of ditAl. A second Tn5 transposon mutant with an insertion in a gene with similarity to reductases of cytochrome P-450 and alkane hydroxylases was identified. However, further experiments aimed at confirming the presence of a cytochrome P-450 in P. abietaniphila provided inconclusive results. iii TABLE OF CONTENTS ABSTRACT » TABLE OF CONTENTS iv LIST OF FIGURES vi LIST OF TABLES viii ABBREVIATIONS AND SYMBOLS ix ACKNOWLEDGMENTS xi INTRODUCTION 1 1. What are resin acids? 1 2. The ecology and diversity of resin acid-degrading bacteria 3 3. Resin acid substrate specificity of aerobic bacteria 5 4. Aerobic pathways for abietane degradation 8 5. Transformation of resin acids in anaerobic environments 12 6. Fungal hydroxylations of resin acids 15 7. Organization of genes for aerobic degradation of aromatic compounds 18 8. Multicomponent ring-hydroxylating dioxygenases 21 THESIS OBJECTIVES 25 MATERIALS AND METHODS 1. Bacterial strains and culture conditions..... 26 2. Tn5 mutagenesis and inverse PCR 26 3. Phenotypic characterization of dit mutant strains 29 4. Biotransformation of diterpenoid by resting cells assays 31 5. Genomic library: Construction and screening 31 6. DNA manipulation and sequencing 32 7. DNA sequence analysis 34 8. Insertional inactivation and construction of transcriptional fusions of dit genes 34 9. Expression of diterpenoid dioxygenase in E. coli 35 10. Purification and identification of the dihydrodiol 36 11. Catechol 2,3-dioxygenase (C230) assays 37 12. Cytochrome P-450 assays 38 13. SDS-PAGE gels and heme-staining 38 iv RESULTS CHAPTER ONE: A gene cluster encoding abietane diterpenoid degradation activity 1. Introduction 39 2. Tn5 mutagenesis, inverse PCR and isolation of cosmid clones 39 3. Cloning and subcloning of the dit gene cluster 43 4. Genetic organization of the dit gene cluster and classification of mutants 44 5. The extradiol ring-cleavage enzyme DitC 47 6. dill) and dilH 50 7. ditB, ditGmd ditl 51 8. dilE and ORF2 52 9. ditF and ORF1 52 CHAPTER TWO: Characterization of a novel aromatic ring-hydroxylating dioxygenase 1. Introduction 56 2. Identification and sequence analysis of the oxygenase genes ditAl and ditA2 57 3. Identification and sequence analysis of the ferredoxin gene ditA3 59 4. Phenotypic characterization of the ferredoxin (ditAS) mutant BKME-91 61 5. Oxidation of 7-oxoDhA by recombinant diterpenoid dioxygenase expressed in E. coli 63 6. Purification and identification of dioxygenase oxidation product 65 7. Evidence of a convergent pathway for abietane degradation 66 8. Analysis of ditAl and dit A3 gene expression and inducer specificity 69 9. Regulation of ditAl and ditA3 expression by DitR 72 CHAPTER THREE: Diterpenoid inducible cytochrome(s) from P. abietaniphila BKME-9 1. Introduction 75 2. Phenotypic characterization of P. abietaniphila Tn5 mutant strain BKME-12 76 3. Inverse PCR product from Tn5 mutant strain BKME-12 768 4. Cytochrome P-450 assays of diterpenoid-induced P. abietaniphila BKME-9 80 5. Identification of a soluble, diterpenoid inducible, heme-containing protein 81 DISCUSSION 84 CONCLUSIONS 100 FUTURE WORK 101 APPENDIX I 1. Overexpression and partial purification of the oxygenase (DitAl A2) component of the diterpenoid dioxygenase Dit A 102 2. Analysis of ditF expression 106 APPENDIX II GenBank submission data 108 APPENDIX III Mass spectra of DhA intermediates 118 REFERENCES 124 v LIST OF FIGURES Fig. 1 Chemical structure of resin acids 2 Fig.2 16S-rDNA phylogenetic tree of resin acid-degrading bacteria 6 Fig. 3 Synthesis of a proposed biochemical pathway for DhA and AbA degradation... 10 Fig. 4 Proposed anaerobic transformation pathways of DhA, AbA and PiA 13 Fig. 5 Summary of hydroxylation reactions achieved by fungi cometabolizing DhA.... 16 Fig. 6 Extradiol and intradiol dioxygenolytic pathways 19 Fig. 7 Comparison of catabolic operons encoding extradiol aromatic degradation pathways 20 Fig. 8 Classification of multicomponent ring-hydroxylating dioxygenases 22 Fig. 9 Organization of multicomponent ring-hydroxylating dioxygenases 23 Fig. 10 Tn5 transposon showing the regions of probe hybridization and the strategy for IPCR. amplification 30 Fig. 11 Physical map of the dit gene cluster indicating fragments cloned from the P. abietaniphila BKME-9 gene library cosmid clone pLC 12 33 Fig. 12 Southern blot analysis of Tn5 insertion and agarose gel electrophoresis of IPCR products from mutant BKME-941 DNA 41 Fig. 13 Proposed pathway for abietane diterpenoid degradation in P. abietaniphila BKME-9 49 Fig. 14 Hydropathy plot of the deduced amino acid sequence of ditF gene product 54 Fig. 15 Alignment of protein sequences of [2Fe-2S] cluster binding domains of a subunits of several classes of dioxygenases 58 Fig 16 Comparison of the ferredoxin, DitA3, to the most similar 4Fe-4S and 3Fe-4S ferredoxins found in the GenBank database 60 Fig. 17 Absorption spectra of 7-oxoDhA and supernatants from cell suspensions incubated with DhA for strains BKME-9, BKME-941 and BKME-91 62 Fig. 18 Removal of 7-oxoDhA and production of metabolites by cell suspensions of E. coli XL1 Blue MR expressing the ring-hydroxylating dioxygenase Dit A 64 Fig. 19 Absorption spectra of 7-oxoDhA and the product of the dioxygenase, 7-oxo-11,12-dihydroxy-8,13 -abietadien acid 67 Fig. 20 GC-FID analysis of the DhA, AbA and PaA biotransformation products of P. abietaniphila BKME-941 {ditAI:: Tn5) 68 Fig. 21 Expression of ditAl and dit A3 in response to various diterpenoids and aromatic compounds 70 Fig. 22 Expression of ditAl and dit A3 in ditR mutant strains BKME-922 and BKME-912 and in ditR mutant strains complemented with pVM220 71 Fig. 23 Physical map of a EcoRV-BgUl fragment containing ditR cloned into pVM220 and nucleotide sequence of three intergenic region between ditE and ditR 74 Fig. 24 DhA transformation products of Tn5 mutant P. abietaniphila BKME-12 77 Fig. 25 Southern blot analysis of Tn5 insertion and agarose gel electrophoresis of IPCR products from mutant strain BKME-12 DNA 79 Fig. 26 Absorption spectra of the soluble fraction of pyruvate- and DhA-grown cultures of wild-type P. abietaniphila 82 Fig. 27 Heme-stained SDS-PAGE gel of the soluble fraction of pyruvate- and DhA-grown cultures of wild-type P. abietaniphila 83 vi Fig. 28 Hypothetical pathway for the extradiol ring cleavage of 7-oxoDhAdiol 86 Fig. 29 The classification of a-subunits from ring-hydroxylating dioxygenases based on the multiple alignment of related proteins 88 Fig. 30 Phylogenetic tree of IclR-type transcription regulators 93 Fig. 31 SDS-P AGE of the partially purified a and P subunits of the dioxygenase Dit A.. 104 Fig. 32 Analysis of the expression of ditF 107 vii LIST OF TABLES Table I Summary of resin acid-metabolizing microorganisms previously identified Table II Bacterial strains and plasmids used in this study Table III Phenotypic characterization of dit mutant strains Table IV Pairwise sequence comparison of deduced amino acid sequences from the dit gene cluster with those of similar proteins Table V Percent similarity of deduced amino acid sequence of IPCR product from mutant BKME-12 with those of similar proteins viii ABBREVIATIONS AND SYMBOLS a.a. amino acid AbA abietic acid bp base pair BpH biphenyl °C degrees Celsius C230 catechol-2,3-dioxygenase Ci Curie Cl2DhA dichlorodehydroabietic acid cm centimeters CV column volumes dCTP deoxycytidine 5'-triphosphate DDT 1,1,1 -trichloro-2,2-bis(p-chlorophenyl)ethane DhA dehydroabietic acid 2,3-DHB 2,3-dihydroxybiphenyl DNA deoxyribonucleic acid dNTP deoxycytidine 5'-triphosphate 8 extinction coefficient EI electron impact g gram GC-FID gas chromatography-flame ionization detector hr(s) hour(s) IpA isopimaric acid IpB isopropylbenzene IPCR inverse polymerase chain reaction J coupling constant kb kilobase-pair kDa kilodalton L liter LB Luria Bertani M molar M + molecular ion mg milligram min minute mL milliliter mM millimolar MS mass spectrum m/z mass to charge ratio N normal NAD(P)H reduced nicotinamide-adeninedinucleotide (phosphate) ng nanogram nm nanometer nM nanomolar NMR nuclear magnetic resonance OD optical density ORF open reading frame ix PaA palustric acid PAGE polyacrylamide gel electrophore PCR polymerase chain reaction Phen phenanthrene PiA pimaric acid Pyr pyruvate SDS sodium dodecyl phosphate TCA tricarboxylic acid TLC thin layer chromatography Ug microgram uL microliter uM micromolar umole micromole U unit UV ultraviolet Vis visible V volt w/v weight per volume ACKNOWLEDGMENTS Foremost, I thank my supervisor Dr. William Mohn for his patience, guidance and support. It was a long and difficult road but you were always there when I needed you. I am also indebted to the members of my supervisory committee Drs. Thomas Beatty, Paul Bicho, John Saddler and John Smit. It's a rotten job but someone has to do it. I also thank Dr. Martin Tanner and Gordon Stewart for their help with the interpretation of the MS and 'H NMR spectral data. I am also grateful to Dr. Jean Armengaud for providing me with the plasmids pAJ130 and pBBRlMCS-2, Dr. Herbert Schweizer for pUCP26/27, pEXlOOT pX1918G(T) and pUCGm and Dr. Thomas Beatty for pSL1190, pUC4-KIXX and E. coli S17-1. I also acknowledge Dr. Lindsay Eltis for his helpful discussions and the use of his laboratory at Universite Laval where I purified DitAl A2, Emma Master and Dr. Martina Ochs for critically reviewing the manuscripts and the Science Council of B.C. for the generous postgraduate scholarship. This work was supported by the Natural Science and Engineering Research Council of Canada, the Council of Forestry Industries of B.C., the Sustainable Forest Management Network Centres of Excellence and the Industrial Research Chair in Forest Products Waste Management. xi INTRODUCTION 1. What are resin acids ? Investigations have elucidated pathways for the degradation of many plant compounds. These studies principally focused on polymers such as cellulose and lignin or low molecular weight compounds like phenolics and flavonoids. Because of their potential commercial and pharmaceutical applications, the microbial degradation products of mono- and triterpenoids have also been extensively researched (Barz and Weltring 1985; van der Werf et al. 1997). This dissertation will focus on the microbial degradation of resin acids, a group of diterpenoids found mainly in the oleoresin of coniferous trees such as pine. Resin acids can be classified into abietanes and pimeranes. Abietanes have an isopropyl side chain at the C-13 carbon atom, whereas, pimeranes have vinyl and methyl substituents at this position (Fig. 1). Although the biodegradation of resin acids is a significant process in the global carbon cycle, it is the research on the of pulp and paper mill effluent detoxification which has been the driving factor behind the biochemical studies of resin acid degradation. Resin acids are extracted from wood during the pulping process and are discharged in wastewater. A significant fraction of the overall acute toxicity of this wastewater can be attributed to the presence of resin acids (Leach and Thakore 1973; Priha and Talka 1986; Walden and Howard 1981). Furthermore, resin acids contribute to the formation of pitch, which can disrupt the paper making process. Thus, understanding the biodegradation process of this class of compounds in biological wastewater treatment systems such as aerated lagoons and activated sludge is important. For other reviews on resin acids and their significance to the pulp and paper industry, the reader is referred to the reviews by Liss et al. (1997) and Martin et al (1999). 1 2. The ecology and diversity of resin acid-degrading bacteria Resin acid-degrading microorganisms are widely distributed in the environment. This is illustrated by the fact that biodegradative activity was found in various samples collected from natural waters (Cote and Otis 1989; Hemingway and Greaves 1973), sediments (Tavendale et al. 1997a; Tavendale et al. 1997b), biological treatment systems for pulp mill effluents (Rogers and Mahood 1974; Servizi and Gordon 1986; Servizi et al. 1986; Taylor et al. 1988; Zender et al. 1994), activated sludge systems (Hemingway and Greaves 1973; Liver and Hall 1994), upflow anaerobic sludge bed reactors (Patoine et al. 1997), forest, agricultural and Arctic soils (Biellmann and Wennig 1971; Mohn et al. 1999a). Consistent with the wide occurrence of resin acid degradation activities, a variety of pure cultures has been isolated from numerous sources after enrichment on resin acids (Table 1). Most of the bacterial isolates listed in Table 1 were isolated from enrichment cultures on mineral media supplemented with resin acids as sole organic substrates. This approach facilitated isolation of microorganisms both degrading and growing on resin acids, but excluded those transforming but not growing on resin acids as sole substrate. Undoubtedly, the latter group also exists. For example, Hemingway and Greaves (1973) showed that among the 69 bacterial isolates from wood sources, few could use resin acids as a carbon source, but eleven species (six Bacillus, Escherichia coli, Flavobacterium sp. Pseudomonas sp. and two unidentified) showed some potential to degrade resin acids. Recently, Yu and Mohn (1999a) isolated from compost a bacterium that mineralized abietane resin acids but additionally required tryptic soy broth for growth. The wide ecological distribution of resin acid-degrading microorganisms may be attributed to the ubiquitous nature of these compounds. Resin acids are released from terrestrial vegetation into the atmosphere, or into water bodies from watershed runoffs and are dispersed to every region of the earth (Mazurek and Simoneit 1997). 3 Table 1. Bacterial and fungal isolates capable of the biotransformation of resin acids. Organism Source0 Substrated Meta- Reference bolisme Bacteria Sphingomonas sp. strain DhA-33 a SBR DhA G (Mohn 1995) Zoogloea resiniphila DhA-35 a SBR DhA G (Mohn 1995) Ralstonia sp. strain B K M E - 6 3 B K M E DhA G (Bicho etal. 1995) Burkholdeha sp. strain IpA51a Forest soil IpA G (Mohn et al. 1999a) Burkholderia sp. strain DhA-54 a Forest soil DhA G (Mohn etal. 1999a) Pseudomonas resinovorans* Wheat field DhA G (Biellmann and soil Wennig 1971) Pseudomonas vancouverensis DhA-51 a Forest soil DhA G (Mohn etal. 1999a) Pseudomonas multiresinivorans IpAl a SBR IpA G (Wilson etal. 1996) Pseudomonas sp. strain IpA-2a SBR IpA G (Wilson etal. 1996) Pseudomonas abietaniphila BKME-9" B K M E DhA G (Bicho et al. 1995) Pseudomonas sp. Not reported DhA G (Biellmann et al. 1973) Flavobacterium resinovorum Pine forest DhA G (Biellmann et al. soil 1973; Biellmann and Wennig 1968) Mycobacterium sp. strain DhA-55 a Forest soil DhA G (Mohn et al. 1999a) Mycobacterium sp. strain IpA-13a SBR IpA G (Wilson et al. 1996) Arthrobacter sp. Lodgepole Methyl DhA G (Levinson and Carter Pseudomonas sp. strain A19-6a a , b pine 1968) B K M E AbA G (Morgan and Wyndham 1996) A Icaligenes sp. strain D11 -13 B K M E DhA SR (Morgan and Wyndham 1996) Alcaligenes sp. Soil AbA G (Cross and Myers 1968) Alcaligenes eutrophus Not reported DhA G (Biellmann et al. 1973) Bacillus psychrophilus Pulp mill DhA C M (Cote and Otis 1989) effluent (3 Proteobacterium strain DhA-71 a Compost DhA C M (Yuand Mohn 1999a) P Proteobacterium strain DhA-73 a E T A S R DhA G (Yu and Mohn 1999a) Pseudomonas sp. strain DhA-91 a Arctic soil DhA G (Mohner al. 1999b) Pseudomonas sp. strain IpA-95a Arctic soil IpA G (Mohn etal. 1999b) Pseudomonas sp. strain IpA-93a Arctic soil IpA G (Mohn etal. 1999b) Sphingomonas sp. strain DhA-96 a Arctic soil DhA G (Mohn etal. 1999b) Fungi Fomes annosus Not reported DhA C M (Ekman and Sjoholm 1979) Corticium sasakii Not reported Methyl DhA C M (Brannon et al. 1968) Mortierella isabellina Culture Resin acids C M (Kutneyero/. 1988) collection Ophiostoma sp. Softwood Resin acids SR (Wang era/. 1995) Lecythophora sp. Aspen wood Resin acids SR (Wang et al. 1995) Chaetomium cochliodes Not reported DhA C M (Yano etal. 1994) a Identified by 16S rDNA sequence.b Formerly Comamonas sp. strain A19-6a. c SBR, sequencing batch reactor; B K M E , bleached kraft mill effluent; ETASR, elevated-temperature activated sludge reactor. d Abbreviations are described in Fig. 1. ° G, use as sole organic substrate; SR, substrate removed from medium; C M , cometabolism of substrate. 4 While resin acid-degrading microorganisms are present in many environments, in most cases, they are probably found at low abundance. In an extensive survey of 21 biotreatment systems of pulp mill effluents, the two dehydroabietic acid (DhA)-degrading species, Pseudomonas abietaniphila BKME-9 and Zoogloea resiniphila DhA-35 were found in ten and three of the systems, respectively (Yu et al. 1999b). However, the abundance of P. abietaniphila and Z. resiniphila in most biotreatment systems was less than 103 cells/ml. In most environments, including biotreatment systems, resin acids are found in low concentrations and only account for a very small portion of the total organic matter present (Yu et al. 1999b). Such low resin acid levels would not be expected to sustain large microbial populations. Resin acid-degrading microorganisms are phylogenetically diverse. Species of both bacteria and fungi can transform resin acids (Table 1). Aerobic bacterial isolates typically use resin acids as sole growth substrates, whereas fungal isolates only transform resin acids, usually by hydroxylation. The analysis of 16S rDNA of resin acid-degrading bacteria illustrates their phylogenetic diversity (Fig. 2). Of the unique aerobic bacterial isolates characterized so far, most are gram-negative Proteobacteria with representatives in the a, J3 and y subclasses. The gram-positive isolates include two Mycobacterium (Fig. 2) and one Bacillus (Table 1) strains. Although most of the isolates characterized to date are mesophiles (Mohn et al. 1999a); thermophilic (Yu and Mohn 1999a) and psychrotolerant (Mohn et al. 1999b) strains have recently been isolated. 3. Resin acid substrate specificity of aerobic bacteria Consistent metabolic patterns emerged from screening the ability of bacteria to metabolize the tricyclic structure of resin acids. These patterns are associated with differing degrees of saturation, isomers, side chains, or chlorine substituents. Several studies (Bicho etal. 1995; Mohn 5 •g j j <D • r " U *j +J CO <U *^ bp' C3 3 I -d co (2 a o c/l c/l O & D O tj o CJ X> "o O • • £ Is c !R 5 e t a c cu 3 cr cu cu _f <u j l j l I l f . l j <u a. to (U 6D •J3 O >- '-5 .SJ' CU <U 3 3 UH - O C C C 6 1995; Mohn et al. 1999a) have demonstrated that the ability of bacterial isolates to grow on DhA coincides with their ability to grow on all abietanes. Another deduction that can be drawn from simple substrate specificity tests is that the pathway(s) responsible for abietane degradation in gram-negative bacteria cannot utilize pimeranes as substrates, whereas the pimerane biodegradation pathway(s) may catalyze abietane degradation (Bicho et al. 1995; Mohn 1995; Wilson et al. 1996). Bacteria isolated from enrichment cultures using either abietic acid (AbA) or DhA use only abietane resin acids. The isopropyl side chain of abietanes at C-13 appears to have a critical role in determining the specificity of the biochemical pathway for this class of compounds. Organisms isolated on pimeranes often can use abietanes. Two Pseudomonas stains, IpA-1 and IpA-2, isolated from isopimaric acid (IpA) enrichment cultures grew on pimeranes (IpA and pimaric acid) and DhA, although growth on the latter was slower (Wilson et al. 1996). Cell suspension induction experiments demonstrated that IpA was required to efficiently induce the degradation of DhA and AbA in strain IpA-1 whereas for strain IpA-2, abietanes could readily induce their own degradation. Interestingly, strain IpA-2 has a diterpenoid-inducible enzymatic system that can degrade pimeranes and abietanes, but this organism can not grow on AbA and grew poorly on DhA. This suggests that strain IpA-2 cometabolizes abietanes in its natural habitat. Recently, a broader group of bacteria (11 isolates) were tested for their specificity for resin acids (Mohn et al. 1999a). Two gram-positive Mycobacterium sp. strains used both pimeranes and abietanes. One strain, Burkholderia sp. IpA-51, was specific for IpA, the substrate used for its enrichment and isolation. Chlorinated derivatives of DhA are formed as a byproduct of pulp bleaching with elemental chlorine. As these compounds are more toxic and more recalcitrant to biodegradation than their precursors (Zanella 1983), their removal from effluents by biological treatment is 7 important. Metabolic studies of chlorinated DhA revealed that, in most instances, a chlorine substituent at the C-14 position hinders degradation of DhA by gram-negative bacteria whereas a single chlorine substitution at C-12 was tolerated (Bicho et al. 1995; Mohn and Stewart 1997). One isolate, Sphingomonas sp. strain DhA-33, was found to remove both the 12- and 14-ClDhA isomers equally, when growing on them. This strain could also remove 12,14-Cl2DhA, when previously induced by growth on CIDhA. Growth of strain DhA-33 on CIDhA was slower than on DhA, with doubling times of 2.7 hrs and 7 hrs for DhA and CIDhA, respectively. Bacteria growing on CIDhA left high residual levels of substrate in the culture medium and, in the case of strain DhA-33, accumulated a metabolite tentatively identified as 3-oxo-14-chlorodehydroabietin. Since chlorinated DhAs are man-made chemicals which appeared only after the practice of bleaching pulp with chlorine, the degradation of CIDhA by the strains studied was probably fortuitous, and likely coevolved with the ability to use DhA as a substrate. As expected, pimerane-degrading bacteria are unable to degrade chlorinated DhA but unexpectedly, the two Mycobacterium sp. did not degrade CIDhA, even though these strains grew on DhA. The inability of abietane-degrading gram-positive bacteria to metabolize CIDhA combined with their ability to degrade pimeranes, probably indicates divergence in the resin acid biodegradation pathways of gram-positive and gram-negative bacteria. Finally, It should be noted that bacteria that can grow on 12,14-Cl2DhA have yet to be isolated. 4. Aerobic pathways for abietane degradation Decades ago, two studies reported on the DhA degradation pathways of Flavobacterium resinovorum (Biellmann et al. 1973a), an Alcaligenes sp., and a Pseudomonas sp. (Biellmann et al. 1973b). These pathways were generated from analyses of the intermediates accumulated using 8 culture media supplemented with metabolic inhibitors. Similarities emerged when comparing pathways from these two studies. For example, in pathway A (Fig. 3), hydroxylation at C-7 to form the alcohol 1 followed by its oxidation to the ketone 2 was observed for all three strains. In contrast, oxidation at C-3 (pathway B) to form the ketone, presumably from the alcohol, followed by decarboxylation (enzymatic or spontaneous) to the corresponding 3-oxodehydroabietin 9 was observed in only F. resinovorum. In the case of F. resinovorum it was unclear which oxidation, C-3 or C-7, proceeded first, as both products were isolated from culture medium (Biellmann et al. 1973a; Biellmann and Wennig 1968). In addition, I note that in the proposed pathway for F. resinovorum one can not determine if the oxidation at C-3 preceded (pathway B) or followed (pathway E) dioxygenation, as neither compound 4 nor 3,7-dioxodehydroabietin were isolated from the culture supernatant. In all three strains, dioxygenation of the aromatic ring led to the formation of a diol presumably from the dihydrodiol 3, to form the 3,7-dioxo-l 1,12-diol 6 in F. resinovorum (Biellmann et al. 1973a) or the 7-oxo-l 1,12-diol 4 in the Alcaligenes and Pseudomonas strains (pathway D) (Biellmann et al. 1973b). The isolation of 2-isopropyl malic acid 8 from culture supernatant suggested that the diols 4 and 6 are degraded via an extradiol ring fission reaction, although the ring cleavage reaction product(s) 5 and 7 were never isolated. The proposed dioxygenolytic pathway of DhA may be analogous to those of the upper degradation pathway of fused ring polycyclic aromatic hydrocarbons (PAHs). In PAH degradation, the extradiol cleavage product is unstable and aromatizes to a hemiketal which spontaneously or enzymatically isomerizes to a /ra«s-o-hydroxyarenylidenepyruvate (Eaton and Chapman 1992). The pathway of DhA degradation may similarly proceed through a hemiketal intermediate, which may explain why the cleavage products (compounds 5 and 7, Fig. 3) were not isolated. However, the 9 C 0 2 + H 2 0 Fig. 3. Summary of proposed biochemical pathways for aerobic abietane degradation or transformation by Flavobacterium resinovorum (Biellmann et al. 1973a), Alcaligenes eutrophus, a Pseudomonas sp. (Biellmann et al. 1973b) an Alcaligenes (Cross and Myers 1968) and biological pulp mill effluent treatment systems (Zender et al. 1994). Compounds in brackets are proposed intermediates. Dashed arrows represent several and/or hypothetical steps in the pathway with unidentified intermediates. 10 hypothetical hemiketal cleavage product(s) of DhA degradation have never been found. In contrast, the degradation of methyldehydroabietate by an Arthrobacter sp. followed a novel route of abietane catabolism (Levinson and Carter 1968) (not shown). In this pathway, methyldehydroabietate is first hydrolyzed to DhA before being converted to 3-oxoDhA. Ring A of the 3-oxo acid was further degraded, while the aromatic ring C remained intact, resulting in the formation of l-carboxy-l,2-dimethyl-6-isopropyltetrahydronaphthalene (not shown). The isolation of the 3-oxo acid from Arthrobacter sp. suggests an enzymatic (not spontaneous) decarboxylation at C-4 (compound 9, Fig. 3) by F. resinovorum, as the 3-oxo acid was shown to survive a similar purification procedure (acidification followed by organic solvent extraction and silica gel or gas-liquid chromatography). The chemical analysis of a biological treatment system receiving effluents from a softwood bleached kraft pulp and paper mill identified four other resin acid transformation products (Zender et al. 1994). These compounds, proposed to be hydration and hydroxylation products of AbA, were identified as 13-abietenic acid 10, abietanic acid 11 and 13P-hydroxyabietanic acid (kinleithic acid) 12 (Fig. 3). The study also provided evidence that AbA was dehydrogenated to DhA and further decarboxylated non-oxidatively to dehydroabietin 13. However, these transformations were based on circumstantial evidence and were not shown to be biologically catalyzed. The transformation of AbA by an Alcaligenes sp. produced three novel compounds (Cross and Myers 1968), which were identified as 5a-hydroxyabietic acid 14, 7P-hydroxyabietic acid 15 and a minor product believed to be a 18 -> 2 or 18 -> 6 epoxy-y-lactone (not shown). Finally, I caution the reader about the uncertainty of the pathways depicted in Fig 3. As many of these metabolites were isolated from culture media, sometimes in the presence of metabolic inhibitors, these intermediates might not be representative of the principal pathway(s) of resin acid 11 degradation. Moreover, it is also conceivable that a branched, rather than a straight pathway can lead to resin acid mineralization by several routes. 5. Transformation of resin acids in anaerobic environments Under anoxic conditions, resin acids can be biotransformed, but there is no conclusive evidence that their carbon skeletons are degraded. Furthermore, these anaerobic transformations have been observed only in complex microbial communities such as freshwater sediments and bioreactors. Resin acids are recalcitrant under a variety of anaerobic conditions (Mohn et al. 1999a) and no pure cultures have been found that can use resin acids as the source of carbon and energy. Because of their hydrophobic nature, resin acids can sorb to suspended solids and settle into environments devoid of oxygen such as sediments. In these instances, how are the resin acids recycled into the biogenesis process? Diterpenoid analysis from contemporary and "aged" samples produced evidence for an oxidative route for anaerobic transformation of resin acids (Simoneit et al. 1985), but also indicated that the transformation products are recalcitrant. The mass spectral analysis of several of these metabolites of anaerobic metabolism with the structural features of abietane and pimerane skeletons have identified aromatized and decarboxylated transformation products. Although anaerobic transformation pathways were pieced together from these structures, the intermediates were not unambiguously classified as products from microbial activities until recently. A biological transformation pathway for DhA in anoxic sediments was recently described (Tavendale et al. 1997a). Deuterated DhA was incubated with anaerobic sediments and compared to a parallel autoclaved control sample. In a 264-day incubation period, dehydroabietin 16 and tetrahydroretene 17 were minor and major transformation products of DhA, respectively (Fig. 4). 12 Fig. 4. Proposed pathways of anaerobic transformation of dehydroabietic, abietic and pimaric acids. The dashed arrow represents several steps of the pathway and the bold arrow represents the probable main path of transformation of DhA. 13 These compounds were previously observed in lake sediments (Wakeham et al. 1980) and probably represent the principal path of anaerobic transformation of resin acids. Tetrahydroretene may be formed in a one-step reaction or may involve the formation of 20-norabietapentaenoic acid (simonellite) 18, a short lived intermediate measured at very low concentration. A very small percentage of tetrahydroretene was converted to retene (1.1%) 19 and methylterahydrophenathrene 20, but the majority of the tetrahydroretene was transformed to unidentified compound(s). The time scale of these experiments clearly indicates that anaerobic transformation of resin acids is slow relative to aerobic degradation. Evidence for the anaerobic biotransformation of pimerane-type resin acids is less conclusive. Anaerobic incubation of lake sediments receiving bleached kraft mill effluent was found to significantly reduce pimaric and isopimaric acids, relative to autoclaved control samples (Tavendale et al. 1997b). Although concentrations of 8-pimarenic 21 (Fig. 4) and 8-isopimarenic acids (not shown) increased slightly during the incubation period, they could only account for a small percentage of the pimeranes removed. The fate of pimerane-type resin acids in anoxic environments remains unresolved. Diterpenoid inventories from recent and dated environmental samples suggest an anaerobic pimerane degradation scheme similar to that for abietane resin acids, with the formation of pimanthrene 22 from pimaric acid in a multi-step process (Wakeham et al. 1980). However, the biological catalysis of this proposed pathway was unproven. Anaerobic biotransformation of resin acids remains poorly understood. The studies on the anaerobic fate of resin acids, described in this section, indicate that aromatization and decarboxylation to alkylated PAHs occurs and that the resulting compounds persist in the environment, as evidenced by the presence of retene and pimanthrene in dated samples. 14 6. Fungal hydroxylations of resin acids Several fungi have been shown to modify resin acids (Table 1). All of the products of fungal attack characterized to date are hydroxylated resin acids (Fig. 5). No complete fungal degradation pathway has been elucidated. For several microbial catabolic pathways, hydroxylation of the substrate is necessary for subsequent degradation steps and assimilation of the carbon as in the case of the 9ct-hydroxylation of the triterpene nucleus (Schoemer and Martin 1980). However, in many instances fungal hydroxylation of terpenoids is regarded as a detoxification or cometabolic process rather than an assimilatory one. Many extractives of wood may act as plant defense compounds aimed at fungal pathogens. Fungal hydroxylation of phytotoxins is a common approach of detoxifying these compounds as in the case for kievitone (an isoflavonoid) (Smith et al. 1980). It is likely that the hydroxylation reactions carried out by resin acid transforming fungi serve to detoxify these compounds as none of these organisms were found to use resin acids for growth, and hydroxylated resin acids are generally more soluble and less toxic (Servizi et al. 1986). The earliest study of fungal metabolism of resin acids reported on the hydroxylation of methyldehydroabietane by Corticium saskii (Brannon el al. 1968). Extended incubations with the substrate yielded 3J3-hydroxy and 33,76-dihydroxymethyldehydroabietane and 33-hydroxy and 33,6p~dihydroxy derivatives from 7-oxo-methyldehydroabietane. Mortierella isabellina was also shown to attack the C-2, C-15 and C-16 positions, producing 2(a and 3 isomers), 15 or 16 monohydroxylated DhA and 2a, 15 or 2a, 16 DhA diols (Kutney et al. 1981a) (Fig. 5). The same authors also reported on the analogous hydroxylations of AbA, IpA, 12-ClDhA, 14-ClDhA, and 12,14-Cl2DhA. Diastereoisomers of the 2a, 16 diol were produced from AbA (Kutney et al. 1982a) whereas 15,16-diol was formed from IpA presumably as a result of dihydroxylation of the vinyl side chain (Kutney et al. 1981b). Mono and dichlorinated congeners of DhA produced the 15 16 same hydroxylated metabolites as DhA with the exception that 2a,15,16-trihydroxy acid was also produced from 14-ClDhA and a 2-oxo metabolite resulted from the incubation with 12,14-Cl2DhA (Kutney et al. 1982b; Kutney et al. 1983a; Kutney et al. 1983b). The different side chain of IpA and the chlorine substituents affected the nature of the metabolites produced by M isabellina probably because of steric hindrance or varying culture conditions and not as a result of the recruitment of new enzymes. Upon investigating the effect of growth phase and immobilization of M isabellina on the hydroxylation of DhA, the authors reported that rapidly growing cultures formed only the 2-hydroxy metabolite whereas only 15- and 16-hydroxy compounds were produced as cultures approached stationary phase (Kutney et al. 1985). From these results, it was postulated that two or three resin acid hydroxylases might be present in M isabellina which are expressed at different stages of growth. Hydroxylase activity was found to be cell associated with no enzyme activity detected in the culture supernatants. Metabolism of DhA by Chaetomium cochliodes followed a similar pattern of hydroxylation as that by M. isabellina (Yano et al. 1994). DhA was first hydroxylated to 15- and (15R),16-hydroxyDhA which were subsequently converted to 15,16- or their 7R-dihydroxy equivalent (Fig. 5). Addition of the metabolic inhibitor a,a'-dipyridyl to the culture medium resulted in the accumulation of 7-oxoDhA with only minor amounts of 15- and 16-hydroxy metabolites produced (Yano et al. 1995). This resulted in the assumption that the chelating agent was inhibiting the enzyme hydroxylating carbons 15 and 16. Oxidations at C-7 were also catalyzed by C. saskii and several bacteria (Fig. 3), emphasizing the importance of this reaction in the catabolism and detoxification of DhA. 17 7. Organization of genes for aerobic degradation of aromatic compounds Because my dissertation reports on the molecular genetics of a dioxygenolytic pathway for diterpenoid degradation, it is important to review what is known about the organization of aerobic pathways for aromatic compound degradation. Since this field has been extensively reviewed (van der Meer et al. 1992 and references therein), it will be covered only briefly in this section. In general, bacteria use a variety of peripheral enzymes to channel their initial aromatic substrates into a limited number of central intermediates such as catechol, protochatechuate and gentisate. These intermediates are frequently further metabolized via dioxygenolytic pathways to TCA cycle intermediates. Two types of dioxygenolytic pathways are possible; the meta (extradiol) and the ortho (intradiol) types (Fig. 6). In ortho-type, the aromatic-ring cleavage occurs between the two hydroxyl groups of the diol, whereas cleavage occurs at a bond proximal to one of the two hydroxyl groups in meta-type reactions. Since only the meta-type cleavage pathways are pertinent to the discussion of my dissertation, ortho-type pathways will not be reviewed further. To simplify the description of the organization of operons for aromatic compound degradation, I have defined the dioxygenolytic pathway as the first three reactions; the ring-hydroxylation, the diol dehydrogenation and the ring cleavage (Fig. 6). The multicomponent ring-hydroxylating dioxygenase complexes comprise of two or three components (see section 8 of the introduction) encoded by three or four genes (Fig. 7). The catabolic genes which encode the first three steps of most dioxygenolytic pathways are clustered into one operon (Fig. 7). This genetic organization not only simplifies the regulation of the expression of the pathway but also permits rapid dissemination of the catabolic potential to other organisms, as these clusters are often located on self-transmissible plasmids (Sayler et al. 1990) and transposable elements (Wyndham et al. 1994). However, there are now a few reported examples where the genes encoding a 18 COOH Step 1 Aromatic ring hydroxylating dioxygenase HOOC OH OH Step 2 Diol dehydrogenase OH OH ^ ^ C O O H meta Step3 Ring cleavage dioxygenase COOH COOH ortho Fig. 6. Dioxygenolytic degradation pathway for benzoate showing the first three enzymatic steps for the extradiol {meta) and intradiol {ortho) cleavage pathways. 19 N c I 3 o fl c/l co u o 5 •§ 3 a CJ "o H o I 1 I JJ & CQ o o JJ PQ CN © § S •§ 3 i JJ & CQ m '5 2 3 s o 5 5 JJ C L S CO u Pi 3 K O 5 .1 d •a o a CO C/l CO u < u & s c o 5 3 o 3 3 § I 3 I 00 £9 u z R o S •8 3 I S" CJ J3 8-z co CO CO u T -S i rS • S .s s * o o •a -a fc fc t t .2 o •o -3 p I 00 (Jo •a 3 tt C o o e <-CU CO t-. C ' 3 ( 2 M <S Til • — T3 co u bO cu O CO £ ' to • H gj CO T3 CO CO CO CO r- co — o co C cu • £ CO "O CU J3 rt cu o 60 >,fl x S o 1 3 CO to a. M.5 u u •rt rt. r-> ni 111* CO CO S a o CO o CJ C o * - ^ CO O T3 cu a. .S 0 "O cu c o •s cj C*H O C o N § to co CU C CU co CU *i co cu cu JS 00 C cu co 0 , - 0 -> o • -oo a 0 3 ^ 75 * (U c o co 1 ^ CJ CU I - _ > 3 O •rt m a, CO CO 3 „ U U J) n j 1— y j 2 eso <u -c 6 H O co CjH O rt SO - cu cu 0 0 S JS <u • - H CO p H [/) PH O . H w 20 dioxygenolytic pathway are not clustered in one operon. The most recent example is from the dioxin degradation pathway of Sphingomonas sp. strain RW1 in which the genes encoding the reductase (redAl), the ferredoxin (fdxl), the oxygenase {dnxAlAl) and the cleavage dioxygenase (dbfB) are not located in one operon (Fig. 7) (Armengaud and Timmis 1997). In another example, only the reductase component of the dioxygenolytic operon of the biphenyl-degrading Comamonas testosteroni B-356 was found 19 kb away from the cluster (Fig. 7) (Bergeron et al. 1994). Although the genetic organization of the degradative pathways of dioxin in Sphingomonas RW1 and biphenyl in C. testosteroni B-356 are atypical, it is my opinion that as degradation pathways for more diverse aromatic compounds are discovered from newly isolated bacterial species, more atypical organization of dioxygenolytic gene clusters will be discovered. 8. Multicomponent ring-hydroxylating dioxygenases The initial step in the bacterial degradation of aromatic compounds is usually the dioxygenation of the benzylic ring, forming a c/s-dihydrodiol or c/s-diol carboxylic acid (Fig. 6). As previously mentioned, these reactions are catalyzed by soluble multicomponent ring-hydroxylating enzyme complexes comprising three or four proteins (Fig. 8). The complex is made up of a nonheme iron-containing catalytic oxygenase and electron transport protein(s) which supply the reducing power to the oxygenase (Fig. 9) (for extensive reviews on the structure of ring-hydroxylating dioxygenases refer to Butler and Mason 1997; Harayama et al. 1992; Mason 1992). The reductase, the first component of the electron transport chain, catalyses the transfer of electrons from NAD(P)H to the ferredoxin, the second component. The ferredoxin in turn transfers electrons to the catalytic terminal oxygenase. The reductases are flavoproteins which in some instances contain an iron-sulfur cluster of the [2Fe-2S]-type. If the reductase protein 21 oo 22 Dihydrodiol Aromatic substrate Fig. 9. Organization of multicomponent ring-hydroxylating dioxygenases showing the flow of electrons from NADH to the catalytic terminal oxygenase. Adapted from Butler and Mason 1997. 23 contains a [2Fe-2S] cluster, the enzyme complex usually lacks the ferredoxin, with one exception being the naphthalene dioxygenase, where a [2Fe-2S] cluster is present in both the reductase and the ferredoxin (Ensley et al. 1982). All the ferredoxins characterized to date contain a [2Fe-2S] prosthetic group of either the plant- or Rieske-type. Aromatic ring-hydroxylating dioxygenases have traditionally been classified based on the variations in the nature of the components as well as the number and size of the subunits of both the oxygenase and reductase (Batie et al. 1992; Butler and Mason 1997). Four of these classes are described in Fig. 8. 24 THESIS OBJECTIVES In spite of the fact that resin acids are a class of compounds which is very problematic to the pulp and paper industry, very little is known about resin acid biodegradation processes in wastewater biotreatment systems. The microbial ecology and activities of wastewater treatment systems as a whole are very complex, and therefore difficult to study. To simplify the task, a single resin acid-degrading bacterium, Pseudomonas abietaniphila BKME-9, was studied. The results from this study may be used as a reference to investigate the more complex ecology of resin acid-degrading microbial populations in wastewater treatment systems. The general objective of my thesis was to elucidate the aerobic resin acid biodegradation pathway of P. abietaniphila BKME-9 using a molecular biology approach. This objective was achieved by (1) identifying metabolic pathway intermediates accumulated by Tn5 transposon mutant strains, (2) cloning and sequencing the genes or gene clusters identified by transposon mutagenesis, (3) identifying which of the genes from the clusters are required for abietane degradation by generating insertional mutations, and (4) by characterizing the function of these disrupted genes by using cell suspension experiments or by expressing the genes in E. coli. 25 M A T E R I A L S A N D M E T H O D S 1. Bacterial strains and culture conditions The bacterial strains and plasmids used in this study are described in Table 2. Escherichia coli strains were grown at 37° C on Luria-Bertani broth (LB) or agar (Difco Laboratories) supplemented with 100 pg of ampicillin per mL, 10 pg gentamicin per mL, 30 pg kanamycin per mL or 12 pg tetracycline per mL. Pseudomonas abietaniphila BKME-9 was grown at 30° C on tryptic soy broth (BBL) or mineral medium supplemented with 1 g/L Na pyruvate, or 0.1 g/L of one the diterpenoids, DhA, AbA or 7-oxoDhA, as previously described (Mohn 1995). Mutants of P. abietaniphila BKME-9 were maintained on 4 pg of gentamicin and/or 30 pg of kanamycin per mL and P. abietaniphila strains harboring derivatives of pUCP26/27 were maintained on 2 pg of tetracycline per mL. These antibiotic concentrations were determined by minimum inhibitory concentrations assays, as published values for Pseudomonas were to high for strain BKME-9. Resin acids were supplied by Helix Biotechnologies, Richmond, Canada. 2. Tn5 mutagenesis and inverse P C R Transposon mutants from biparental matings of P. abietaniphila strain BKME-9 with E. coli S17-1 containing pSUP2021 (Simon et al. 1983) were selected on minimal medium containing pyruvate and kanamycin. Kanamycin resistant colonies were subsequently screened for the inability to grow on minimal media containing DhA (0.1 g/L) as carbon source using replica plating. The Southern blot analysis of selected mutants was done on restriction endonuclease digested DNA samples (-2.5-5 pg) electrophoresed overnight on 0.5 % agarose at 25 V, blotted onto Nytran nylon membranes (Schleicher and Schuell) using a 0.4 M NaOH/0.6 M NaCl transfer 26 Table 2. Strains and plasmids used in this study Strain or plasmid Genotype or description Reference or source Strains Pseudomonas abietaniphila B K M E - 9 wild-type (Bicho etal. 1995) BKME-12 mutant with TnJ insertion in region with similarity to NAD(P)H this study oxidoreductascs BKME-941 ditAl ::Tti5 this study BKME-91 ditA3::xylE-Gmr this study BKME-92 ditAl ::xylE-GmT this study BKME-93 ditC::GmT this study BKME-94 ditI::xylE-Gm' this study BKME-95 ditH::xylE-Gmr this study BKME-96 ditF::xylE-Gmr this study BKME-97 ditRv.Kxti this study BKME-98 ditD::xylE-GmT this study BKME-99 ditB::xylE-Gmr this study BKME-912 ditA3::xylE-GmT ditR::Kmr this study BKME-922 ditA 1: :xylE-Gm' ditR: :Km r this study BKME-91 rescued BKME-91 mutant with pVM120 this study (pVM120) BKME-912 rescued BKME-912 mutant with pVM220 this study (pVM220) BKME-922 rescued BKME-922 mutant with pVM220 this study (pVM220) Escherichia coli DH5a endAl hsdR17 (rk' mk") supE44 thi-1 recAl gyrA (Nalr) Gibco BRL relAl A(lacZYA-argF) U169 deoR (<t>80d/acA(/acZ)M15) XL1 B L U E M R A(mcrA)l%3 A(mcrCB-hsdSMR-mrr)173 endA supE44 thi-1 Stratagene recA 1 gyrA 96 relA 1 lac S17-1 recA pro thi hsdR with integrated RP4-2-Tc::Mu- (Simons etal. 1983) Km::Tn7;Tra +,Tr r, Sm r Plasmids pUC19 Cloning vector; Ap r (Yanisch-Perron et al. 1985) pSL1190 Cloning vector; Ap r Pharmacia pSUP2021 conjugable Tn5 mutagenesis vector; Ap r K m r (Simons etal. 1983) SuperCosl cosmid vector; Ap r Stratagene pEXlOOT sacB conjugable plasmid for gene replacement; Ap r (Schweizer and Hoang 1995) pX1918G or G T xylE-Gmr fusion cassette-containing plasmid; Ap r , Gm r (Schweizer and Hoang 1995) pBBRlMCS-2 conjugable broad-host-range vector; K m r (Kovachefa/. 1994) pUCP26/27 conjugable broad-host-range vector; Ap r , Tc r (Schweizer 1991) pAJ130 fdxl and redA2 encoding type IIA electron-transfer proteins (Armengaud et al. cloned into pVLT35 1998) pUC4-KIXX Plasmid containing the kanamycin resistance cassette from Tn5; Pharmacia Ap r , K m 1 27 Table 2. continued pUCGm Plasmid containing the gentamicin resistance cassette; Ap r , Gm r (Schweizer 1993) pCRII T A cloning vector Invitrogen pIPCRM 12 pCRII containing IPCR product from BKME-12 mutant this study pIPCRM41 pCRII containing IPCRM41 product from BKME-941 mutant this study pLCP450 SuperCosl cosmid library clone containing IPCRM12 sequence this study pLC12 SuperCosl cosmid library clone containing dit gene cluster this study p V M l 5.8 kb EcoRl fragment ofpLC12 into EcoRl of pSLl 190 this study pVM2 9.8 kb EcoRl fragment from pLC12 into EcoRl of pUC19 this study pVM3 3.2 kb Sacll fragment of pLC 12 into Smal of pUC 19 this study pVM4 3.6 kb EcoRl-Smal fragment from pVM2 into EcoRl-Smal of this study pUC19 pVM5 4.3 kb Smal fragment from p VM2 into Smal of pUC 19 this study pVM10 891 bp Nael fragment from pVM4 into Smal of pEX 100T this study p V M 101 Hindlll x y / £ - G m r cassette from pX 1918G into unique Hindlll of this study pVMlO pVM20 Kpnl-BamUl PCR product containing ditA 7 and ditA2 in this study pBBRlMCS-2 p V M 120 891 bp Nael fragment from pVM4 into Smal of pUCP27 this study pVM220 1368 bp EcoRV-Bglll fragment containing ditR into Smal of this study pUCP26 solution and baked at 80° C. Blotted membranes were hybridized overnight at 42° C using a 1861 bp Hind IIVBamH I probe from the Tn5 transposon labeled by nick translation (Gibco BRL) with [a32P]-dCTP (New England Nuclear). Membranes were washed at 42° C in a 0.1 X SSPE/0.1% (w/v) SDS solution until background signal was eliminated. Templates for inverse PCR (IPCR) were prepared from ~1 ug of DNA digested with enzymes that do not cut Tn5 (Aal II, EcoK I, Kpn I, Mlu I, Sac I or Sst I) or with enzymes that cut at a single Tn5 site (BamH I, Sal I, Sph I, or Xma I). Following digestion, enzyme reactions were heat inactivated at 65° C for 15 min, phenol/chloroform extracted and the DNA was precipitated with ethanol. The digested DNA preparations were ligated overnight at 16° C in 200 pl reactions with 200 U of T4 DNA ligase (New England Biolabs). DNA flanking the Tn5 transposon was amplified using a Ericomp thermocycler in 100 pl reactions containing -500 ng of 28 template DNA, 20 mM Tris-HCl pH 8.4, 50 mM KCI, 1.5 mM MgCl, 200 uM of each dNTP, 0.5 uM of each primer. Hot-start IPCR was performed by initially denaturing the samples at 95° C for 3 min before adding 2.5 U of Taq DNA polymerase (Gibco BRL). The amplification was in two steps; the first five cycles were 95° C denaturing for 30 s, 60° C annealing for 1 min and 72° C extension for 4 min. followed by 30 cycles with 55° C annealing. The plasmid pRZ705, which contains Tn5 (obtained from W.S. Reznikoff, University of Wisconsin, Madison), was used as control template for the IPCR reaction. Outward extending primers from the Xma I, Sal I and Sph I sites of Tn5 were designed to amplify the DNA flanking the one side of the transposon (Fig. 10). The sequence of the primers were: The universalTn5 (Rich and Willis 1990) 5'-GGTTCCGTTCAGGACGCTAC-3' (complementary to bases 37 to 18 and to bases 5784 to 5803 of the transposon) was used in conjunction with one of the following primers: XmaITn5 5'-AGGCAGCAGCTGAACCAA-3' (complementary to bases 2557 to 2539), RSalITn5 5'-ATGCCTGCAAGCAATTCG-3' (complementary to bases 2722 to 2740), LSalITn5 5'-AACCAGCAGCGGCTATCC-3' (complementary to bases 2614 to 2631) and SphITn5 5'-AGCCGAACTGTTCGCCAGG-3' (complementary to bases 2051 to 2069). Primers were synthesized by the Nucleic Acid and Protein Service (NAPS) unit at the University of British Columbia on a Perkin Elmer ABI synthesizer. 3. Phenotypic characterization of dit mutant strains Mutants of P. abietaniphila BKME-9 were characterized for their ability to grow on 0.1 g/L of either DhA, AbA, 7-oxoDhA or 1 g/L Na pyruvate in a mineral medium. Medium was inoculated (0.1%) with a culture grown overnight on pyruvate and monitored for growth for 3 days. Growth was determined by microscopic examinations with comparisons to positive (wild-29 co c o *** CD -o 2 a 5n c <u co « • § 2 >^ -B S o T3 C co c o N It O CL x> JS o e 3 O C/3 <a e o c CO c/1 C o '5b £ <u X i •4-* o x; C o O a . c CO \— c 'o >> s o jS "Q c/i C o "is c op '55 <u •a a> a n> O c o '« "3, e (* U & 'ST t3 . c H a> JS •!-» o £ ca u -00 T3 co a> o E u X ! O C/3 o 6 T 3 C CO T3 X i oo •c C/3 C o >> E o a . CU o a CO H »1 S 30 PH type strain BKME-9) and negative controls (no substrate). 4. Biotransformation of diterpenoid by resting cells assays Cells suspensions of P. abietaniphila were grown overnight at 30° C in 250 mL of mineral medium supplemented with 1 g/L Na pyruvate, washed once in 10 mM phosphate buffer pH 7.5 and suspended in 50 mL of the same medium at a final OD6io of-3.0. Resin acids were dissolved in methanol and added to the cell suspension. Cell suspensions were incubated on a rotary shaker at 30° C. Samples (1.5 mL) were taken at regular intervals and immediately frozen at -20° C. Thawed samples were analyzed by GC-F1D for the production of pathway intermediates, using the method previously described for DhA analysis (Mohn 1995), except that samples (0.5 mL) were acidified with one drop of 1 N HCl prior to ethyl acetate extraction to improve the recovery of acidic intermediates. UV-Vis absorption spectra of culture supernatants fluid were measured with a Cary IE spectrophotometer (Varian). 5. Genomic library: Construction and screening Genomic DNA from P. abietaniphila BKME-9 was isolated and purified as previously described (Burns et al. 1989). The DNA was partially digested with Mbo I and ligated to BamtU-digested SuperCosl cosmid arms, as described by the supplier (Stratagene). The ligated DNA was packaged in vitro using the Gigapack III Gold packaging extract (Stratagene). E. coli XL1 Blue MR was transfected with the packaged DNA, and library clones were selected on LB medium containing 50 pg of ampicillin per mL. The cosmid genomic library was screened by colony lift using Nytran nylon membranes (Schleicher & Schuell). The immobilized DNA was hybridized to the IPCR product labeled with [a32P]-dCTP (New England Nuclear) using the nick translation 31 system from Gibco BRL. Cosmid DNA from mini lysate of positive clones was analyzed by Southern hybridization, as described above, to confirm the presence of the IPCR sequence in the cosmid insert and map it. 6. DNA manipulation and sequencing Plasmid DNA was isolated by the standard alkali lysis method and restriction endonuclease digestions were performed by standard procedures (Ausubel et al. 1992). Ligation mixtures were incubated at 15° C overnight or at room temperature for 2-3 hrs with T4 DNA ligase (New England Biolabs) and used to transform E. coli cells made competent by the RbCl method of Hanahan (1983), or by electroporation using the protocol supplied with the BioRad gene pulser. P. abietaniphila BKME-9 was transformed by electroporation as previously described (Farinha and Kropinski 1990). Plasmids pUC19 or pSL1190 and E. coli DH5oc were used for the subcloning of pLC12 cosmid fragments needed for DNA sequencing. DNA fragments were purified from agarose gels with QIAquick spin columns (Qiagen) and templates for DNA sequencing were purified with QIAprep spin columns (Qiagen). Successive unidirectional deletions of DNA were prepared for sequencing large fragments using the double stranded nested deletion system from Pharmacia Biotech. Oligonucleotide primers synthesized at the NAPS unit were used to sequence DNA regions not covered by the deletions. DNA sequences were determined by the NAPS unit using AmpliTaq dye terminator cycle sequencing (Applied Biosystem) and Centri-Sep columns (Princeton Separation) to purify the extension products. A schematic representation of the subcloning strategy is depicted in Fig. 11. 32 Fig. 11. Physical map of the dit gene cluster indicating fragments cloned from the P. abietaniphila BKME-9 gene library cosmid pLC12. Fragments of DNA subcloned for homologous recombination are depicted by black rectangles. The location of the various insertions is illustrated by an arrow with the endonuclease and base pair position depicted on the kb scale. ND indicates location of nested deletion of clones used in subcloning the DNA fragment. FPCR indicates the fragment of DNA cloned using PCR. The nucleotide sequence reported in this study have been submitted to the GenBank database under accession no. AFT 19621 and is located in appendix II. 33 7. DNA sequence analysis Clone Manager for Windows (version 4.01) and Primer Designer (version 2.0) were used for sequence analysis and PCR primer design. ClustalX (version 1.5b) and PHYLIP (available from the Department of Genetics, University of Washington) were used to align sequences and generate the phylogenetic trees. Deduced a. a. sequences of putative ORFs were analyzed for similarity to GenBank databases entries using the BLASTX and BLASTP programs (Altschul et al. 1990) available on the National Center for Biotechnology Information server via the internet. Searches for PROSITE protein signature consensus sequences and prediction of transmembrane regions were done using ProScan and TMpred programs available on the internet server of the Swiss Institute for Experimental Cancer Research. 8. Insertional inactivation and construction of transcriptional fusions of dit genes The pEXlOOT gene replacement vector containing the sacB counter-selectable marker (Schweizer and Hoang 1995) was used for the insertional inactivation of dit genes. All DNA fragments required for insertional inactivation were blunt-ended with Klenow polymerase or mung bean exonuclease and subcloned into the unique Sma I site of pEXlOOT. The location of the fragments and the restriction enzymes used to subclone the genes of interest into pEXlOOT are shown in Fig. 11. To create xylE transcriptional insertions/fusions, the xylE-Gvri cassette isolated from pX1918G (without transcriptional terminator) or pX1918GT (with transcriptional terminator) was inserted into the genes in unique restriction endonuclease sites identified in Fig. 11. The cassette containing no terminator was inserted only in those genes suspected of being in a polycistronic transcript (dilA3, ditB, ditD and ditH) in order to minimize downstream polar effects. The Kmr cassette isolated from a Sma I digest of pUC4-KIXX was used to disrupt the 34 putative regulatory gene ditR, and to allow double mutations in strains carrying the xylE-Gwl fusions. The putative meta cleavage dioxygenase gene, ditC, was inactivated by insertion of the Gmr gene (no xylE) isolated from a BamH I digest of pUCGm, to avoid a possible complementation artifact of the ditC mutation by catechol 2,3-dioxygenase (C230). As several attempts failed to clone ditF using restriction enzymes, it was PCR amplified from the plasmid pVM5 by using primers SCP-1 (5' -TCG AGGATGTCTGGCTG-3') and SCP-2 (5'-GCTGAGCAAGGTGCTGT-3') and cloned into the Sma I site of pEXlOOT. Homologous recombination of the mutated alleles into the chromosome of P. abietaniphila BKME-9 was accomplished by conjugation using the E. coli mobilizing strain SI7-1. Colonies containing integrated plasmids were selected on mineral medium supplemented with pyruvate and 4 pg of gentamicin per mL. Isolated colonies, which appeared after 48 hrs at 30° C, were plated on the same medium supplemented with 5% sucrose. Successful gene replacement was confirmed by colony PCR using 17-mer primers and an annealing temperature of 58° C (Zon et al. 1989). 9. Expression of diterpenoid dioxygenase in E. coli The pUC-based plasmid pVMlO, which was constructed to knockout di(A3, was also used for expression of the ferredoxin. The oxygenase (a and P subunits) genes were cloned into the broad-host-range vector pBBRlMCS-2 by PCR. A 2108 bp fragment containing ditAl, its putative ribosomal binding site and ditA2 was amplified using primers VM100 (5'-CGGGGTACCGGCTCGGAGTA-3') and VM101 (5'-CGCGGATCCTTAGAGGAATACCGC-3') which introduced Kpn I and BamH I at the 5' and 3' ends, respectively. The PCR product was cloned into pBBRlMCS-2, which was previously digested with the same endonuclease, to produce pVM20. For expression of the enzyme, plasmids were introduced into E. coli strain XL1 35 Blue MR, and 100 mL of prewarmed LB broth containing the appropriate antibiotic(s) were inoculated (1%) with an overnight culture and grown to logarithmic phase (OD6io -0.6). Cultures were chilled on ice, harvested, washed once in 10 mM Na phosphate buffer (pH 7.5) and suspended in 20 mL of 0.1% glycerol mineral medium at a final OD6io of -3.0. DhA and 7-oxoDhA were dissolved in methanol and added to cell suspensions at final concentrations of 333 pM and 318 pM, respectively. Samples (triplicate of 0.5 mL) were taken at 1-hr intervals, acidified with 1 drop of 1.2 N HCl and immediately frozen at -20° C. The removal of the substrate and the production of the dihydrodiol was monitored by GC-FH), as for P. abietaniphila cells suspensions. 10. Purification and identification of the dihydrodiol A 500-mL cell suspension of E. coli expressing the dioxygenase was incubated overnight (37° C, 180 rpm) in mineral medium with 50 mg of 7-oxoDhA. The acidified (pH 3.0) culture supernatant was extracted twice with ethyl acetate, dried with anhydrous N a 2 S 0 4 and concentrated under vacuum in a rotoevaporator. The dihydrodiol of 7-oxoDhA, which precipitated out of solution during ethyl acetate concentration, was purified by preparative thin layer chromatography (0.5 mm silica gel 60 A with fluorescent indicator, Whatman) using benzene:methanol:acetic acid (79:20:1, vol/vol/vol) as developing solvent and methanol as the elution solvent. UV-Vis absorption spectra were recorded on a Cary IE spectrophotometer. GC electron impact (EI) mass spectrometry was performed with a Varian 3400 equipped with a Varian Saturn 4D ion trap mass spectrometer and a DB-5MS capillary column (30 m x 0.25 mm ID and 0.25 pm film thickness, J&W Scientific). The GC oven temperature program was 70° C for 2 min then 10° C/min to 280° C with an injector and detector temperature of 260 and 290° C, 36 respectively. Prior to 1 pi injection, samples were derivatized by sparging with ethereal vapor of diazomethane to form methyl esters. High resolution EI mass spectra were recorded with a Kratos MS50 at 70 eV and 150° C. Proton (*H) nuclear magnetic resonance (NMR) spectra were recorded with a Bruker-WH400. 11. Catechol 2,3-dioxygenase (C230) assays For C230 activity assays, P. abietaniphila strains were grown to mid-log phase (OD6io 0.6 to 0.7) in 125-mL of mineral medium supplemented with 1 g/L Na pyruvate. Cultures were chilled on ice for 15 min, harvested and washed twice in 10 mM potassium phosphate buffer (pH 7.5) at 4° C. Antibiotics were excluded from the mineral medium, as they reduced the growth rate of the cultures and affected the C230 specific activity. The washed cells were suspended in 10 mM KPO4 buffer (pH 7.5) and adjusted to a final OD6io of 0.6 before induction with 0.5 mM of either DhA, AbA 7-oxoDhA, isopimaric acid (IpA), pyruvate (Pyr) or 0.1 mM of 12,14-Cl2DhA, biphenyl (BpH), isopropylbenzene (IpB) or phenanthrene (Phen). These concentrations were in excess and far above their aqueous solubility, with the exception of pyruvate. The cell suspensions (20 mL) were induced for 8 hrs at 30° C on a rotary shaker at 150 rpm. Triplicate C230 enzyme assays were performed on whole cells in 1 mL of 10 mM KPO4 buffer (pH 7.5). C230 activity was assayed spectrophotometrically at 30°C by measuring the formation of 2-hydroxymuconic semialdehyde at 375 nm (s = 44 mM"1 cm"1) for 1 min. Protein concentrations of cell suspensions were determined using the micro bicinchoninic acid protein assay kit (Sigma) and bovine serum albumin as the standard (Smith et al. 1985). 37 12. Cytochrome P-450 assays Cultures (1 L) for cytochrome P-450 assays were grown to stationary phase in mineral medium supplemented with either 1 g/L pyruvate (uninduced) or 0.2 g/L DhA (induced). The cells were harvested, washed once in 0.85% NaCl and suspended in 5 mL of 10 mM Tris-Cl (pH 8.0) and ImM DDT. The cells were broken with one pass through a French pressure cell at 10 MPa and the lysates were centrifuged at 12 000 g for 20 min. Air-oxidized, sodium dithionite-reduced and carbon monoxide (CO)-sparged absorption spectra of the soluble fractions were recorded on a Cary IE spectrophotometer. A culture of Rhodococcus rhodochrous strain 116, which contains a 2-ethoxyphenol-inducible cytochrome P-450 (Eltis et al. 1993), was used as a positive control for the P-450 assay and the heme stain (see below). 13. SDS-PAGE gels and heme-staining SDS-PAGE was performed as described (Laemmli 1970) using a 5% stacking- 10% separating polyacrylamide gels. The gels were stained with Coomassie brilliant blue R250. Heme-staining of SDS-PAGE gels was performed using dimethoxybenzidine hydrochloride (Sigma) according to the method of Francis and Becker (1984) except that protein samples were incubated at 50 °C for 15 min prior to electrophoresis. 38 RESULTS CHAPTER ONE: A gene cluster encoding abietane diterpenoid degradation activity 1. Introduction The genes encoding catabolic pathways for aromatic compounds in prokaryotes are commonly found in a cluster or an operon (Fig. 7). Early in my project, I identified a gene (ditAl), from P. abietaniphila BKME-9, that is required for DhA degradation. This gene had sequence similarity to ring-hydroxylating dioxygenases (Martin and Mohn 1999a). From this result, it was reasonable to anticipate that the genes encoding the degradation pathway of DhA would be organized in a similar manner as the catabolic genes of aromatics degradation pathways. For this reason, a large fragment of DNA containing the dioxygenase gene was examined. This chapter describes the cloning, sequencing and functional analysis of several genes found on this large DNA fragment. 2. Tn5 mutagenesis, inverse PCR and isolation of cosmid clones Transposon mutagenesis was used to obtain Tn5-insertion mutants of P. abietaniphila BKME-9 which were no longer capable of growing on DhA as a sole carbon source. These mutants were subsequently screened for the accumulation of biodegradation pathway intermediates using a cell suspension assay. One of the DhA" isolates, strain BKME-941, was found to accumulate a 7-oxoDhA intermediate (Fig. 3, compound 2). The identity of this metabolite was confirmed by comparison of its GC retention time and mass spectrum to those of a pure analytical standard. A modified inverse PCR method (Martin and Mohn 1999a) (Fig 10) was used to isolate the DNA sequences flanking the Tn5 insertion in BKME-941. This PCR product 39 was used to (1) rapidly obtain a partial sequence of the disrupted gene, and (2) as a probe to screen a wild-type cosmid library of P. abietaniphila BKME-9. Southern blot analysis of EcoR I, Kpn I, Mlu I, Sac I or Sst I digested BKME-941 DNA indicated that the Tn5 insertion had occurred in genomic fragments >14 kb (data not shown). Aat II was found to give a fragment of adequate size for IPCR, with a predicted product of -2 kb (-7.8 kb minus 5.8 kb) (Fig. 12 A). Although an IPCR product of -2 kb was amplified from the Aat II digested DNA the yield of the reaction was low (Fig. 12 B). This low yield was generally observed in other IPCR reactions with anticipated products > 2.5 kb. The limiting factor in those reactions was likely the low frequency of intramolecular ligation of long fragments in template preparation, and not a result of poor PCR amplification. Tn5 probe hybridization to BKME-941 DNA digested with restriction enzymes that cut the transposon once showed that Sal I, Sph I, or Xma I were potential enzymes for IPCR whereas BatnH I yielded fragments that were too long for IPCR amplification (Fig. 12 A). Dark bands on the Southern blot represent Tn5 probe hybridization to the left side of the transposon which is complimentary to the entire probe, and light bands to the right side which is complimentary to only one end of the probe, with the exception of Sph I where the probe hybridizes primarily to the right side of Tn5 (Fig. 10). I predicted that amplification of the left side of Sal I digested DNA would yield a -316 bp product (-3000 bp minus 2684 bp); whereas, amplification of the right side would yield a product of -1366 bp (-4500 bp minus 3134 bp). Only one band resulted from the digestion with Xma I, and it was suspected that the fragments co-migrated during electrophoresis. From this Xma I band, IPCR products of-2484 bp (-5000 bp minus 2516 bp) for the left side and -1698 bp (-5000 bp minus 3302 bp) for the right were predicted. As expected, bands of-1.4 kb (IPCR41) and -1.7 kb (IPCR411) were amplified from BKME-941 templates digested with Sal I and Xma I, 40 1 2 3 4 2> Fig. 12. (A) Southern blot analysis of Tn5 insertion. Restriction endonucleases are BamHI (lane 1), Xmal (lane 2), Sail (lane 3), Sphl (lane 4) and Aatll (lane 5) ( B ) Agarose gel ( 0 . 8 % ) electrophoresis of IPCR products from mutant BKME-941 DNA showing products for DNA flanking both sides of the transposon (Aatll, lane 1) and the right side of the transposon (Sail, lane 2 and Xmal, lane 3). M: Molecular weight marker. 41 respectively (Fig. 12 B). Although the IPCR yielded some small non-specific products, no attempt was made to optimize the reaction since amplified fragments of the predicted size were easily cloned using the TA cloning system (Invitrogen). DNA sequencing of the cloned IPCR products using Ml 3 primers established the presence of short Tn5 DNA sequences flanking the amplified genomic sequence in addition to the restriction enzyme site created by the ligation for template preparation. The sequence analysis of IPCRM41 revealed that the transposon had inserted into a gene with similarity to genes encoding the a subunit of ring-hydroxylating dioxygenases. Interestingly, this mutant had also lost the ability to grow on abietic acid, a nonaromatic diterpenoid (Table 3). Screening of the P. abietaniphila wild-type cosmid library using the 1.4 kb IPCRM41 inverse PCR product as a probe produced nine distinct positive E. coli cosmid clones. No dioxygenase activities were detected when these positive clones were tested for the production of indigo from indole and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid from 2,3-dihydroxybiphenyl, which are two colorimetric assays for ring-hydroxylating and ring-cleavage dioxygenases, respectively. Moreover, these E. coli cosmid clones did not metabolize DhA or 7-oxoDhA in resting cell suspension assays. However, two of the library clones (pLC12 and pLC162) were able to metabolize 7-oxoDhA, but not DhA, in cultures growing on LB broth. The IPCR procedure described here is a rapid and simple alternative method to isolate DNA flanking Tn5 insertions. This method is particularly useful in those cases when a fragment of suitable size for cloning or IPCR cannot be generated using enzymes that do not cut within Tn5 (Fig. 10). I have found this to be the case with several Tn5 insertions in P. abietaniphila BKME-9. These IPCR products can be used to quickly obtain sequence information on the region of insertion of the transposon without tedious subcloning of mutant genomic DNA. In addition, 42 amplification of only one side of the transposon eliminates the potential problem of using a non-contiguous probe generated by the previously described IPCR technique as illustrated for Aat II ligated templates on the left side of Fig. 10. Table 3. Phenotypic characterization of Pseudomonas abietaniphila mutants Strain Gene mutated Growth substrate3 DhA AbA 7-oxoDhA pyruvate BKME-9 wild-type + + + + BKME-94 diti - - - + BKME-941 ditAl - - - + BKME-95 ditH - - - + BKME-96 ditF - - - + BKME-97 ditR + + + + BKME-98 ditD + + + + BKME-93 ditC - - - + BKME-99 ditB + + + + BKME-91 dit A 3 - - - + BKME-91(pVM120) dit A3 +b +b + a +, growth; -, no growth b Growth restored but slower (48 h) than wild type (24 h) to reach stationary phase 3. Cloning and subcloning of the dit gene cluster The mutagenesis of P. abietaniphila BKME-9 with the Tn5 transposon followed by the screening of the P. abietaniphila gene library produced the cosmid clone pLC12 which contains the dit gene cluster described in this chapter. Subcloning of the pLC12 cosmid allowed the sequencing of a 16.7 kb DNA fragment containing 13 complete and 1 partial ORFs (Fig. 11 and appendix II for complete DNA sequence and amino acid sequence of putative gene products). This fragment was subcloned as two EcoRl fragments of 5.8 and 9.8 kb (pVMl and pVM2), and DNA from pVM2 was further subcloned as 3.6-kb EcoRl-Smal (pVM4) and 4.3-kb Smal-Smal fragments (pVM5) (Fig. 11). Custom designed primers were used to sequence DNA regions not 43 covered by the deletions in order to generate a contiguous DNA sequence. The complete sequence of ORF2 was obtained by sequencing directly from the cosmid pLC12 with custom primers designed from known sequence. To assure high quality DNA sequences, both strands of the 16.7 kb fragment were sequenced with each sequence overlapping the next. 4 . Genetic organization of the dit gene cluster and classification of mutants The dit DNA cluster of P. abietaniphila BKME-9 comprises ORFs with sequence similarity to catabolic and regulatory elements of biodegradation pathways, as well as a putative substrate transport system (Table 4). In order to determine the functional role of these ORFs in the diterpenoid degradation pathway, insertional mutations of nine putative genes were constructed by homologous recombination. The mutants were classified for their ability to grow on DhA, AbA and 7-oxoDhA, three abietane diterpenoid substrates which support the growth of wild-type P. abietaniphila BKME-9 (Bicho et al. 1995). Insertional mutations in six ORFs resulted in the loss of growth on all three diterpenoid substrates, whereas, mutations in three ORFs did not affect the growth of the strains on abietanes (Table 3). Interestingly, all mutants which lost the capacity to grow on DhA also failed to grow on the nonaromatic diterpenoid AbA as well as the pathway intermediate 7-oxoDhA (Table 3). These results indicated that a common pathway may be used for the biodegradation of these three diterpenoids. Furthermore, the loss of growth of the mutants on all three substrates also suggested that the mutated genes did not encode enzymes required for the transformation of AbA to DhA or DhA to the pathway intermediate 7-oxoDhA. Because 14C-labelled abietanes are not commercially available and are difficult to synthesize in the laboratory, I could not perform substrate uptake assays. I therefore decided not 44 CJ 3 1X5 u S <L> OO . & I 0S* 2 CJ 3 ! • § ! 1 55 w | Q B 9 > 1> O o . 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In addition, because the second chapter of my dissertation is devoted to the characterization of the aromatic ring-hydroxylating dioxygenase, the description of the genes encoding the aromatic ring-hydroxylating dioxygenase enzyme (ditAlA2A3) and their regulator (ditR) is reported in chapter two. The other open reading frames found in the vicinity of these genes are described in the following sections. 5. The extradiol ring-cleavage enzyme DitC. The gene ditC encodes an extradiol ring cleavage dioxygenase. A 1.2-kb fragment containing ditC was cloned into pEXlOOT where it was under the control of the lac promoter. This plasmid construct was transformed in the heterologous host E. coli which resulted in a strain capable of the extradiol cleavage of 2,3-dihydroxybiphenyl (2,3-DHB) as indicated by the formation of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid which shows up as yellow colonies when sprayed with a solution of 2,3-DHB. This ring cleavage dioxygenase activity was not observed when cosmid library clones containing ditC were tested with 2,3-DHB. This result might be explained by a lack of ditC expression from its native promoter in E. coli or by an undetectable activity due to low cosmid copy number combined with low activity towards 2,3-DHB. Amino acid sequence analysis of DitC revealed that it is a two domain type I extradiol dioxygenase which contains the consensus sequence, from the PROSITE database, for extradiol dioxygenases between residues 240 and 261. Phylogenetic analysis (data not shown) of DitC indicates that it belongs to the 1.3 family of dioxygenases which includes enzymes with preference for bicyclic substrates (Harayama and Rekik 1989). This classification of DitC also conforms to the phylogenetic scheme proposed by Eltis and Bolin (Eltis and Bolin 1996), since DitC has approximately 30% identity with several enzymes of this family (Table 4). However, DitC 47 appears to represent a new subfamily since it has less than 54% identity to all extradiol dioxygenases found in GenBank. Cell suspensions of the ditC mutant strain BKME-93 oxidized DhA and produced a yellow-colored medium. The UV-Vis absorbance spectrum of the supernatant showed maxima at X 261 and 360 nm. GC analysis of the supernatant medium of cell suspensions showed the transient accumulation of eight compounds. From the GC-MS analysis of the methyl-ester derivatized metabolites, the following tentative structures were assigned to four of the compounds (Fig. 13 and Appendix III for the MS spectra); IV, 7-hydroxyDhA (M+ 330, base peak [bp] 237); V, 7-oxoDhA (Jvf 328, bp 253); VII, 7-oxo-ll,12-dihydroxyDhA (M* 374, bp 299); VIII, 3,7-dioxo-11,12-dihydroxyDhA (M+ 388, bp 313); The identity of compound V was confirmed by using a pure analytical standard, whereas compound IV was synthesized from the sodium borohydride reduction of V. A compound with the identical molecular ion as VII was previously characterized as an intermediate of DhA degradation (Biellmann et al. 1973) and is presumably the oxidation product of the dihydrodiol intermediate characterized from P. abietaniphila (see chapter two). The tentative identification of compound VIII was deduced from the combination of its ion fragmentation pattern and a previous report on the formation of a 3,7-dioxo diol degradation intermediate of DhA (Biellmann et al. 1973). Compound VIII had a similar ion fragmentation pattern as VII, with the addition of a mass of m/z 14 to the M4" and base peaks. This structure assignment is also in agreement with the ion fragmentation pattern reported for the addition of a oxo functional group to DhA (Brownlee and Strachan 1977). The structure assignment of compounds VII and VIII was tentative and relied on the interpretation of the MS data in comparison to what is known about the MS characteristics of previously identified metabolites. These intermediates are produced transiently and in small quantities in cell suspension 48 4 9 assays and may be unstable (see Discussion). Therefore, their isolation and purification for further structure analysis may be difficult. 6. ditD and di tH The two ORFs ditD and ditH encode putative proteins that have weak sequence identity to both HpcE and HpaG (Table 4), two dual function 5-oxo-l,2,5-tricarboxylic-3-penten acid decarboxylase/isomerase enzymes responsible for the fourth and fifth steps in the extradiol catabolic pathway of homoprotocatechuate in E. coli (Prieto et al. 1996; Roper et al. 1993). The analysis of DitH using the BLASTP program revealed that its highest sequence identity was to an ORF of unknown function from Archaeoglobus fulgidus (Table 4). Inspection of the neighboring ORFs on the A. fulgidus genome did not identify genes potentially involved in aromatic catabolic pathways. Analysis of the multiple sequence alignment showed that the sequence similarity of most proteins in the GenBank database to DitH was confined to the C-terminal region (data not shown). The dual function HpcE and HpaG isomerase/decarboxylase enzymes are thought to have evolved from a gene duplication since their respective N-terminal domains show a high sequence similarity to their C-terminal halves (Roper and Cooper 1993). In contrast, the C- and N-terminal halves of DitH did not show significant similarity to each other. In addition, the N-terminal sequence of DitH showed no significant similarity to other proteins in GenBank. DitH may have evolved from the fusion of a gene encoding a decarboxylase/isomerase to another gene as described for pcaL (Eulberg et al. 1998). The predicted translation product of ditD had similarity to several putative gene products of unknown function and shared 30% identity of its aligned amino acid to DitH (Table 4). The putative stop codon of ditC is located one nucleotide upstream of the ditD start codon and the predicted ditE start codon overlaps the putative stop codon of 50 ditD by 2 nucleotides. Therefore, ditCDE are presumably co-transcribed. The coding sequences of ditH and ditG also overlap by 2 nucleotides and these two genes are most likely co-transcribed. 7. ditB, ditG a n d diti The three putative proteins encoded by ditB, ditG and diti showed similarity to proteins of the short chain alcohol dehydrogenase/reductase (SDR) superfamily (Joernvall et al. 1995) (Table 4). The genes encoding the cleavage dioxygenase DitC and the ferredoxin DitA3 are separated by ditB (Fig. 11). This location of ditB in the gene cluster suggests that it may encode a dihydrodiol dehydrogenase since the genes encoding this enzyme are frequently located adjacent to their ring-cleavage dioxygenase counterpart (Fig. 7). However, its putative amino acid sequence shows little sequence similarity to known dihydrodiol dehydrogenases of aromatic degradation pathways (Table 4). Cell suspensions of E. coli harboring pVM4, a plasmid containing di(A3BC under the control of the lac promoter, did not produce a yellow color from 7-oxo-l 1,12-dihydrodiolDhA, the product of the ring-hydroxylating dioxygenase (see chapter two, Fig. 18). Furthermore, a mutation in ditB did not impede DhA degradation (Table 3). However, because the location of the xylE-GmT cassette insertion was close to the C-terminus (17 residues away), it may not have prevented the expression of a functional enzyme. Alternatively, the ditB mutation may have been complemented by another dehydrogenase present in strain BKME-9. The diti gene was found to encode a protein required for the growth of strain BKME-9 on abietanes (Table 3) but its role in the catabolic pathway was not elucidated. 51 8. ditE and ORF2 The analysis of the deduced amino acid sequences of ditE and ORF2 indicated similarity to membrane-bound permease proteins of the major facilitator superfamily (MFS) (Table 4). However, these two putative permeases showed no significant a. a. sequence similarity to permeases of the aromatic acid:H+ symporter family which are frequently associated with aromatic acid catabolic pathways (Pao et al. 1998). Computer-assisted transmembrane topology predictions with TMpred and hydropathy plots of the deduced amino acid sequences identified 12 potential transmembrane helices in the ORF2 gene product and 11 in DitE (not shown). The protein encoded by ditE contains a ~138-residue hydrophilic C-terminal segment of unknown function. Pairwise a. a. sequence comparison between DitE and TetV, a tetracycline efflux pump protein (De Rossi et al. 1998), indicated 23% identity between aligned residues. Interestingly, wild-type P. abietaniphila BKME-9 is hypersensitive to tetracycline, with an minimum inhibitory concentration of < 0.05pg/ml. This raised an interesting question: is DitE a permease involved in diterpenoid transport which fortuitously transports tetracycline? Comparison of the deduced amino acid sequence of ORF2 to GenBank databases showed very weak similarity only to proteins with undetermined function (Table 4). From the predicted topology of the translated permease-like ditE gene product and its genetic locus, I postulate that it may be involved in transport of diterpenoids into the cell, but I provide no further evidence for this. 9. ditFmd ORF1 The gene ditF encodes a 397-amino acid protein with similarity to 3-ketoacyl-CoA thiolases and sterol carrier proteins, SCP-X. SCP-X are multi-function eukaryotic proteins with thiolase activity encoded in the N-terminal domain, which also promote the exchange of a variety 52 of lipids and sterols between membranes in vitro (Seedorf et al. 1994a). The sterol carrier activity is encoded in the 143-amino acid C-terminal domain of the protein (Seedorf et al. 1994b), a domain which is not present in DitF. Although the predicted size of DitF corresponds to the average size of thiolases (-400 residues), it does not contain the two highly conserved and biologically important cysteine residues believed to form an acyl-enzyme intermediate and involved in deprotonation in the condensation reaction. In addition, ditF does not encode a PROSITE thiolase signature sequence; although, one region (residues 340 to 356) shows 7 of the 11 conserved residues of the thiolase consensus sequence (not shown). From this sequence analysis, I concluded that ditF probably does not encode a thiolase enzyme. The hydropathy plot of DitF suggests that it has a topology similar to membrane fusion proteins (MFP) (Dinh et al. 1994) (Fig. 14). A short hydrophobic segment of 22 amino acids at the N-terminal region of the protein (96-WLMMTQGVMLIAAAALSVLSSL-l 17), which could be predicted as a transmembrane helix, is followed by a moderately hydrophobic region and a long stretch of hydrophilic residues followed by a second short hydrophobic region in the C-terminus. Although the size of DitF (397 residues) corresponds closely with the average size of MFPs (422 +13% residues), it shows no similarity to MFPs at the primary sequence level. However, known members of the MFP family also show limited a.a. similarities which may indicate that the comparison of secondary and tertiary structures would be more appropriate. The function of this putative protein was not resolved but its similarity to proteins involved in the metabolism of sterol suggests that it may have a role in the catabolic pathway of resin acids. Disruption of ditF resulted in the inability of the mutant strain, BKME-96, to grow on DhA (Table 3) or consume DhA in a cell suspension (data not shown). This result was unlike those with other mutants that had lost the ability to grow on DhA but could consume the substrate in cell suspension assays. Preliminary 53 I I I I 100 I > 200 I I i I 300 Residue Number & o.o 1-0-5 <-1.0 100 Fig. 14. A. Hydropathy plot of the deduced amino acid sequence of ditF gene product and B. Average hydropathy plot of twenty sequenced membrane fusion proteins (reproduced from Dinh et al. 1994). The plot in panel A was generated using the method of Kyte and Doolittle (1982). 54 data on the analysis of the expression of this gene in P. abietaniphila is presented in Appendix I. Although the preliminary results established the requirement of DitF for abietane degradation, its precise function in the pathway was not resolved. The deduced translation product of the partial open reading frame ORF1 was found to have similarity to Co A ligases (Table 4). Since the putative ORF1 gene was not mutated in P. abietaniphila, its involvement in the resin acid degradation pathway was not determined. However, tdtL, a gene encoding a protein with 85% sequence identity to ORF1, was found to be required for the growth of Pseudomonas sp. A19-6a on AbA (Cam Wyndham, Dept. of Biology, Carleton University, Ottawa, personal communication). This result suggests that ORF1 may be involved in resin acid degradation. In summary, by Tn5 transposon mutagenesis and inverse PCR, a 16.7-kb DNA fragment was cloned from P abietaniphila BKME-9 and sequenced. This fragment was found to contain 13 complete and one partial ORFs. From the nine putative genes that were disrupted by insertional inactivation, six were required for the growth of strain BKME-9 on DhA, AbA and 7-oxoDhA. This chapter also identified the protein encoded by one of those genes, ditC as an extradiol ring-cleavage dioxygenase. 55 CHAPTER TWO: Characterization of a novel aromatic ring-hydroxylating dioxygenase 1. Introduction Ring-hydroxylating dioxygenases are central to the biodegradation of aromatic compounds. The first step in the aerobic degradation of aromatic rings is usually the formation of a dihydrodiol by a dioxygenase enzyme (Fig. 6). A large body of literature exists which describes ring-hydroxylating dioxygenases isolated from Pseudomonas species. All of the enzymes described are multicomponent systems comprising of two to four proteins (Fig. 8). Variations in the composition of these proteins have led to their classification into three groups (Batie et al. 1992). The second and third class of dioxygenases are three component enzyme complexes made up of a ferredoxin reductase, a ferredoxin and a two-subunit oxygenase. The ferredoxins characterized to date all contain a [2Fe-2S] iron-sulfur cluster of either the plant-type, as in pyrazon and dibenzofuran dioxygenases (Bunz and Cook 1993; Sauber et al. 1977), or Reiske-type, as in toluene dioxygenase (Subramanian et al. 1985). The sequence analysis of the dit gene cluster described in chapter one identified, among others, genes encoding a putative ferredoxin and the a and (3 subunits of a dioxygenase. Unlike most ring-hydroxylating dioxygenases, the gene encoding this putative ferredoxin was not clustered with the genes encoding the terminal oxygenase or the reductase (Martin and Mohn 1999b). Furthermore, the primary sequence analysis indicated that this putative ferredoxin was unlike any other ferredoxins previously characterized in gram-negative Proteobacteria. Therefore, I set out to determine if this putative ferredoxin, together with the previously identified oxygenase, are components of a member of new class of diterpenoid ring-hydroxylating dioxygenases. 56 2. Identification and sequence analysis of the oxygenase genes ditAl and ditA2 Two ORFs, designated ditAl and ditA2 (Fig. 11), were similar to the genes encoding the a and (3 subunits of the oxygenase components of several bacterial ring-hydroxylating dioxygenases. The DNA sequence of the inverse PCR product (IPCRM41) was in perfect agreement with the sequence of ditAl, confirming that this gene was corresponding to the one disrupted in strain BKME-941. The deduced amino acid sequences of DitAl and DitA2 consisted of polypeptides of 469 (DitAl) and 201 (DitA2) amino acids, with calculated molecular masses of 52.5 and 22.9 kDa, respectively. From the primary amino acid sequence alignment of DitAl and a naphthalene dioxygenase (NDO) for which the three-dimensional structure was recently solved (Kauppi et al. 1998), I identified the highly conserved consensus sequences for the coordination of a [2Fe-2S] Rieske-type cluster (Fig. 15) and a catalytic non-heme iron. The [2Fe-2S] cluster-binding sequence (Cys-X-His-Xn-Cys-X2-His) was located at residues 91 to 114, and the catalytic iron coordination residues were His219, His224 and Asp399 (corresponding to His 208, His213 and Asp362 of NDO). Although these features, which are common to the a subunit of ring-hydroxylating dioxygenases, are present in DitAl, it has only weak overall sequence similarity to other proteins of this family. The a subunits of biphenyl dioxygenases from several Rhodococcus sp. strains showed the highest sequence similarity to DitAl, exhibiting up to 30% identity (Table 4). Both ditAl and ditA2 are translated from the ATG start codon and are preceded by a potential ribosomal binding site. A 25 bp stem-loop located 43 bp downstream of the ditA2 gene probably serves as a rho-independant transcription termination site, indicating the likely end of an operon. The segments of cloned DNA flanking ditAlA2 did not contain genes similar to those encoding electron transport proteins typically associated with ring-hydroxylating dioxygenases. 57 BphAl.RHAl BphA.LB400 CmtAb.Fl NdoB.NCIB9816 TftA.ACllOO DxnAl.RWl TdnAl.UCC22 DitAl BKME-9 C R H R G M R I C R A D G G N A K S F T C S Y H C R H R G M R I C R S D A G N A K A F T C S Y H C P H R G A T V C R E R S G N S K N F Q C F Y H C R H R G K T L V S V E A G N A K G F V C S Y H C R H R G A L L C P F S K G N Q K F H V C R Y H C R H R G N T L C L A D R G N A K S F R C S Y H C S H R G A S V C R E H R G N A A G F T C P Y H C P H R G M R I S T A D C G N T Q I H K C I Y H Fig. 15. Alignment of protein sequences of [2Fe-2S] binding domains from a subunits of several classes of dioxygenases. Amino acids in bold are residues involved in [2Fe-2S] binding. The protein and strain designations, substrate for the enzyme, and GenBank accession numbers are as follows: BphAl.RHAl, biphenyl, Rhodococcus sp. RHA1 (D32142); BphA.LB400, biphenyl, Burkholderia cepacia LB400 (M86348); CmtAb.Fl, /?-cymene, Pseudomonas putida Fl (U24215); NdoB.NCIB9816, naphthalene, Pseudomonas putida NCIB9816 (M23914); TftA.AC1100, 2,4,5-trichlorophenoxyacetic acid, Burkholderia cepacia AC1100 (U11420); DxnAl.RWl, dioxin, Sphingomonas sp. RW1 (AJ223219/223220); TdnAl.UCC22, aniline, Pseudomonas putida UCC22 (D85415); DitAl.BKME-9, dehydroabietic acid, Pseudomonas abietaniphila BKME-9 (AF119621). 58 3. Identification and sequence analysis of the ferredoxin gene ditA3 Alignment searches using the BLAST programs (Altschul et al. 1990) identified one ORF from the dit cluster which encoded a protein with similarity to both [4Fe-4S]- and [3Fe-4S]-type ferredoxins. This ORF, designated as ditA3, was located 9.2 kb upstream of ditAl and is transcribed from the same strand. Although five potential methionine start codons are possible for this ferredoxin gene, it is likely that the start codon encoding a 78 a.a. protein (the smallest of the five possible ferredoxins) is the correct codon, since the protein alignments to similar ferredoxins did not extend to the N-terminus of the longer possible DitA3 ferredoxins (Fig. 16). Examination of the nucleotide sequence preceding this ORF identified a possible ribosomal binding site (GGAGA) and a putative a54-like promoter consensus sequence, TGGAGCN5TTGCA (Dixon 1986), 89 bp upstream of the Met start codon. Percent a.a. identities of the putative DitA3 ferredoxin to [4Fe-4S] or [3Fe-4S] ferredoxins are low, ranging from 22 to 36% for the ferredoxins listed in Fig. 16. Analysis of the amino acid composition of DitA.3 reveals that it contains five cysteine residues. This protein is unusual in that its putative consensus sequence for [Fe-S] cluster coordination consists of three Cys and one Tyr residues (Cys-X2-Tyr-X2-Cys-X„-Cys) rather than the typical four Cys (Fig. 16). Deviations from the consensus sequence have been observed in some proteins where Asp can act as a fourth ligand (Adams and Zhou 1997) or in the case of Ala substitution where the protein adopts a [3Fe-4S] form (O'Keefe et al. 1991). It is impossible to predict if the Tyr residue in DitA3 can act as a cluster coordinating ligand; thus, the cluster geometry will have to be determined experimentally. The ditC gene which encodes a diterpenoid meta ring-cleavage dioxygenase genes was located 872 bp downstream of the dit A3 gene (Fig. 11). This data suggested that this ferredoxin and the terminal oxygenase, DitAl A2, previously identified, are components of a diterpenoid ring-hydroxylating dioxygenase which 59 < n o r - r - o r < i o > L O « » i H H C N t r ) t - o o a ) CM CS C9 CS CS CS <CS CS CS <^f<! CD H H H H H H > > U H H H M a H 9£ 8 5 „ .. > > H H _ " s fi E E H b H b ,3 e . 3 At <H *5 <H +J Ft •H •H • r l U - r l H 0> H 0> • CI> • CQ • CD CO I CO O <S I CD CO I CO <B CJ) XI M 4 i n i J J i ? • ' D ' d i C ' o • < O D • • • "o • • -H •U -P J J 21 O" Ov Ov C Q 11 ' » 5 ) H © _S .2 H i ^ c» ,<SJ T 3 60 together with the cleavage dioxygenase DitC form a dioxygenolytic pathway. 4. Phenotypic characterization of the ferredoxin (ditA3) mutant BKME-91 To determine the possible role of the putative ferredoxin in diterpenoid degradation, a chromosomal mutation in dit A3 was constructed by allelic exchange, to insert a xylE-Gvcf transcriptional fusion cassette containing no terminator sequence (Schweizer and Hoang 1995). DhA was added to cell suspensions of BKME-9 (wt), BKME-941 (ditAl :.Tn5) or BKME-91 (dilA3::xylE-GmT), and the metabolites were analyzed by GC-MS and UV-Vis absorption. The strain carrying the ferredoxin mutation exhibited the same phenotype as the strain with the a subunit Tn5 mutation (Fig. 17 and Table 3). GC-MS analysis of the medium from both mutant strains identified 7-oxoDhA as the most abundant metabolite. UV-Vis absorption spectra of culture supernatants from the two mutants were nearly identical, with a major peak at 260 nm. But, comparison to pure 7-oxoDhA dissolved in mineral medium yielded a spectrum in which this major peak was centered at 253 nm peak (Fig. 17). This difference was probably due to residual DhA in the medium of the mutant strain cell suspensions, as observed by GC-FID. The spectra clearly indicate the appearance of a peak in the 300 to 310 nm region, indicating the formation of a ketone. To rescue the ferredoxin mutation and discount possible polar effects caused by the insertion of the xylE-GmT cassette, a 891 bp Nae I fragment was cloned from pVM4 into the Sma I site of the broad-host vector pUCP27, resulting in pVM120 containing dit A3 under control of the lac promoter. Although strain BKME-91 harboring pVM120 did not completely revert to wild-type phenotype, it regained the ability to grow, albeit slower, on DhA (Table 3). Interestingly, the rescued mutant showed the transient accumulation of a yellow colored compound(s) which had the same absorption spectrum as the supernatant from the ditC mutant 61 3 230 280 330 380 Wavelength (nm) Fig. 17. Absorption spectra of 7-oxoDhA (—) and supernatants from cell suspensions incubated with DhA for 10 hrs; wild-type, BKME-9 (— ); ditAl::Tn5, temiinal oxygenase mutant strain BKME-941 (— ); ditA3vjcylE-Grd, ferredoxin mutant strain BKME-91 (— ). 62 strain cell suspension culture fluids. This result suggested that the xylE-GmT insertion impaired the expression of ditC located downstream (Fig. 11) and probably explains why the rescued mutant strain did not fully regain the wild-type phenotype. 5. Oxidation of 7-oxoDhA by recombinant diterpenoid dioxygenase expressed in E. coli Diterpenoid dioxygenase activity was reconstituted in E. coli from a two-plasmid expression system similar to the one described by Armengaud et al. (1998). Plasmids pVM20, containing the genes encoding the oxygenase (ditAlA2), and pVMlO, containing the gene encoding the ferredoxin (ditAS), were introduced into E. coli XL1 Blue MR. When a cell suspension of this strain was incubated with 7-oxoDhA, the 7-oxoDhA was removed, and two metabolites were detected by GC-FID (Fig. 18). GC-MS analysis of the methyl ester derivatives showed that a third peak detected (not shown) was an artifact, resulting from the incomplete methylation of an hydroxyl group, as seen by a difference in mass of+14 for the molecular ion and the base peak compared to the major metabolite. Interestingly, substrate transformation was only observed in cell suspensions supplemented with 0.1% glycerol. Since the putative reductase component of the diterpenoid dioxygenase was not cloned, it is likely that a reductase of the E. coli host substituted for the missing ferredoxin reductase in actively growing cultures as previously observed (Bergeron et al. 1994; Kurkela et al. 1988). Parallel experiments demonstrated that the E. coli strain expressing the diterpenoid dioxygenase is unable to transform DhA. A control strain expressing only dit AS, E. coli XL1 Blue MR (pBBRlMCS-2/pVM10), did not remove 7-oxoDhA in the same assay. Surprisingly, a control strain expressing only ditAlA2, E. coli XL1 Blue MR (pVM20/pEX100T), removed approximately 5% of the 7-oxoDhA (Fig. 18) and formed proportional amounts of the metabolites described above (not shown). 63 1 4 0 0 0 0 2 4 6 8 Time (hrs) Fig 18 Removal of 7-oxoDhA by cell suspensions of E. coli XL1 Blue MR expressing DitA3 (pVM10/pBBRlMCS-2) (•), DitAl A2 (PEX100T/pVM20) (O) or DitAl A2 plus Dit A3 (pVM10/pVM20) (A); and formation of the dihydrodiol (•) and a minor metabolite (A) by the latter. 64 Additionally, I used plasmid pAJ130 encoding a functional ferredoxin (Fdxl) and its reductase (RedA2) from the dioxin dioxygenase of Sphingomonas RW1 (Fig. 8) (Armengaud et al. 1998) to evaluate the functionality of a class-IIA dioxygenase electron supply with the diterpenoid dioxygenase. Cell suspensions of E. coli (pVM20/pAJ130) expressing the genes encoding the class-IIA electron transfer proteins and DitAl A2 did not transform 7-oxoDhA. 6. Purification and identification of dioxygenase oxidation product I expected Dit A to transform 7-oxoDhA to an nonaromatic dihydrodiol. Consistent with this expectation, the evidence provided by the UV-Vis (Fig. 19), MS (appendix III) and "H NMR spectra indicate that the major metabolite produced by the diterpenoid dioxygenase is 7-oxo-ll,12-dihydroxy-8,13-abietadien acid (Fig. 13, compound VI). The substrate (7-oxoDhA) had the following characteristics: Rf value of 0.39 by TLC in benzene:methanol:acetic acid (79:20:1). UV-Vis (methanol) 213, 253, 301 nm; GC-MS (methyl ester), molecular ion [M (% relative intensity)] at m/z 328 (21) and major fragment ions at m/z 296 (15), 269 (11 ) and 253 (100); "H NMR (CD3OD), 5 1.25 (7=6.94 d 6H,) 6 1.29 (s IH), 51.34 (s IH), 5 7.39 (J=8.22 d IH), 5 7.48 (7=1.98 7=2.11 dd IH), 5 7.81 (7=2.09 d IH). The major metabolite had the following characteristics: Rf value of 0.35 by TLC in benzene.methanol.acetic acid (79:20:1). UV-Vis (methanol) 215, 310 nm; GC-MS (methyl ester), molecular ion [M (% relative intensity)] at m/z 358 (62) and major fragment ions at m/z 343 (10), 326 (31), 283 (89) and 284 (100); high resolution MS, apparent molecular ion at m/z 330.18231 (7) corresponding to a formula of C20H26O4 and a base peak at m/z 269.15532 (100) corresponding to C i 8 H 2 i 0 2 ; 'H NMR (CD3OD), 5 1.07 (7=6.81 d 3H), 5 1.12 (7=6.80 d 3H), 5 1.27 (s 3H), 5 1.35 (s 3H), 6 4.24 (7=6.08 m IH,) 5 4.38 (7=4.99 d IH), 6 6.24 (7=3.53 m IH). 65 UV-Vis spectrum of the purified metabolite clearly shows the loss of aromaticity (Fig. 19) as evidenced by the loss of the peak at 253. Further evidence for the production of the diene came from the 'H NMR spectra which show the loss of all three aromatic protons seen in 7-oxoDhA (6 7.39, 7.48, 7.81) and the appearance of one alkene proton on C-14 (8 6.24) and two methine protons adjacent to the OH groups (8 4.24 and 4.28). In addition, the CH 3 signal(s) for the isopropyl group of 7-oxoDhA occurs as a doublet at 8 1.25 but is split into two doublets at 8 1.07 and 1.12 for the metabolite, indicating the loss of aromaticity. The mass spectra of 7-oxoDhA and the major metabolite is in agreement with the EI mass fragmentation scheme of this family of compounds as previously described (Brownlee and Strachan 1977; Enzell and Wahlberg 1969). GC and high resolution mass spectra of the major product showed a compound with an M 4 of -18 (H20) relative to that of the expected dihydrodiol structure. High resolution mass spectral analysis by chemical ionization with NH 3 also did not produce the expected molecular ion. Dehydration would be expected given the unstable nature of this dihydrodiol. 7. Evidence of a convergent pathway for abietane degradation Strains with a mutation in genes coding for this DitA (ditAl or ditA3) accumulated the pathway intermediate 7-oxoDhA in cell suspension assays with DhA (Fig. 20 A). Furthermore, the disruption of genes encoding the aromatic ring-hydroxylating enzyme also resulted in the loss of growth on the nonaromatic substrate AbA (Table 3), I hypothesized that P. abietaniphila aromatized AbA to DhA prior to aromatic-ring attack. To test this hypothesis, the ditAl::Tn5 mutant strain BKME-941 was incubated in cell suspension assays in the presence of AbA and palustric acid (PaA), two nonaromatic abietane diterpenoids. The intermediate 7-oxoDhA was produced from both substrates (Fig. 20 B and C), and DhA was produced from PaA (Fig. 20 C). 66 210 260 310 360 Wavelength (nm) Fig. 19. Absorption spectra of 7-oxoDhA and the product of the dioxygenase, 11,12-dihydroxy-8,13-abietadien acid. 67 A Oh JU J 10 1 B Oh 15 lO 24 h 15 area GC peak * \o2H ^ 0 2 H - l - l ' 10 15 10 15 C Oh LLJL 24 h uu 15 lO 15 Time (min) Fig. 20. GC-FID analysis of the dehydroabietic acid (A), abietic acid (B) and palustric acid (C) biotransformation products of P. abietaniphila BKME-941 (ditAL:Tx\5) at 0 hrs and 24 hrs of incubation. 68 Although it is likely that DhA is an intermediate in the conversion of AbA to 7-oxoDhA, DhA was not observed when using AbA as substrate (Fig. 20 B). Two additional compounds, one produced from AbA and the other from PaA were observed (Fig. 20 B and C). Although they were not identified, I suspected that they might be 7-oxo derivatives of AbA and PaA, as previously identified (Cross and Myers 1968). Approximately 90% of the carbon was recovered as substrate or identifiable metabolites, after 24 hrs of incubation with DhA or PaA. The carbon mass balance was poor for AbA, with approximately 40% recovery. However, abiotic controls also showed a loss of 22% of the AbA carbon. The transformation of AbA and PaA to DhA or isomerization of PaA to AbA did not occur in the abiotic incubations of the substrates in mineral medium. These spontaneous reactions have been previously reported under some conditions (Stoltes and Zinkel 1989). These results unambiguously demonstrated that under the experimental conditions used, the aromatization of AbA and PaA to DhA and/or 7-oxoDhA is a biological process. 8. Analysis of ditAl and ditA3 gene expression and inducer specificity The expression of the genes encoding the a subunit and ferredoxin components of the diterpenoid dioxygenase was analyzed by measuring catechol 2,3-dioxygenase (C230) activities of ditAlwxylE (strain BKME-92) and ditA3:.xylE (strain BKME-91) transcriptional fusion strains. Wild-type P. abietaniphila BKME-9 cultures grown on pyruvate and subsequently induced with diterpenoids showed no endogenous C230 activity (data not shown). Pyruvate-induced cultures of strains BKME-91 and BKME-92 showed basal levels of C230 expression (Fig. 21) which were not significantly different from those of uninduced cultures (Fig. 22). The specificity of ditA 1 and dit A3 induction was investigated by using AbA, DhA and 7-oxoDhA, which can serve as growth 69 Fig. 21. Expression of ditAl and dit A3 in response to various diterpenoids and aromatic compounds. Potential inducers were added at the following concentrations: 500pM DhA, AbA 7-oxoDhA, Pyr and IpB and lOOpM Cl2DhA, BpH, IpA and Phen. Error bars indicate standard deviation; n = 3. 70 I a O) E c E 120 100 80 60 & 40 > u (0 O CO CM O 20 0 ditAl ::xyIE i 60 50 40 30 20 10 0 ditA3::xylE i ditR- ditR- ditR+ ditR+ ditR- ditR- ditR+ ditR+ Fig. 22. Expression of ditAl and ditA3 in ditR mutant strains BKME-922 and BKME-912 (ditR) and in ditR mutant strains complemented with pVM220 (ditR+), a plasmid containing ditR and its predicted promoter from the EcoRW-BgUl fragment illustrated in Fig. 4. Cultures were not induced (black bars) or induced with 500 pM of 7-oxoDhA (gray bars). Error bars indicate standard deviation; n = 3. 71 substrates for strain BKME-9, as well as a chlorinated abietane (12,14-Cl2DhA) and a pimerane (IpA), which do not support the growth of strain BKME-9. All five diterpenoids induced ditAl and ditA3 expression (Fig. 21). Since I have demonstrated that AbA and DhA are transformed to 7-oxoDhA by ditAl and ditA3 mutants (Fig. 20), it is plausible that only 7-oxoDhA, the substrate for the dioxygenase, is the sole inducer of the dit A genes. However, considering the high C230 levels observed with the non-metabolizable diterpenoids Cl2DhA and IpA It is likely that ditAl and dit A3 expression was induced by AbA and DhA. Induction of the aromatic ring-hydroxylating dioxygenase appears to require diterpenoids, as the aromatic compounds biphenyl (BpH), isopropylbenzene (IpB) and phenanthrene (Phen), which are not growth substrates for P. abietaniphila, did not induce ditAl or dit A3 expression (Fig. 21). 9. Regulation of ditAl and ditA3 expression by DitR An ORF with similarity to regulatory proteins was identified (Table 4) between dit A3 and ditAl, 4.4 kb downstream of the ferredoxin gene and 3.8 kb upstream of the a subunit gene (Fig. 11). This ORF, designated ditR, is located 79 nucleotides downstream of ditE, and its expression may be controlled by its own promoter (Fig. 23). Analysis of the DNA sequence preceding the putative ribosomal binding site (RBS) shows -10 and -35 elements closely matching the E. coli promoter consensus sequence. I speculate that the inverted repeats which overlap the -35 sequence possibly serve as a binding site for DitR for the purpose of autoregulation. Comparison of the deduced amino acid sequence of DitR to the GenBank databases entries revealed low sequence identity to IclR-type transcription regulators (Sunnarborg et al. 1990) (Table 4). In addition to the sequence similarity, I also identified a potential helix-turn-helix (HTH) DNA binding motif by using the method of Dodd and Egan (1990), further suggesting that ditR encodes 72 a regulatory protein. Like regulators of the IclR-family, the a. a. sequence of the putative HTH motif (53-SVDLARVLGINPSTCFNT LR-71) of DitR is located in the N-terminal region of the deduced protein. In order to determine if DitR regulates expression of the dioxygenase at the transcriptional level, ditR was inactivated in the C230 reporter strains BKME-91 and BKME-92. A Kmr cassette was inserted in ditR and the selection of Gmr, Km' double mutants yielded strains BKME-912 (ditA3..xylE-Gm\ ditR.KnY) and BKME-922 (ditA 1 ::xylE-Gmr, ditR.Km1). C230 assays of strain BKME-912 showed that the 7-oxoDhA-induced expression level of dit A3 was similar to noninduced levels (Fig. 22). Electroporation of pVM220, a d/7/?-containing plasmid (Fig. 23), into strain BKME-912 restored the transcriptional regulation of dilA3 as shown by its wild-type expression level (Figs. 21 and 22). The expression of ditAl in uninduced cultures was increased three-fold in the absence of a functional ditR gene (Fig. 22). As for BKME-912, electroporation of pVM220 into BKME-922 restored the regulation of ditA 1 expression almost to wild-type levels. These results indicate that ditR encodes a inducer-dependent transcriptional activator ofditA3 and possibly a repressor of ditAl and possibly coordinates the expression of the ferredoxin and the oxygenase components of the dioxygenase. 73 ditE R-ditR ditF pVM220 EcoRV BglU -35 -10 Fig. 23. Physical map of the EcoR V-Bgl H fragment containing ditR cloned into pVM220 nucleotide sequence of thre intergenic region between ditE and ditR. The putative -10 and promoter elements are underlined and the putative +1 base is indicated with an asterisk, inverted repeat overlapping the -35 region is marked with arrows. 74 CHAPTER THREE: Diterpenoid inducible cytochrome(s) from P. abietaniphila BKME-9 1. Introduction Cytochromes P-450 are important enzymes in the metabolism of endogenous and exogenous hydrophobic compounds. In eukaryotes, cytochromes P-450 are known to play a role in the metabolism of endogenous steroid hormones, as well as exogenous compounds such as drugs and carcinogens. In prokaryotes, cytochromes P-450 are mainly involved in the catabolic degradation of compounds such as monoterpenes (Koga et al. 1985; Peterson et al. 1992; Ullah et al. 1983), herbicides (Nagy et al. 1995; O'Keefe et al. 1991), aromatic and nonaromatic cyclic compounds (Karlson et al. 1993; Warburton et al. 1990), cholesterol (Horii et al. 1990), fatty acids (Ruettinger and Fulco 1981) and alkanes (Asperger et al. 1984). However, the function of prokaryotic cytochromes P-450 are not limited to catabolic reactions. Recently, a cytochrome P-450 from a Streptomyces sp. was shown to catalyze multiple steps in the biosynthesis of the antibiotic, doxorubicin (Walczak et al. 1999). In view of the variety of substrate transformations that this enzyme family is able to catalyze, it would not be surprising to find that a cytochrome P-450 was involved in resin acid degradation. A second Tn5 transposon mutant of P. abietaniphila BKME-9 which had lost the capacity to grow on DhA was characterized. Sequence analysis from the inverse PCR product from this Tni mutant indicated that strain BKME-9 may harbor a cytochrome P-450 which is involved in DhA degradation. Furthermore, a gene (idtD) with sequence similarity to genes encoding cytochrome P-450 enzymes was recently identified in the resin acid-degrading bacterium Pseudomonas strain A19-6a (Dr. Cam Wyndham Carleton University, personal communication). The insertional inactivation of this putative cytochrome P-450 gene, demonstrated that it is 75 required for the growth of Pseudomonas strain A19-6a on abietic acid. Therefore, this third chapter reports on the results of experiments aimed at determining if P. abietaniphila uses a cytochrome P-450 enzyme for the degradation of resin acids 2. Phenotypic characterization of P. abietaniphila Tn5 mutant strain BKME-12 The Tn5 transposon mutagenesis of P. abietaniphila produced a mutant strain which lost the ability to grow on DhA and accumulated metabolites not previously observed from the mutation analysis of the dit cluster. Cell suspension assays of this strain, designated BKME-12, showed the transient accumulation of two metabolites when analyzed by GC-FID (Fig. 24 A). Several attempts were made to purify the two metabolites by high pressure liquid (HPLC) or thin layer chromatography (TLC) (data not shown). In every instance, what was identified as a single peak (HPLC) or a single spot (TLC), resolved as two peaks by GC-FID. GC-MS analysis of the methyl esters of the metabolite(s) produced two peaks with the mass spectra shown in Fig. 24 B. Although I could not confidently determine the identity of the metabolites, I suspected that a hydroxylated metabolite was transiently produced by the mutant strain. The more polar intermediate with a relative retention time (RRT) to DhA of 1.27 had abundant ions of m/z 312 and m/z 237 (Fig 24B ). These two fragments possibly represent the loss of H 2 0 (m/z 18) from hydroxylated DhA (DhA has a M + of m/z 314 and a base peak of m/z 239). A weak ion at m/z 331 in the spectrum of this metabolite may be the true M^ (Fig. 24 B). The second metabolite with a RRT to DhA of 0.66 had a base peak at m/z 239 with no discernable M + with a higher mass. Although I suspected that the observation of two peaks only by GC was an artifact of the analysis, it was impossible to confirm this suspicion from the MS data since a definite structure of the two compounds was not established, which might explain the unstable nature of the intermediate. 76 500000 450000 400000 to 350000 « 300000 2 250000 a O 200000 o 150000 100000 50000 0 0 B rrt 0.66 rrt 1.27 20 40 60 T i m e (hrs) 80 & JiJ i  iljililjiiiiujii, !i.Ji!,i!i!ul, wiiiijuiiaii!!!., Fig. 24. A. Cell suspension of P. abietaniphila Tn5 mutant strain BKME-12 showing the removal of DhA (A) and the production of two metabolites with relative retention times to DhA of 0.66 (•) and 1.27 (A ) and B. electron impact mass spectra of the metabolites as methyl esters. 77 3. Inverse PCR product from Tn5 mutant strain BKME-12 Southern blot analysis of restriction enzyme digested BKME-12 DNA indicated that the Tn5 insertion was located in mutant genomic EcoR I Kpn I, Aat II, Sac I or Sst I DNA fragments >8 kb (data not shown). Although EcoR I-digested DNA was found to produce a fragment of adequate size for IPCR amplification (predicted product of-2.5 kb), repeated attempts failed to amplify the Tn5 flanking region of EcoR I-digested self ligated BKME-12 DNA templates. Tn5 probe hybridization to strain BKME-12 DNA digested with restriction enzymes that cut the transposon once showed that Sal I, Sph I, or Xma I were potential enzymes for IPCR, whereas BamH I yielded fragment(s) that were too long for IPCR amplification (Fig. 25 A) (refer to Fig. 10 for a description of the IPCR principle). From the band pattern of the Southern blot, I predicted that amplification of the left side of Sal I-digested DNA would yield a -2100 bp product (-4700 bp minus 2684 bp); whereas, amplification of the right side would yield a product of-100 bp (-3200 bp minus 3134 bp). IPCR products from Sph I-digested templates would potentially yield a -1020 bp product (-3100 bp minus 2080 bp) for the left side and -460 bp (-4200 bp minus 3738 bp) for the right side of the Tn5 transposon. As expected, bands of -2.1 kb and -1.1 kb were amplified from Sal I- and Sph I-digested self-ligated BKME-12 DNA templates (Fig. 25 B). The amplified fragments were cloned in the TA vector, producing pEPCRM12, and sequenced using Ml3 primers. DNA sequence analysis revealed that the Tn5 transposon insertion was located in a region with sequence similarity to the NADH-oxidoreductases of cytochrome P-450 and alkane hydroxylase enzymes (Table 5). This oxidoreductase is probably not the one associated with the Dit A enzyme as the Tn5 mutants strains BKME-941 and BKME-12 accumulated different intermediates. The wild-type genomic library was screened by colony lift hybridization using the IPCR product from the Sal I prepared PCR template. One colony was 78 Fig. 25. Isolation o f D N A flanking the Tn5 transposon insertion from the P. abietaniphila mutant strain B K M E - 9 1 2 Panel A. Southern blot analysis o f mutant strain B K M E - 1 2 genomic D N A hybridized to the ///ndlll-itamHI D N A fragment from Tn5 (see Fig. 8). The restriction enzymes used were: Lane \,Aatll; lane 2, Sphl; lane 3, BamHI; lane 4, Smal; lane 5, Sail. Panel B. Agarose gel electrophoresis o f inverse P C R product from Sphl (lane 1) and Sail (lane 2) digested, self-ligated B K M E - 1 2 D N A template amplified with primers UTn5, SphITn5 and LSalITn5. The numbers on the left o f the blot and the right o f the gel (lane 3) represent molecular size markers in kb. 79 identified and this cosmid clone, designated pLCP450, was analyzed by Southern blot to confirm the presence of the IPCR sequence in the cosmid insert and to map it. No further subcloning or sequencing was done on this cosmid library clone. Table 5. Comparison of the deduced amino acid sequence of the inverse PCR products from the Tn5 mutant BKME-12 with those of similar proteins found in GenBank. Primer used for sequencing Identity of similar gene product % identity GenBank accession no. Universal Tn5 Rhodocoxin reductase3 41 P43494 RedA2b 34 AJ002606.1 Terpredoxin reductase3 32 P33009 Putidaredoxin reductase3 31 P16640 Rubredoxin reductase0 23 P17052 LSalITn5 no significant similarity NA NA SphITn5 several retinol dehydrogenases ND U33500d 3 Reductase component of cytochrome P450 monoxygenase b Reductase component of type-IIA dioxin dioxygenase c Reductase component of alkane hydrolase d Sequence with highest identity NA, not applicable; ND not determined 4. Cytochrome P-450 assays of diterpenoid-induced P. abietaniphila BKME-9 In order to determine if P. abietaniphila harbors a cytochrome P-450 enzyme, visible absorption spectra of soluble fractions of P. abietaniphila cells grown on pyruvate or DhA were measured. The soluble fraction of pyruvate-grown cells did not produce spectra typical of a cytochrome P-450 or cytochrome c (Fig. 26 A). In contrast, the air-oxidized and dithionite-reduced spectra of the soluble fraction of DhA-grown cells were similar to those of a cytochrome c, with a, (3 and y peaks at 550, 521 and 414 nm, respectively (Fig. 26 B). The absorption of the reduced soluble fraction from DhA-grown cells did not show a typical cytochrome P-450 type 80 spectrum but a small shoulder at 450 nm appeared upon sparging with carbon monoxide (indicated by an arrow in Fig. 26 B). The significance of this shoulder was unclear, but I suspected that another inducible cytochrome, possibly a c-type, might have masked the absorption spectrum of a possible cytochrome P-450. 5. Identification of a soluble, diterpenoid inducible, heme-containing protein To test the hypothesis that a c-type cytochrome was masking the absorption spectrum of a possible cytochrome P-450, crude lysates and soluble fractions of pyruvate- and DhA-grown cells were resolved by SDS-PAGE and heme-stained. Using this method, I assumed that c-type as well as P-450-type cytochromes would be detected. To test this assumption, the cytochrome P-450-containing Rhodococcus rhodochrous strain 116 grown on 2-ethoxyphenol (Eltis et al. 1993) was used as a positive control. The P-450 cytochrome from R. rhodochrous was easily detectable by visible light spectroscopy (data not shown). But, the P-450 from R. rhodochrous was not observed on a heme-stained SDS-PAGE gel on which myoglobin, a positive control for the heme-stain was detected (data not shown). A single cytochrome, with an apparent molecular mass of -20 kDa, was detected only in DhA-grown cells (Fig. 27). This cytochrome was detected in both the crude lysate and the soluble fraction. No further experiments were carried out to determine the nature and function of this putative cytochrome and to find the putative cytochrome P-450. In summary, initial results aimed at determining the presence of a cytochrome P-450 in P. abietaniphila BKME-9 were presented. However, the results from the cytochrome P-450 assays, Tn5 mutant phenotype and inverse PCR sequence could not, with certainty, confirm the presence of a cytochrome P-450 enzyme in P. abietaniphila BKME-9 81 Fig. 26. Visible absorption spectra of soluble fractions from pyruvate-grown (A) and DhA-grown (B) cultures of P. abietaniphila. Air-oxidized (—), sodium dithionite-reduced (—), FfcCvoxidized (— ) and sodium dithionite-reduced, CO-sparged (— ). The arrow in panel B identifies the shoulder formed in the spectrum upon sparging the sample with CO. 82 Pyruvate-grown DhA-grown Fig. 27. Heme-stained SDS-PAGE gel of crude lysate (lane: 2, 3, 6 and 7) and the soluble fraction (lane: 4, 5 and 8) of pyruvate- and DhA-grown cultures of P. abietaniphila. The amount of sample was adjusted such that lanes 2, 4, 6, 8 and lanes 3, 5, 7 were loaded with equal amounts of cellular material based on the optical density of the cultures. Lane 1: 1.5 pg of horseheart cytochrome c. The numbers on the left are molecular mass markers in kDa. 83 DISCUSSION This is the first report of the cloning and characterization of a gene cluster encoding enzymes that catalyze abietane diterpenoid degradation. Although the cluster described does not encode the complete pathway for abietane mineralization, it encodes several of the enzymes required for growth of P. abietaniphila BKME-9 on abietanes (Table 3). The organization and sequence uniqueness of the catabolic genes in the dit cluster is unusual. Pairwise comparison of the deduced amino acid sequences encoded by dit genes to similar proteins in databases showed at best 41% residue identity, with most proteins sharing identities of 30% or less (Table 4). This meant that I could not deduce the function of most genes from their sequences, as is often the case for gene clusters encoding aromatic degradation pathways in which a. a. sequence identities are >30%. Furthermore, a comparison of the genetic organization of gene clusters encoding aromatic hydrocarbon oxidation pathways to that of the dit cluster demonstrated no similarity in the order or relative location of homologous genes (Figs. 7 and 11), which suggests that these clusters are not closely related. In most instances, the three or four genes encoding the ring-hydroxylating dioxygenases for aromatic hydrocarbons are located in one transcriptional unit (Fig. 7), which results in coordinate gene expression (van der Meer el al. 1992). The genes encoding the diterpenoid oxygenase subunits, ditAl and ditA2 are located on a separate transcriptional unit from the genes encoding the electron transport components, DitA3 and from its putative NAD(P)H-ferredoxin oxidoreductase (Fig. 11). The scattered genetic organization of a multicomponent oxygenase was recently reported for dibenzo-/?-dioxin (Armengaud et al. 1998) and naphthalene/phenanthrene dioxygenases (Laurie and Lloyd-Jones 1999). Both studies demonstrated the substrate-dependent expression of the oxygenase component but did not show 84 coordinate or constitutive expression of the electron transport proteins. The oxidation of alkanes by the multicomponent hydroxylase of an Acinetobacter sp. is another example where the gene encoding the catalytic hydroxylase component AlkM is distantly located on the chromosome from the genes encoding its electron transport proteins, RubA and RubB (Gralton et al. 1997). In this instance, alkM expression is regulated by alkanes (Ratajczak et al. 1998) but expression of its electron transport proteins is constitutive (Geissdorfer et al. 1995). The several examples of cloned dioxygenases which are functional in E. coli in the absence of their respective ferredoxin and/or reductases (Bergeron et al. 1994; Kurkela et al. 1988; Laurie and Lloyd-Jones 1999) implies the presence of non-specific "housekeeping" proteins which may provide the electrons, via NAD(P)H, to the catalytic oxygenase. Although this phenomenon might also occur in the natural host of dioxygenases, I have demonstrated that the expression of the ferredoxin component of the diterpenoid dioxygenase DitA3 is inducible and coordinated with DitAl expression. The P. abietaniphila strain BKME-93 with a mutation in the gene encoding the extradiol ring cleavage dioxygenase (ditC) produced a yellow colored supernatant from DhA in cell suspension assays. Although the compound(s) producing the yellow color was not identified, I suspect that it was formed from the spontaneous oxidation of 7-oxo-l 1,12-dihydroxyDhA to 7-oxoDhA-l 1,12-quinone (Fig. 28). Two GC peaks with mass spectra similar to compounds VII and VIII (Fig. 13) but with the loss of m/z 2 for the NT and the base peak were identified in the supernatant liquid of strain BKME-93 cell suspensions. These peaks may be the quinone oxidation products of the diols or may have resulted from the loss of H 20 (m/z 18) from the M + of a secondary alcohol. The unambiguous structures of these two compounds were not determined, but the oxidation of the diol to a yellow compound would be similar to the previously described spontaneous oxidation of 1,2-dihydroxynaphthalene to the yellow coloured 1,2-naphthaquinone 85 diol ' C 0 2 H spontaneous • ' ' C 0 2 H meta cleavage (DitC ?) quinone f e l l o w ?) CIS hemiketal trans ' C 0 2 H ' C 0 2 H ' C 0 2 H spontaneous spontaneous isomerization (DitD or D i t H ?) (enzymatic or spontaneous) C O O H ' C 0 2 H Fig. 28. Hypothetical extradiol cleavage pathway for the degradation of the 7-oxo-l 1,12-diolDhA intermediate based on the analogous pathways for the degradation of fused ring PAHs. 86 (Patel and Barnsley 1980). The products of several extradiol ring cleavage enzyme reactions are yellow because they form a semialdehyde. The E. coli strain carrying pVM4, a plasmid containing a DNA fragment which encodes a putative dehydrogenase and the extradiol dioxygenase (Fig. 11), did not produce a yellow cleavage product from 7-oxo-l 1,12-dihydrodiolDhA. This result suggests that ditB does not encode the dihydrodiol dehydrogenase (see Fig. 6). However, the ring cleavage product of the DhA degradation pathway may be analogous to the products formed as intermediates in the degradation of fused-ring polycyclic aromatic hydrocarbons (PAH). In several of these pathways, the unstable cleavage products do not produce a yellow compound, but spontaneously rearomatize to hemiketals (Eaton and Chapman 1992). A hypothetical pathway representing this spontaneous rearrangement of the 7-oxo-l 1,12-diolDhA intermediate is described (Fig. 28). I described the cloning and expression of a representative of a new class of ring-hydroxylating dioxygenases. This diterpenoid dioxygenase, DitA, is like other ring-hydroxylating dioxygenases in its basic subunit structure, and these subunits have recognizable similarity to those of other dioxygenases. Like other dioxygenases, DitA catalyzes hydroxylation of an aromatic-ring, forming a dihydrodiol. However, DitA is distinct in several ways. First, the phylogenetic analysis of the protein sequence of the a subunit of DitA clearly indicates that DitAl does not cluster with a subunits of the class I, II or III of dioxygenases, but rather, forms a distinct branch (Fig. 29). Furthermore, DitA does not belong to any of the oxygenase classes proposed by Batie et al. (1992), which are distinguished by their variation in terminal oxygenase composition and their electron-transport components (Fig. 8). The ferredoxin component of all dioxygenase enzymes reported in the literature contain a [2Fe-2S]-type cluster, which functions as the electron supply to the oxygenase. However, the ferredoxin component of the DitA appears to 87 Fig. 29. The classification of a-subunits from ring-hydroxylating dioxygenases based on the multiple alignment of related proteins. The phylbgenetic tree (unrooted) was constructed using the PHYLIP protein distance and neighbor-joining methods, and confidence levels were determined by bootstrap analysis. Numbers on the branches represent percent confidence of 100 replicate analyses. The scale bar indicates percent divergence. The sequence abbreviations, enzyme substrate, species and GenBank references are as follows: Aniline.YAA, aniline, Acinetobacter sp.strain Y A A (D86080); TdnAl.UCC22, aniline, Pseudomonas putida UCC22 (D85415); XylX-mt2, toluate, Pseudomonas putida mt2 (M64747); BenA.BD413, benzoate, Acinetobacter calcoaceticus BD143 (M76990); TftA.AC1100,2,4,5-trichlorophenoxyacetic acid, Burkholderia cepacia AC1100 (U11420); CmtAb.Fl, p-cymene, Pseudomonas putida F l (U24215); NdoBJMCIB9816, naphthalene, Pseudomonas putida NCIB9816 (M23914); NahA3.BS202, naphthalene, Pseudomonas putida BS202 (AF010471); PahAc.OUS82, polyaromatic hydrocarbon, Pseudomonas putida OUS82 (D16629); NahAc.G7, naphthalene, Pseudomonas putida GI (M83949); PahA3.PaKl, naphthalene, Pseudomonas aeruginosa PaK.1 (D84146); DDtAc.DNT,.2,4-dinitrotoluene, Burkholderia sp. strain DNT (U62430); NtdAc.JS42,2-nitrotoluene, Pseudomonas sp. strain JS42 (U49504); BedCl.ML2, benzene, Pseudomonas putida ML2 (L04642);. TodCl .F l , toluene, Pseudomonas putida ¥1 (J04996); BnzA.BE81, benzene, Pseudomonas putida BE-81 (M17904); TcbAa.P51, chlorobenzene, Pseudomonas sp. strain P51 (U15298); BphAl .RHAl , biphenyl, Rhodococcus sp. strain RHA1 (D32142); BpbAl.P6, biphenyl, Rhodococcus globerulus P6 (X80041); BphAXB400, biphenyl, Burkholderia cepacia LB400 (M86348); CumAl.IPOl, isopropylbenzene, Pseudomonas jluorescens IP01 (D37828); BphAl.KKS102, biphenyl, Pseudomonas sp. strain KKS102 (D17319); BphA.B356, biphenyl, Comamonas testosteroni B356 (U47637); XylCl .RBl , substrate unknown, Cycloclasticus oligotrophy RBI (U51165); DxnAl.RWl, dioxin, Sphingomonas sp. strain RW1 (AJ223219/223220); DitAl.BKME-9, dehydroabietic acid, Pseudomonas abietaniphila BKME-9 (AF119621). 88 be of the [4Fe-4S] or [3Fe-4S] cluster type and has little sequence similarity to [2Fe-2S] ferredoxins. Finally, unlike most known dioxygenases for which the genes encoding the components of the enzyme are found in the same transcriptional unit, DitA is encoded by genes at three independent loci on the genome of P. abietaniphila. This unusual genetic organization of dioxygenase genes was also recently reported for a type-IIA dioxin dioxygenase from Sphingomonas sp. strain RW1, where the genes encoding the three components of the enzyme are separated by >40 kb (Armengaud et al. 1998). The protein sequence alignment of the a subunits of aromatic dioxygenases showed limited similarity between the putative catalytic domain (C-terminal) of DitAl and that of other dioxygenases. The residues presumed to line the substrate pocket are not conserved between naphthalene dioxygenase (NDO) and DitAl, with the exception of the three active site iron ligands (His219, His224 and Asp399) and possibly Phe389 (Kauppi et al. 1998). This lack of similarity is expected, as the substrate specificity of dioxygenases is thought to be principally determined by the C-termini of the a subunits of the terminal oxygenases (Kimura et al. 1997; Parales et al. 1998). Most of the similarity of DitAl to other dioxygenases is limited to the Rieske domain, located between residues 48 and 170 (38-158 of NOD), and the region surrounding the putative ligands to the iron at the active site. The Asp216 residue, shown to be essential for toluene dioxygenase activity (Jiang et al. 1996), and proposed to be a key residue in the electron transfer between the Rieske center and the iron at the active site (Kauppi et al. 1998) is also present in DitAl. In all likelihood, the path of electron transfer and oxygen activation at the active site of the diterpenoid dioxygenase is the same as for other classes of dioxygenases. However, given the atypical nature of the ferredoxin component of the diterpenoid dioxygenase, one would expect the binding site on DitAl for Dit A3 to be distinct from the binding sites on previously 89 characterized terminal oxygenases for their respective [2Fe-2S] ferredoxins. In fact, the residues in the NDO a subunit (Lys97, Gly98, VallOO, Glnl 15, Serl 16, Prol 18 and Trp211), thought to form a depression for interaction with the [2Fe-2S] ferredoxin, are not present in DitAl. These residues are also not fully conserved in a subunits of oxygenases from other classes, perhaps reflecting different evolutionary adaptations of the oxygenases to interact with their respective ferredoxins. The peculiar organization of the genes encoding the diterpenoid dioxygenase, and the sequence of the ferredoxin proved to be problematic in the cloning of the genes encoding this enzyme. Upon identifying the dilAlA2 genes, several unsuccessful attempts were made to locate the ferredoxin component by PCR and Southern blots, based on the expected conservation of the residues of the [2Fe-2S] cluster ligands and the ferredoxins observed in previously characterized dioxygenases. When enzyme assays with surrogate ferredoxins from type-IIA and IIB dioxygenases coupled to DitAl A2 also failed (data not shown), I resorted to sequencing the DNA in the vicinity of ditAl A2, in the hope of finding the electron transport component(s) of the enzyme. This resulted in the sequencing of the dit gene cluster. The discovery of a putative [4Fe-4S] or [3Fe-4S] ferredoxin gene in the proximity of a ring-cleavage dioxygenase gene (Fig. 11) suggested that this ferredoxin might be a component of the diterpenoid dioxygenase, despite not being phylogenetically related to [2Fe-2S]-type ferredoxins of known ring-hydroxylating dioxygenases. The results from this study clearly established that the ferredoxin, Dit A3, is a functional component of the diterpenoid dioxygenase. This is the first report of a [4Fe-4S]- or [3Fe-4S]-type ferredoxin functioning as an electron-transport protein of a multicomponent dioxygenase, although such ferredoxins have been shown to supply electrons to multicomponent P-450 monooxygenases in Streptomyces spp. (O'Keefe et al. 1991; Trower et al. 1992). 90 Interestingly, several proteins in the GenBank with similarity to DitA3 are found in Archea and gram-positive organisms (including thermophiles and anaerobes), but proteins with such similarity have not been found in Proteobacteria (Fig. 16). The significance of this observation is unclear, but it suggests either an ancestral origin of DitA3 or acquisition of the gene from a distantly related organism. The presence in E. coli of the diterpenoid dioxygenase lacking the reductase (only DitAlA2A3) or lacking the reductase and the ferredoxin (only DitAl A2) both resulted in expression of the functional enzyme, although the activity was very low in the latter case (Fig. 18). This result has been observed for other multicomponent dioxygenases (Bergeron et al. 1994; Kurkela et al. 1988) and suggests relatively low specificity for electron-transport components of some multicomponent oxygenase systems. This characteristic, and the location of ditA3 and ditAJA2 and the putative gene encoding the reductase on separate transcriptional units, suggest that the electron transport proteins of the diterpenoid dioxygenase might be shared with other redox systems, possibly to maximize the catabolic potential while limiting its genetic burden. Harayama et al. (1992) proposed that this tolerance between redox and oxygenase partners might also function as an evolutionary process for multicomponent oxygenases. Although some potential catabolic and evolutionary benefit may result from multipurpose electron transport proteins, it raises the question of coordination of expression of the genes: Are these electron transport proteins expressed constitutively or are they regulated? The discovery of a new class of ring-hydroxylating dioxygenase for which the genes encoding the three components are unlinked confirms that this characteristic is not restricted to the type-IIA dioxygenases (Armengaud et al. 1998). The identification of homologues of DitA by examining other resin acid-degrading 91 bacteria, or by large scale sequencing projects, might reveal that this genetic organization is more common than previously thought. The identification o f DitR as a regulator of ditAl and dit A3 expression demonstrated the coordinate expression o f genes encoding two components o f a dioxygenase, which are not linked in the genome. Phylogenetic analysis o f the protein encoded by ditR indicates that it belongs to the IclR-like family o f transcription regulators (Fig. 30). This family includes IclR, a repressor o f the glyoxylate bypass operon in E. coli (Sunnarborg et al. 1990), GylR, an activator of the glycerol operon in S. coelicolor (Smit and Chater 1988) and regulators o f aromatic metabolism such as PobR and PcaR, two activators of p-hydroxybenzoate hydroxylase enzymes from an Acinetobacter sp. (DiMarco et al. 1993) and P. putida (Romero-Steiner et al. 1994), and H p p R and MhpR, two regulators of 3-(3-hydroxyphenyl)propionic acid degradation in R. globerulus and E. coli (Barnes et al. 1997). The search for a soluble, non-toxic inducer o f P C B degradation for use in P C B bioremediation has led to the hypothesis that plant terpenes may be the "natural" substrates for biphenyl biodegradation enzymes, or their ancestors, since biphenyl is not naturally abundant (Focht 1995). This raises the interesting question: Is the diterpenoid dioxygenase described in this study ancestral to biphenyl dioxygenases? It was previously demonstrated that P. abietaniphila BKME-9 will not grow on biphenyl as a sole organic substrate (Mohn el al. 1999a). I have not tested the possible cometabolism o f D h A and biphenyl by P. abietaniphila or the hydroxylation of biphenyl by the cloned D i t A enzyme, although these activities are unlikely. A structure-function analysis o f potential inducers of P C B biodegradation by Arthrobacter sp. strain B I B suggested that isoprenoids were able to induce P C B degradation, with the most potent inducer being an aromatic isoprenoid (p-cymene), which resembles the aromatic region of the D h A molecule (Fig. 92 MhpR.Ecol QJ. Fig. 30. Phylogenetic tree of IclR-type transcription regulators. The unrooted tree was generated with sequences aligned with ClustalX and using the PHYLIP protein distance and neighbor-joining methods. The number on the branches represent boostrap values of 100 replicate analysis. The accession numbers are as follows: HppR.Rglo, (U89712); YAGI.Ecol, (P77300); KdgR.Ecol, (P37728); GylR.Scoe (PI 5360), IclR.Ecol (AE000475), PcaR.Ropa (AF003947), PcaU.Acin (L05770), PobR.Acin (L05770), MphR.Ecol (P77569). 93 1) (Gilbert and Crowley 1997). However, the induction specificity experiments (Fig. 21) clearly demonstrated that the expression of the dioxygenase was induced only by diterpenoids and that other aromatic compounds, such as isopropylbenzene and biphenyl, were incapable of inducing the synthesis of this enzyme. Therefore, with the exception of the weak a.a. sequence similarity, DitA appears to be distantly related to BpH dioxygenases. The diterpenoids used in this study are insoluble in aqueous solutions at concentrations above -20 pM (Liss et al. 1997). Since the gene expression experiments were conducted at inducer concentrations above this saturation level, I assume that the soluble concentration of diterpenoid available for induction was much lower than the nominal concentration, but maximal. Although ditA3 expression was 40-60% that of ditAl in the reporter constructs, this comparison may not apply to the wild-type strain since I have observed that in some instances, the insertion of the xylE::GmT, with or without a terminator, into a gene affected the expression level of that gene (data not shown). This effect might be due to disruption of the regulation of the genes or alteration of the stability of the mRNA being transcribed. DitR is an activator of ditA3 and a possible repressor of ditAl expression. This difference in the mechanism of regulation of the two components of the same enzyme may reflect the independent evolution of the enzyme and the regulation system (de Lorenzo and Perez-Martin 1996). Interestingly, the strain lacking a functional ditR maintained its ability to grow on abietanes (Table 3). The constitutive expression of ditAl combined with the residual expression level of ditA3 may account for this phenotype. Furthermore, the finding that ditR mutant strains reproducibly responded, albeit at low expression levels, to the 7-oxoDhA inducer compound (Fig. 22), suggests that expression of ditAl and dit A 3 is controlled by at least one additional mechanism. Examination of the nucleotide sequence upstream of dit A 3 identified a putative o54-promoter consensus sequence 89 bp upstream of the 94 putative ATG start codon of ditA3. Although I have no data confirming the involvement of the alternative sigma factor, a5 4, other than the existence of this sequence, it is possible that the regulation of ditA3 expression might also involve a a54-dependent regulator (Shingler 1996). The bacterial degradation of aromatic compounds is frequently initiated by their conversion to diol intermediates followed by cleavage of the aromatic ring. However, the initial attack on the aromatic diterpenoid DhA by P. abietaniphila occurs at two regions of the molecule, much like bile acids and steroid degradation by another Pseudomonas sp. (Leppik 1989). Previous studies reported that the hydroxylation at C-3 or C-7 precedes aromatic ring oxidation in the DhA biodegradation pathway of Pseudomonas sp., Flavobactehum resinovorum and Alcaligenes sp. (Biellmann et al. 1973a; Biellmann et al. 1973b). Interestingly, 7-oxoDhA has been detected in effluent treatment systems (Zender et al. 1994), suggesting that the abietane pathway reported here is widespread. In light of the results presented in this study, the presence of 7-oxoDhA in biotreatment systems might potentially be used as an indicator of the biomass inhibition or sludge health with respect to the ability to degrade resin acids. If this convergent route for abietane biodegradation is typical, it may have an important implication in the pulp and paper industry. Any adverse condition preventing the degradation of DhA in a biotreatment system would prevent removal of all abietane diterpenoids, which may result in toxic effluent discharges into the environment. The initial oxidation at C-7 is consistent with the possible presence of a membrane-bound hydroxylase acting during uptake of the substrate, as in the alk system (van Beilen et al. 1994). The aromatic ring-hydroxylating dioxygenase, DitA, appears to be central to the biodegradation of abietanic diterpenoids, since a mutation in ditAl or ditA3 inhibits growth of strain BKME-9 on the nonaromatic abietane, abietic acid (Table 3). It appears that 7-oxoDhA is a key intermediate in the degradation of all abietanes by strain BKME-9, since 95 the dioxygenase-deficient strains lacked the ability to grow on AbA and PaA and accumulated 7-oxoDhA when incubated with those substrates (Fig. 20). The conversion of AbA and PaA to 7-oxoDhA previous to aromatic-ring attack represents a novel mechanism by which cyclic dienes are aromatized previous to ring dioxygenation. The most analogous pathway resembling this aromatization reaction is for the degradation of cyclohexane carboxylic acids (Trudgill 1984). Attack of the cyclohexane carboxylic acids is initiated by the formation of 4-oxocyclohexane which is the substrate for the desaturase and the formation of p-hydroxybenzoate, which is further degraded via a dioxygenolytic pathway. Most described resin acid degraders which grew on DhA also had the ability to grow on nonaromatic abietanes but not on pimeranes (Mohn et al. 1999a). This is consistent with a convergent pathway similar to the one in P. abietaniphila BKME-9 existing in those organisms. This study did not find mutants which failed to grow on AbA but grew on DhA (Table 3). This suggests that the gene(s) encoding the enzyme(s) responsible for the aromatization of abietanes remain(s) unidentified. Previous work demonstrated that the ability of several members of the Proteobacteria (including BKME-9) to metabolize chlorinated DhA was correlated with their ability to metabolize DhA (Mohn and Stewart 1997), suggesting that 12-ClDhA is degraded by the same enzyme system used to metabolize DhA. If this hypothesis holds true, it might indicate that the diterpenoid dioxygenase is capable of oxidative dechlorination. The potential dechlorination activity of the dioxygenase is of importance since chlorinated DhA isomers, which are found in pulp and paper bleaching effluents, are more toxic and persistent than DhA (Mohn and Stewart 1997; Zanella 1983). Dioxygenase-catalyzed dechlorination was previously reported for the biphenyl 2,3-dioxygenase of Burkholderia cepacia LB400 (formerly Pseudomonas cepacia), using chlorobiphenyl substituted at the 2,2' positions (Haddock etal. 1995). 96 Bacteria are able to degrade or transform numerous compounds by various cytochrome P-450 enzyme-mediated reactions (Sariaslani 1991). The reactions characterized to date include, hydroxylation, N- and S-oxidation, epoxidation, dechlorination, O-dealkylation, and desaturation. Like ring-hydroxylating dioxygenases, cytochromes P-450 are multicomponent enzyme systems which are classified on the basis of the number of proteins required for activity. The majority of prokaryotic cytochrome P-450 enzymes are classified as type I, as defined by their requirement for a reductase, a redoxin and a cytochrome P-450 to reconstitute enzyme activity. The sequence analysis of the inverse PCR fragment isolated from the Tn5 mutant strain BKME-12 implied that P. abietaniphila harbors a DhA-inducible multicomponent monooxygenase system (Table 5). However, the phenotype of the mutant suggested that a hydroxylated intermediate was produced by strain BKME-12, which did not agree with the speculated identity of the disrupted gene as a cytochrome P-450 hydroxylase. To determine if P. abietaniphila expressed a DhA-inducible cytochrome P-450, assays were carried out on pyruvate-and DhA-grown cells. The results from the P-450 assays were inconclusive. The visible absorbance spectra showed the presence of a DhA-inducible cytochrome resembling those of the type-c (Fig. 26). This cytochrome(s) is most likely one which showed on the heme-stained SDS-PAGE gel (Fig. 27) since: (1) its molecular mass of -20 kDa is smaller than cytochrome P-450 proteins, which have a mass between 38 and 52 kDa (Asperger and Kleber 1991) and (2) the positive control P-450 from the crude lysate of R. rhodochrous did not show up on the heme-stained PAGE gel. Thus, the presence of a DhA-inducible P-450 enzyme was neither confirmed nor disproved from the results of the cytochrome P-450 assays and heme-stained gels. It is possible that a cytochrome P-450 protein was induced by DhA but its absorption spectrum was masked by another more abundant cytochrome (Fig. 26). Recently, a mutant strain of 9 7 Pseudomonas sp. A19-6a (Table 1) which can degrade DhA but not AbA was isolated (CA. Morgan and R.C. Wyndham, Dept of Biology, Carleton University, Ottawa, personal communication). This insertional mutation was located in an open reading frame, designated tdtD, with similarity to genes encoding cytochrome P-450 monooxygenase enzymes. This result led to the hypothesis that a cytochrome P-450 dependent desaturation reaction may aromatize AbA to DhA, which would subsequently be degraded through a common dioxygenolytic pathway (Fig. 13). By transposon mutagenesis, CA. Morgan and R.C. Wyndham (personal communication) cloned a cluster of genes encoding the putative P-450 monooxygenase gene tdtD. This cluster contained an additional six open reading frames that potentially encode a CoA ligase (tdtL), a putative regulator (tdtR), an unidentified gene product (tdtS), an isomerase (tdtA), a dehydrogenase (tdtB), and a thiolase (tdtC). Insertional inactivation of tdtL demonstrated that it was essential for abietic acid catabolism. Interestingly, the translation product of tdtL, the last gene of the tdt cluster of Pseudomonas sp. A19-6a, and the partial sequence of ORF1 (Fig. 11) of P. abietaniphila BKME-9 share 85% identity of their aligned a.a.. Therefore, the dit and tdt gene clusters may be contiguous in both Pseudomonas spp, as the strains are closely related phylogenetically (CA. Morgan and R.C. Wyndham, personal communication). Microbial cytochromes P-450 catalyze the hydroxylation of monoterpenes such as camphor (P-450cam) (Koga et al. 1985), a-terpineol (P-450terp) (Peterson et al. 1992) and of triterpene-type compounds such as steroids (Berg et al. 1976). Therefore, it is reasonable to predict the involvement of a cytochrome P-450 enzyme in diterpenoid degradation. However, the proposed desaturation reaction is unusual for prokaryotic P-450s. Desaturation reactions have been reported for eukaryotic cytochrome P-450 isozymes which catalyze the formation of steroid hormones (Corbin et al. 1988), and in one example the rat liver microsome 6(3 steroid 98 hydroxylases were proposed to carry out both a desaturation and a hydroxylation reaction (Nagata et al. 1986). Thus it is conceivable that bacterial P-450s may catalyze desaturation reactions but this activity would be novel. Finally, the phenotype of the P-450 mutant of Morgan and Wyndham (growth on DhA but not AbA) does not correspond to the phenotype observed for my P-450 ferredoxin reductase mutant strain BKME-12 (no growth on DhA). The reason(s) for this difference in phenotypes is unknown. Possible explanations may be that the reductase of P. abietaniphila strain BKME-9 and the cytochrome P-450 of Pseudomonas sp. A19-6a belong to different enzyme systems or that the mutation in the accessory reductase does not abolish P-450 activity and the phenotype observed is from a polar effect of the Tn5 transposon insertion. 99 CONCLUSIONS This thesis describes the characterization of a gene cluster from P. abietaniphila BKME-9 encoding a novel dioxygenolytic degradation pathway for abietane diterpenoids. A 16 kb fragment of DNA which contains 13 complete and one partial ORFs was cloned and sequenced. By using insertional mutations, transcriptional fusions or by expressing the proteins in E. coli the function of several of the genes was determined. These genes include ditAl,ditA2 and ditA3, encoding a member of a new class of ring-hydroxylating dioxygenase; ditC, encoding an extradiol ring cleavage dioxygenase; and ditR, which encodes a regulatory protein modulating the expression of dit genes. The gene diti, encoding a putative dehydrogenase, and ditH, encoding a putative decarboxylase/isomerase are required for the growth of P. abietaniphila on abietanes but their functions were not determined. This study also reports the structure of catabolic intermediates and proposes a pathway for the degradation of abietane-type resin acids. I provide evidence that nonaromatic abietanes, such as palustric and abietic acids are transformed to 7-oxoDhA which is subsequently degraded via a dioxygenolytic route. This pathway is consistent with a previously proposed pathway for DhA degradation by a Pseudomonas sp. (Biellmann et al. 1973) and with the observation that DhA-degrading bacteria grow on all abietanes (Mohn et al. 1999a). Genetic evidence for the presence of a diterpenoid transport system and a cytochrome P-450 enzyme is provided but further experimental work will be needed to confirm these conclusions. 100 F U T U R E W O R K The function of several ORFs of the dit cluster remains unresolved. For example does ditB encode the 7-oxo-ll,12-dihydrodiol dehydrogenase (Fig. 12)? Do ditD or ditH carry out the isomerization reaction of the proposed hemiketal (Fig. 28)? Does ditE encode a permease which transports abietane diterpenoid? What is the function of ditF? Furthermore, I have hypothesized that a a54-type transcription regulator and a cytochrome P-450 enzyme may be involved in the biodegradation pathway but these genes and proteins remain uncharacterized. A number of phylogenetically diverse aerobic bacteria that grow on resin acids have been characterized. It would of interest to determine if all of these organisms utilize the same biochemical pathway. Evaluations of the presence and functional expression of ditAl A2 and ditA3 homologues within these organisms might help answer this question. Such information could be useful for ecological studies of pulp and paper effluent biological treatment systems, if conserved molecular probes targeting these genes could be used to study phylogenetically diverse guilds of resin acid degraders. Simple substrate specificity tests of resin acid-degraders demonstrated that in gram-negative bacteria, the abietane degradation pathway cannot utilize pimeranes as substrates, whereas the pimerane biodegradation pathway(s) may catalyze abietane degradation (Bicho et al. 1995; Mohn 1995; Wilson et al. 1996). The isopropyl side chain of abietanes at C-13 appears to have a critical role in determining the specificity of the biochemical pathway for this class of compounds. With the exception of the isolation of a few pimerane degrading strains (Wilson et al. 1996), our knowledge of bacterial pimerane degradation is nonexistent. 101 APPENDIX I The aim of this appendix is to report the work from my Ph.D. that is incomplete but should be formally written as a reference for possible follow up experiments. The methods and results of this section were combined and the results are not discussed. 1. Overexpression and partial purification of the oxygenase (DitAlA2) component of the diterpenoid dioxygenase DitA The genes encoding the ferredoxin and the NAD(P)H-reductase components of the DitA dioxygenase are not contiguous on the chromosome of P. abietaniphila. Therefore, before ditA 3 was identified, I set out to isolate the two electron-transfer proteins using a dioxygenase O 2 -consumption enzyme assay. The principal objective of the purification of the oxygenase component of the dioxygenase was to use it in enzyme assays in conjunction with protein fractions from DhA-grown P. abietaniphila crude lysate. A similar approach was successfully used to isolate the reductase and the ferredoxin components the dibenzofiiran 4,4a-dioxygenase from Sphingomonas sp. strain RW1 (Biinz and Cook 1993). However my attempt at isolating the ferredoxin and reductase components of the DitA dioxygenase using this approach was not successful. Therefore, this section will report only on the cloning, expression and partial purification of the a and P subunits of the diterpenoid ring-hydroxylating dioxygenase. Cloning and expression of the a and P subunits. To overexpress the a and P subunits of the oxygenase, the ditAlA2 genes were PCR amplified from pVMl and cloned into the T7 expression vector pET22B(+). The primers 5'-GGAATTCCATATGCTAACAAGAACAA-3' and 5'-CGCGGATCC7T4GAGGAATACCGC-3' introduced a start (underline) and stop (italic) 102 codons as well as Nde I and BamW I sites (in bold), which were subsequently used to directionally clone the PCR fragment into the pET vector. The resulting plasmid (pVMctP) was transformed into E. coli BL21(DE3). Time course experiments and solubility of the expressed proteins was tested and it was found that expression at 25°C for 5 hrs was optimal. Furthermore, the expression of the proteins dropped significantly when cells from frozen stock cultures were used or when the inoculum was allowed to reached stationary phase before transfer to a large volume culture. For expression of the proteins, a colony from newly transformed E. coli BL21(DE3) cells was grown at 30°C to an OD 6i 0 of 2.0 in 1 L baffled flasks without reaching stationary phase. The cultures were chilled on ice to 25°C, induced with 0.1 mM of IPTG, and incubated for an additional 5 hrs. Partial purification of the a and P subunits. With the exception of the preparation of the crude lysate, the entire purification procedure was carried out under anaerobic conditions (N2 atmosphere) using a glove box. The crude lysate was prepared by suspending -33 g of cells (wet weight) into 67 mL of 25 mM Hepes (pH 7.3) containing 10% glycerol, 1 mM PMFS, 1 mM MgCl2, 2 mM CaCl2 and 0.1 mg/mL DNase I. The cells were broken by passing the suspension through a French pressure cell at 13 MPa. The cell lysate was then centrifuged at 230 000 g and 4°C for 1 hr. The supernatant was filtered through a 0.45 pm (pore size) membrane and sparged with argon before the first injection. The supernatant liquid (Fig. 31, lane 1) was loaded onto an AKTA (Pharmacia Biotech) protein liquid chromatography system equipped with a Resource Q anion exchange column (Pharmacia Biotech) equilibrated with 25 mM Hepes (pH 7.3) plus 10% glycerol (buffer A). The unbound fraction was washed out from the column with 2 column volumes (CV) before eluting the proteins with a linear gradient of 50 to 300 mM NaCl over 15 103 Fig. 31. SDS-PAGE of the successive purification steps of the a and p subunits of the P. abietaniphila ring-hydroxylating dioxygenase, DitA, expressed in E. coli. Lane 1, crude lysate; lane 2 and 3 , Resource Q anion exchange; lane 4 and 5, Phenyl Sepharose hydrophobic interaction (unbound fraction); lane 6 and 7, Superdex 200 molecular sieve; lane 8 and 9, Mono Q anion exchange; lane 10 and 11, Phenyl Sepharose hydrophobic interaction (lane 10, fraction eluting at 5 % ammonium sulfate and lane 11, fraction eluting at 0% ammunium sulfate). Lanes were loaded with 10 ug protein except for lanes 3 , 5, 7 and 8 which were loaded with 5 pg protein. The numbers on the left designate molecular mass markers in kDa. 104 CV. The progression of the chromatography was monitored at X 323 and 455 nm. The fractions which contained the a and (3 subunits eluted between 150 and 230 mM NaCl, as determined by SDS-PAGE (Fig. 31, lane 2 and 3).These fractions were pooled and concentrated anaerobically in the glove box using an Amicon ultrafiltration unit. The concentrate was made to 5% ammonium sulfate, filtered through a 0.45 pm (pore size) membrane, and loaded onto a Phenyl-Sepharose hydrophobic interaction column (22.5 mL bed volume) equilibrated with buffer A containing 5% ammonium sulfate. The two subunits did not bind to the Phenyl-Sepharose (Fig. 31, lane 4 and 5). The unbound fractions were pooled and concentrated by Amicon ultrafiltration to a volume of <2 mL before loading onto a Superdex 200 HR 26/60 molecular sieve column (320 mL bed volume) equilibrated with buffer A containing 150 mM NaCl. The eluting fractions containing the two subunits were pooled (Fig. 31, lane 6 and 7), concentrated and frozen in liquid nitrogen. Up to this stage of the purification, the protein yield was 7.6 %. The protein preparation was thawed and loaded on a Mono Q anion exchange column (Pharmacia Biotech) equilibrated with buffer A. The unbound fraction was washed out with 2 CV and the a and P subunits eluted at -300 mM NaCl during a linear gradient of 200 to 350 mM NaCl over 22 CV (Fig. 31, lane 8 and 9). The fractions containing the oxygenase subunits were pooled, concentrated and made to 10% ammonium sulfate for a last purification step by chromatography over the Phenyl-Sepharose column. Since the protein smeared during the chromatography with a linear gradient of ammonium sulfate, a step gradient was used. The steps were: 5 CV at 10% (NFL^SCu then 5% (NH4)2S04 in 0CV with a hold for 5 CV then 0% (NH4)2S04 in 0CV with a hold for 5 CV. The SDS-PAGE profile of a fraction eluting at 5% and 0% (NFLO2SO4 (Fig. 31, lane 10 and 11) showed that although some further purification was achieved, the subunits eluted over a large volume which meant that they were not binding appropriately to the column matrix. The starting 105 ammonium sulfate concentration was raised to 12.5% with no improvement in the chromatography (data not shown). Although DitAl A2 were not purified to homogeneity, the partially purified proteins could be used in oxygraph enzyme assays. 2. Analysis of ditF expression The expression of the ditF gene was analyzed by measuring C230 activities of ditFv.xylE (strain BKME-96) transcriptional fusion strains, as described in Materials and Methods. Although ditF'xs required for the growth of strain BKME-9 on DhA (see chapter one, Table 3), diterpenoid-(DhA, AbA, 7-oxoDhA, Cl2DhA and IpA) induced cultures of strain BKME-96 did not show significant increases in C230 activity when compared to pyruvate-induced cultures (Fig. 32). To determine if DitF is required for its own expression, the ditF gene was cloned by PCR under the control of the lac promoter of the broad-host-range plasmid pUCP26, and the resulting plasmid (pUCPditF) was transformed into strain BKME-96. This complemented strain showed an increased C230 activity when induced with 7-oxoDhA (induction with other diterpenoids was not tested), indicating that DitF is required for its own expression. The significance of these results is unclear, but they possibly indicate that DitF is involved in either the transport of the substrate into the cell or the regulation of dit gene expression. To determine if DitR regulates ditF expression, C230 activity was analyzed in DitR" strains (ditR::Km, ditFv.xylE) harboring the plasmid pUCPditF. The results were similar to that of dit A3 expression in that DitR appears to positively regulate ditF expression, and the expression of ditF increased slightly in the response to the inducer 7-oxoDhA. 106 0.5 0 1 ET£HL Fig. 32. Analysis of ditF expression in a xylE transcriptional fusion strain (white) complemented with pUCPditF (gray) and in ditR mutant complemented with pUCPditF (black). Cultures were induced with 500 pM 7-oxoDhA. Error bars indicate standard deviation; n=3 107 APPENDIX II GenBank submission data L O C U S A F 119621 15986 bp D N A B C T 26-MAR-1999 DEFINITION Pseudomonas abietaniphila B K M E - 9 Diti (diti), dioxygenase DitA oxygenase component small subunit (ditA2), dioxygenase DitA oxygenase component large subunit (ditAl), DitH (ditH), DitG (ditG), DitF (ditF), DitR (ditR), DitE (ditE), DitD (ditD), aromatic diterpenoid extradiol ring-cleavage dioxygenase (ditC), DitB (ditB), and dioxygenase DitA ferredoxin component (ditA3) genes, complete eds; and unknown genes. ACCESSION A F 119621 NID g4455069 VERSION AF119621.1 GI:4455069 K E Y W O R D S . SOURCE Pseudomonas abietaniphila. ORGANISM Pseudomonas abietaniphila Bacteria; Proteobacteria; gamma subdivision; Pseudomonas group; Pseudomonas. R E F E R E N C E 1 (bases 1 to 15986) AUTHORS Mohn,W.W., Wilson,A.E., Bicho,P. and Moore,E.R.B. T I T L E Physiological and Phylogenetic Diversity of Bacteria Growing on Resin Acids JOURNAL Syst. Appl. Microbiol. (1999) In press R E F E R E N C E 2 (bases 1 to 15986) AUTHORS Martin,V.J.J, and Mohn,W.W. T I T L E A Novel Aromatic Ring-Hydroxyladng Dioxygenase from the Diterpenoid-Degrading Bacterium Pseudomonas abietaniphila B K M E - 9 JOURNAL J. Bacteriol. (1999) In press R E F E R E N C E 3 (bases 1 to 15986) AUTHORS Martin,V.J.J, and Mohn,W.W. T I T L E Direct Submission JOURNAL Submitted (12-JAN-1999) Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd., Vancouver, B.C. V6T 1Z3, Canada F E A T U R E S Location/Qualifiers source 1.15986 /organism="Pseudomonas abietaniphila" /strain="BKME-9; ATCC700689" /db_xref="taxon:89065" /db_xref="ATCC:700689" CDS <1..1335 /note="ORFl; hypothetical protein; similar to Co A ligases" /codon_start=l /transl_table=ll /product="unknown" /protein_id=" AAD21073.1" /db_xref="PID:g4455082" /db_xref="GI:4455082" /transIation="EFLRHQIADTGTHLV!CEADYLSRISAIADQLTO S D D S A F E R K P Q P S D L A C L I Y T S G T T G P S K G C M I S Y N F M C N L A R L Q L R A G P A S E D D V T I T P L P L F H M N A L C V S I I A S I L V G A R A A I L P R F S V S N F W P E V E R S G A T I A S I L G G M G G L L A Q A P D N E A M L R C R G Q I H T A R G N P Y T E E T K Q I W T ^ P J ^ G T R L V G G N G Y G L T E A C V V T S L A A G E Y A A P G S S G K R I A D F D V R I V D E Q D N E W G G T P G E I W R P Q R P D I M F Q G Y W P J I P E D T Q K L M J ^ D R K X D Y L R R R G E N I S S F E M E A A F A T H P A L S E V A V T t A W S D K G E D D V X V T A V L H E N T E L A P E A L F H W A A D T V P Y Y A L P R Y I E F R T S L P K N P Q G R V L K Y L L R D E G K T A T T W D L D D T D I K V A K R " gene complement(1378..2235) /gene="ditl" CDS complement(1378..2235) /gene="ditl" 108 /note="hypothetical protein; short-chain dehydrogenase/reductase family (SDR)." /codon_start=l /transl_table= 11 /product="DitI" /protein_id=" AAD21071.1" /db_xref="PID:g4455080" /db_xref="GI:4455080" /translat ion="MFTYVAVv^IHSKNNKKGAAMSLLRGQVALITGAGGGIGRSV A E L D E A S G A A V A E E L K A L G A Q A L F I R T D V S S K A D I Q A A 1 D A A W H F G G L D I L V N N A F A P T P N V L L E E K T D D N / L E R T L N S T V W A A W W S M K A A F P H M Q A ^ A W G L T R S A A S E W G R F N I R V T s l A I A P T A M G A T P H K L A E E W V W F L A S E M S R F V T G E T L H V T J G G L H L P G Y N S R P K D I P A R E Y " gene complement(3145. .3750) /gene="ditA2" CDS complement(3145.3750) /gene="ditA2" /codon_start= 1 /transl_table= 11 /evidence=experimental /product="dioxygenase DitA oxygenase component small subunit" /protein_id=" AAD21061.1" /db_xref="PID:g4455070" /db_xref="GI:4455070" /translat ion="MSDTQLAEKPRVWQQRGADHVQPGSPL^ A A R L A T D L I Y T W L R H T R T A A E L S T T I W S V Q H Y H D D Y R S I M G R 1 L R L S G K S A W A E D P P S R T R R L \ O T N [ W \ ^ E T E K P D E F I W S Y L L L T R S R F K ^ T P N L A V F L " gene complement(3801..5210) /gene="ditAl" CDS complement(3801..5210) /gene="ditAl" /codon_start= 1 /transl_table=ll /evidence=experimental /product="dioxygenase DitA oxygenase component large subunit" /protein_id=" AAD21063.1" /db_xref="PID:g4455072" /db_xref="GI:4455072" /translat ion="MLTRTSPVXSDGTKVADLIYPST^ E I P N S G D F M W D L G S D S V I V A R D K E G E V H V T L N V C P H R G M R I S T A D C G N T Q I H K C I ^ N G D F I G S P V D R E C N ^ G K M L P K E Q L G L R K A R W L Y G G L V F A T \ W V T ) G P T F D E F L G D A K W Y Y D M L F L R S D K G M E V L G P P Q R F I W A N V v K T A G E Q S A A D ^ A D L S P E M Y G V ^ I S S P H G H A L R C r o L A R K I K R L T G L D P E S L T V E E K L Q A L P P A G M T A D M V E Q L A R > ^ S E D Q L K \ ^ T S M P P Q V G G M F P M L F G F V Y T P Q P D G T W G S N i T L H A Y W Q G r n ) A S P E L R E K M L K Q T I Q L F G T S G M V E Q D D S D T W P H M T L A A K G A M G R K S T L K Y Q A V T E T G A P E G W G P G I V N E G F T K D D T Q W ^ W W T . Y W H E L M T A P V G E D Q " gene complement(5311 ..6321) /gene="ditH" CDS complement(5311..6321) /gene="ditH" /note="hypthetical protein; C-terminus similar to isomerase/decarboxylase." /codon_start= 1 109 /transl_table=ll /product="DitH" /protein_id=" AAD21070.1" /db_xref="PID:g4455079" /db_xref="GI:4455079" /translation="MRLWFTQDDGIERAGALID>TO Q A L E L A R T L L R K S P T A S V 1 A R N E W L R A P I Q P P P Q M R D C S C F E L H L R Q A F A A A R R A R A L R T E D P E ATLKAMNTPvADERVITiTFhJTlQPIY^ I S R D N A R D H I V G F T I F N D M S A R D A Q A A E M P G l v l L G P A K S K D F D T A N I M G P C L V T A D E L G D P Y D L >JMVARVNGEEWGRGOTRDMR\VT<FEDV1AHISRSETLYPGEFLGSGT D R V E L E V D G I A C C A P R W A M R S R F E R L T D " gene complement(6318..7052) /gene="ditG" CDS complement(6318.7052) /gene="ditG" /note="hypothetical protein; short-chain dehydrogenase/reductase family (SDR)." /codon_start=l /transl_table=ll /product="DitG" /protein_id="AAD21069.1" /db_xref="PID:g4455078" /db_xref="GI:4455078" Araris lat ion="MSSSRQDQALEDSPLQAJlVlVvTGGFG\^GTALGQYXSGRGAJ<lVALLDRAETSQ T R V S D D A N V L A L G G V D L T S V E S A R S A F A Q V A E H F G R I D G L W V A G L Y Q M N L R T A V I A S Q A V L E H L L A H D G G R I V N r c E L K D R G I T V N A V L P S I J U D T P T N R T D M P D A D F S R W RV" gene 7596..8789 /gene="ditF" CDS 7596..8789 /gene="ditF" /note="hypothetical protein; similar to non-specific Jipid-transfer protein and 3-ketoacyl-CoA thiolase." /codon_start=l /transl_table= 11 /product="DitF" /protein_id=" AAD21068.1" /db_xref="PID:g4455077" /db_xref="GI:4455077" / translation= u NBvrFDHRYEKDVAITGIGQSEVGRPSSKSANlRLTLEACLEAIA^ C \ V T G D N N N G D P F S P V G P S A L K S A L G L N W A V F G A G Y E G P G P L A G V ^ R T I T E A S A R Q H N K Q A G A L S A K T Q G R D S S H A W Q W ^ Q L A Q I A L T C R A N A Q R N P K A I Y R T P N T J ^ ^ K D T R J H Q P I R J E A M G A A L D Q P W S \ \ T J Q I S L T Q M A A F D V G R M M W L T N f l W L E A L G L C S T G Q S G A F W G G E P J A L T C K ^ A E G R Q T R P H T V A A V A A G G G P L G G S L L L A R D " gene complement(8811..9632) /gene="ditR" CDS complement(8811.. 963 2) /gene="ditR" /note="transcription regulator (IclR family)" /codon_start= 1 /transl_table=ll /product="DitR" /protein_id=" AAD21072.1" 110 /db_xref="PID:g4455081" /db_xref="GI:4455081" /translation="MPRTQTDSNAPQRPDTAETTTDVQ A R \ ^ G I W S T C F W L R T L \ T v l E D V V D F M > L S K J l Y S A G L G L A ARLRvTVTLVVTlRMGPDRIVlVSSAASPTDvTiroMAEGQRLPILM D F E A I R W A R P L P L K V Y R Q D V A L A A E R G W A V D D G W S I G I L A I A A P W Q R D E E G I E E I G R T L V T F C A E L S K V L F " gene comp!ement(9712..11355) /gene="ditE" CDS complement(9712.. 11355) /gene="ditE" /note="hypothetical transmembrane transport protein of the facilitator superfamily." /codon_start=l /transl_table= 11 /product="Dit£" /protein_id="AAD21067 1" /db_xref="PID:g4455076" /db_xref="GI:4455076" /translation="MSDLSGLSPDEPAWRAQPASAWAPIRmSFRVv^ W I M T L L S T S P L M V S L V Q T A I S L P A F L F G L P G G V 1 A D L V D D M L S D W S L L A F T F L L G T G S A L S M S A V v T V l W W I A Y \ V l v I A A S W \ a i T V C L A L V I V L V A A F T P R Q T P H A L P A E N F A A G L R S G W Y I R H S A V l . V S A L K Q V F I F T L S A S A L W A L L P L V A K H E L G M D A G G Y G L L M G F L G A G A V A G A L N L T A M Y T I R F P L R V L I I A G TAGFALATLVAALSHSRWAVCGTLMIAGMSVVMAVmTO L M G A M A I G G A I W G T L A E Y L G L Q N S L \ ^ A A V T L G V G L I L T R S G R L E L G Q E A D Y A S A I Q P T D R L L W E Q L N P G P G P V S I E L A Y R V A P E D R A W I A V A Y A V G Q S P J I R N G A K N W R L Y H D L D E D G H Y V E R F I \ ^ S W L D Y L R Q Q S R T T Q A D Q L L E A R L S G M R C D A Q I S R R Y I H Q P L A G A P E N N " gene complement(l 1352.. 12233) /gene="ditD" CDS complement(l 1352.. 12233) /gene="ditD" /codon_start= 1 /transl_table= 11 /product="DitD" /protein_id="AAD21066.1" /db_xref="PID:g4455075" /db_xref="GI:4455075" /translation="MRLLRVEYRAECFWALLDADAAYLQRrRGPFAEWAGEYASRGIAALALDKVGIA L A Q C R J L P P L E P G A R W G V G L N Y L S H L Q H L G S T A P E H P L G Y I K P E S A L V G A S D D I Q Y P A L T A Q L D YEVELVA\aARALGDEPCASACLLGYTCGNDISARDAGKJUGRLDLLTQKAMDRTTPVGPWW T L D E I G V G Q P H L D I E L S \ ^ E C R Q F D H m Q M ^ DGRFLQPGDRVEVRJEGIGSMSNRVSQPRVLTTARSVGRLGQPVSP" gene complement(12235.. 13176) /gene="ditC" CDS complement(12235.13176) /gene="ditC" /codon_start=l /transl_table=ll /evidence=experimental /product="aromatic diterpenoid extradiol ring-cleavage dioygenase" /protein_id="AAD21065.1" /db_xref="PID:g4455074" /db_xref="GI:4455074" 111 /translation="MDIIGIGYLGFETPDLDAWREYGPQv^ P G K \ O D R L A Y I G w T i A M G R Q A F D A A r c ^ F Y G Q K W T P R S F M P G R P H G G F L A N E R G A G H W V 1 T P E Y T D E L E D F X T R I M G F H W Y G A G A G K G R T G F F R S R L N H H T S H D I A Y G H G V G R M G V Q H V G L F V N S r R D V G E T Y D I V K K R ^ QDPHLSFYHFTPSGFAFETIAEIEPWQGDPFELNPECLSLW ASKR" gene complement(13214..13975) /gene="ditB" CDS complement( 13214.. 13975) /gene="ditB" /codon_start=l /transl_table= 11 /product="DitB" /protein_id="AAD21064.1" /db_xref="PID:g4455073" /db_xref="GI:4455073" / t r a n s l a t i o n = " M A G R L Q E K V A ^ I T G T G G G Q G R V A A L K F A R E G A V V V G C D T N A G A N ^ T A A L M R A E G L K L H G S A P V T J L G D P E Q A K A W I E A A V Q E H G P J D V X Y N N A S A A K F G P V S E L S f f i D W E r o L L F Y T T K Y A W N H L A E Q R G W N I S S T A G W G I R A V S I S P G F I A T P G T T A F M E W K V U A A L L D G V ^ VTDGGLLAI" gene complement( 14047.. 14283) /gene="ditA3" CDS complement( 14047.. 14283) /gene="ditA3" /codon_start=l /transl_table= 11 /evidence=experimental /product= "dioxygenase DitA ferredoxin component" /protein_id=" AAD21062.1" /db_xref^"PID:g4455071" /db_xref="GI:4455071" / t rans la t ion="MSSEKPQFKWADRSRCCGYGLCAAVCPSIYKLDDDGLVTLDDDRVPPELEDEA R E G A A A C P A E A I W L E P L D T P D N A A " CDS 14663.. 15943 /note="ORF2" /codon_start=l /transl_table=ll /product="unknown" /protein_id=" AAD21074.1" /db_xref="PID:g4455083" /db_xref="GI:4455083" / trans la t ion="MTSSKSEAAIDFTEFRSA\ \^ILILSVAGVAISINAALLYGFGTLVWLNQAFGWTR P E L Q A C I T F L F G G A V T S L Q L V G W F N L R Y G I K R W V I S L L L L V X G Y L A T T Q L T H P I V G M G T L A W V V T Q L L S L V V Y T R N R G L A L A I G L S G ^ ^ A I L M ^ W L L P L T L L W R L P G V E V i K J l S E L A E K A A D H L L L T L P G V S F R E G M R S G K F W V S S W G M W S T I P L L Q S K G L S A A D A G L I F S G F G I S L I V G P J V 1 L I G Y L L D R L W P A V A A A S L M M P A V G C M I Y L S G T T D F Q M L L M A A M L V G F G A G A E F D I A A F L V A R W G L R E Y G R L F G V T 4 Q G L N T V A S A L A P L L F A F M L S R S G D Y S A M L V Y C M A C C L I G P L L L L M L G R A P R F Q G A A L A A S S " B A S E C O U N T 3137 A 4911 C 4703 G 3235 T ORIGIN 1 GAATTCCTGC GCCATCAGAT CGCGGACACC GGCACACATC TGGTGATCTG CGAAGCGGAT 61 TATCTTTCAC GCATCAGCGC AATCGCTGAC CAGCTCACGG ATGTCCACCG CGTTCTCTAT 121 CGCAATGCCA GCGGCCAGCA GGTCACTTCA CCTTCAGCTC CTTTCCCTAT CGAATCACTG 181 GATGCCCATC GCGGCTCCGA TGATTCGGCC TTCGAGCGCA AGCCGCAGCC ATCCGACCTT 112 241 GCCTGCCTGA TCTACACCTC CGGCACCACC GGACCTTCGA AAGGCTGCAT GATCAGTTAC 301 AACTTCATGT GCAACCTCGC CCGGCTGCAA CTGCGAGCAG GCCCGGCGAG CGAGGACGAT 361 GTGACGATCA CACCCCTGCC GCTGTTCCAC ATGAATGCGC TGTGCGTCAG CATCATCGCC 421 AGCATTCTGG TAGGCGCCCG CGCCGCCATC CTGCCGCGTT TTTCGGTGTC CAACTTCTGG 481 CCGGAAGTCG AACGTTCGGG CGCGACCATC GCCTCGATCC TCGGCGGCAT GGGTGGCCTG 541 CTCGCTCAGG CCCCGGATAA CGAAGCGATG TTGCGCTGCC GAGGACAGAT TCACACCGCC 601 AGAGGCAACC CCTACACCGA AGAAACCAAA CAGATATGGC GTGAACGCTT TGGCACCCGG 661 CTGGTGGGAG GAAATGGCTA CGGATTGACC GAGGCATGCG TGGTCACCTC ACTGGCGGCC 721 GGCGAATACG CAGCGCCGGG GTCATCAGGC AAACGCATCG CGGATTTCGA TGTGCGCATC 781 GTCGATGAAC AGGACAATGA AGTTCCGGGT GGGACGCCGG GCGAAATCGT GGTCAGACCG 841 CAACGCCCGG ACATCATGTT CCAGGGTTAC TGGCGCCGTC CCGAGGACAC CCAGAAACTG 901 ATGCGCAACA TGTGGTTCCA CACAGGAGAC GTCGGCAAGT TCGATGACGA AGGCTTTTTC 961 TACTTCGTCG ACCGCAAGAA GGATTACCTG CGCCGCCGAG GCGAGAACAT CTCCAGTTTC 1021 GAGATGGAGG CCGCGTTTGC CACACATCCT GCATTGTCGG AAGTGGCGGT ACATGCGGTG 1081 CCGTCGGACA AGGGCGAGGA TGACGTAAAA GTCACTGCGG TGTTGCACGA GAACACCGAA 1141 CTCGCGCCAG AAGCGTTGTT CCACTGGGCG GCGGACACGG TCCCTTACTA CGCGCTTCCC 1201 CGCTACATCG AGTTCCGCAC CAGCCTGCCG AAAAACCCTC AGGGTCGGGT GCTCAAGTAC 12 61 CTGTTGCGCG ATGAAGGGAA AACGGCGACT ACCTGGGATC TGGACGACAC CGATATCAAG 1321 GTCGCGAAAC GCTGAACGGC GAAGCGGCGT GTCTGACACG CCGCGTCCCC TTATGAATCA 1381 ATACTCCCGA GCGGGAATAT CCTTGGGTCT GGAGTTGTAG CCAGGCAAAT GCAGGCCGCC 1441 ATCGACGTGC AAGGTCTCAC CCGTCACGAA GCGCGACATC TCGGATGCAA GGAACACCAC 1501 CACCGGGCCG ATATCTTCTT CCGGTTCCCC GCACCGTCCC AGTGGCCGCA ATCCAGCCGA 1561 CATCTCGGCG AATCCCGGGT TTTCTTCCGC CAGCTTGTGA AAGGTCGCGC CCATGGCGGT 1621 GGGGGCAATC GCGTTGACGC GGATGTTGAA ACGTCCCCAC TCGGAAGCGG CGCTGCGGGT 1681 CAGGCCGACC ACGGCGGATT TCGCCGTGTT GTAGTCCCCG TGCAGCCACG CGCCGATCTC 1741 GGTGTCGATG GAGTAGAAGT TGATGATCTT GCCGCCGCCT CGTGCCTGCA TGTGCGGGAA 1801 CGCTGCTTTC ATCGACCACC AGGCGGCCCA GACGGTCGAG TTCAGCGTGC GCTCGAGCAT 18 61 GTCGTCGGTT TTTTCCTCCA GCAGCACGTT AGGCGTGGGC GCAAATGCAT TGTTCACCAG 1921 GATATCCAGC CCTCCGAAAT GCGTGACGGC TGCATCGATG GCCGCCTGGA TATCCGCTTT 1981 GCTACTGACA TCGGTCCTGA TGAACAGGGC CTGCGCACCA AGCGCCTTAA GCTCCTCGGC 2041 AACCGCTGCA CCGCTGGCTT CGTCGAGCTC CGCGACCACC ACCGATGCAC CCTGTTTGGC 2101 GAAACTCAAC GCCACACTTC GTCCGATACC ACCGCCAGCG CCGGTGATCA GCGCCACTTG 2161 TCCTCGCAGC AGGCTCATAG CCGCTCCTTT TTTGTTGTTG TTCGAGTGAA TTTCCCATGC 2221 CACATATGTG AACATTATCC GATATAGTGT CTAAATAAAA CCGATAAAAA ACACTCGCTC 2281 AGCCAGACAA AAAGGATAAG CCGGATGAGT ATGTTTCGCC GCCGTGTCGT GATCGTTTCC 2341 AGTGGCCAGA TAGAGGGCGG CGAAGTGCGT GCCGCTCTGG AGGACGATTT TCACCACTTT 24 01 CGCGTATCCC TGCGCCATGA ACACGGCGTA CTGAGCCAGG TCACCGGCCA GGCCCTGAGG 2461 TTTCCTTACT CTGCCTGCCC CGAGGCAATC GAGCCGTTGC ACGCACTGTC AGGCATGGTG 2521 TTGTCACCCA TTGCCCATTC AGTCACCCGA GAAACGGATG CCCGGCATCA ATGCACGCAC 2 581 CTGCTGGATC TCGCGGGGCT GGCTGTCGCA GCGGCGCTAC GGCCGGCACA GGTCCGCCGT 2641 TACGACATTC AGGTGCCCAT GCGCCTGGAG GGACGAACGC AGGCCATGCT GCAGCTCAAT 2701 GGCCAACCGT TGCTGGAGTG GCAGGTCAAA GGCGCGATCA TCGAAGATCC ACAACCGTTC 2761 TCGGGTGTGA ACATTCGCGA GGGCATGGCC CGCTGGGCAC TGAATACACT GACCCCGGAC 2821 CTTGCCGAGG CGGCACTGTT GCTGCGGCGC TGCACCATGA TCTCCATCGG CAAGACTCAT 2881 GATCTGGATA GCCAGATTCA CGCCTCCAGC ACCGATCGCT GCTTCAGCCA GCAGACGATC 2941 AGAGCGCCGT ACGCATTGCG TATTAAGGGC TCGACGTGGG ATATCGCGAG CAACGAGACC 3001 CTCTGCGCAG CTGACGCGGA CTGGCTGAAG GAATGCACGC AGACGACTTG CTGATCGGTG 3061 CCAGAGCCAG CCACAAAAAA GCCTGCTGAA CCGATCAGCA GGCTAAAGGT GCGACACGAT 3121 GAACGCTTTC AATGATTTGG ACGTTTAGAG GAATACCGCC AGGTTCGGGG TCCCCAATAC 3181 GGCCTGATCG AGAAT GAC T T CACGTCGGGC GAGCTTGAAA CTGTCGCCAT CCACACGCAG 3241 CAGATCGCGC CGCTCCCCGC TTATGATGTC GACGTGGTGA TCCTTGAAGC GGCTCCGGGT 3301 GAGGAGCAGA TAGCTCGTCA CGATGAACTC GTCCGGCTTC TCGGTTTCCT C GAC GAACAC 3361 GTTGGTGACC AGCCGCCGCG TGCGCGACGG CGGGTCTTCG GCCCAGGCGC TTTTACCGGA 3421 CAGACGCAGT ATGCGACCCA TGATGGATCG ATAGTCGTCG TGGTAGTGCT GCACGCTGCG 3481 AACGATGGTG GTGGAAAGTT CAGCGGCCGT ACGGGTATGG CGCAGCGGTA CGGTGTAGAT 3541 GAGGTCCGTC GCCAGGCGCG CGGCCCAGTC CTGCAGGCGG ATCTGGTCCA GCAGCGTCGC 3601 TTCTTCATAA AGAAACGTGA CCACTTCGTT GTAAACAGGC GAGCCGACAG GCACTCGCTT 3661 GATACCCAGC GGCGAGCCGG GTTGCACATG GTCGGCGCCG CGCTGCTGGA CGTCGACCCG 3721 CGGTTTTTCC GCCAATTGAG TGTCAGACAT GAATGTATCC CCGAATCTTA TTGTTGTTGA 37 81 ATCACGGAAC GCGAACGCCG TTACTGATCT TCACCGACAG GCGCGGTCAT CAACTCATGC 3841 CAGTACAGCC ACCAGTGCCA CTGGGTGTCG TCCTTGGTAA AACCTTCATT GACAATTCCG 3901 GGGCCGGGCC AACCTTCCGG GGCGCCGGTT TCGAACACCG CCTGATACTT GAGCGTGCTC 113 3961 TTGCGGCCCA TCGCGCCCTT GGCAGCCAGC GTCATGTGCG GCCAAGTGTC AGAGTCATCC 4021 TGCTCGACCA TCCCGGAGGT GCCGAACAGC TGAATGGTCT GCTTGAGCAT CTTCTCGCGC 4081 AGCTCGGGAG AGGCGTCCTT CTCGGCAAAG ATCCAGTTCA CGAACTCCAG CTTGTCCGGT 4141 CCTTGTGGCA CATAGGCATG CAAGGTCATG GAGCCGACCA CCGTGCCGTC TGGCTGGGGA 4201 ATGTAGACGA AGCCAAAGAG AATGTTGGGG AACATCCCGC CCACTTGAGG CGGCATGCTG 4261 GTAAGCACTT TGAGCTGATC TTCGCTGAGG TTGCGCGCCA ATTGCTCGAC CATGTCAGCG 4321 GTCATGCCCG CCGGTGGCAG GGCCTGAAGT TTTTCTTCGA CGGTCAGCGA TTCGGGGTCC 4381 AGGCCGGTCA GACGCTTGAT CTTGCGCGCC AGGTCGATGC AGCGCAGCGC ATGGCCGTGA 4441 GGCGAACTGA TCTCCACGCC ATACATTTCG GGACTCAGGT CGGCACTCTG TCCGTTTTCG 4501 TCCTTTTTCG CATAGTTGCC GACTTCGCCC AACCAGCGGT GAAGCGTCAG CGTGTGGAAG 4561 CCATCGGCGG CAGATTGTTC ACCCGCCGTC TTCCAGTTGG CATTGACGAT GAAGCGTTGC 4 621 GGCGGGCCCA ACACCTCCAT ACCTTTGTCC GATCGCAGGA ACAGCATGTC GTAATAC CAC 4681 TTGGCGTCGC CCAGAAACTC GTCGAACGTC GGGCCGTCAA CGTTCCACGT GGCGAATACC 4741 AGGCCGCCGT ACAGGGTAAC CCTGGCTTTT CTCAGACCCA GCTGTTCTTT GGGCAGCATC 4801 TTGCCGTGCA TGCACTCGCG ATCGACTGGC GAGCCGATGA AGTCACCGTT GGGACGGAAT 4 861 GCCCAGCCGT GGTAGATGCA TTTATGGATC TGGGTGTTGC CGCAATCGGC GGTACTGATG 4921 CGCATGCCAC GGTGCGGGCA GACGTTGAGG GTGACGTGGA CTTCGCCTTC CTTGTCGCGG 4981 GCGACGATGA CCGAGTCCGA ACCCAGGTCC CGGACCATGA AGTCTCCGGA GTTGGGGATT 5041 TCCGATTCGT GCCCGAGAAG AACCCAGATC TTGCCGAAGA TCTTCTCCAT TTCAATTTCG 5101 TAGATCTCGC GGTCAGACAG CACACGCATC TGCACTTCGC GAGTCGAGGG ATAGATAAGG 5161 TCAGCGACCT TGGTTCCATC GGACAGTACG GGGCTTGTTC TTGTTAGCAT ATTGGCCTCC 5221 AGTTCAGGGT GTCACGGCTA CTCCGAGCCG GTTGAATATA AGACAGATTC ATAAAATATG 52 81 TATAGCACTG TCGTTTGTCT CGCAGACAAA TCAATCAGTG AGCCGTTCAA ACCTGCTGCG 5341 CATTGCCCAC CTGGGTGCGC AGCACGCTAT TCCGTCGACC TCCAGCTCGA CCCGATCTCC 54 01 AGGCTTCAGA TAGCGCAGTT GCTCCAGGCC GCAGCCGTTG CCGACCGTTC CCGAGCCCAG 5461 GAACTCGCCC GGATAGAGCG TCTCGGAGCG TGATATGTGG GCGATGACGT CTTCGAACCG 5521 CCAGCGCATG TCTCGTGTGT TGCCCCTGCC CCACTCCTCA CCGTTGACCC TGGCAACCAT 5581 ATTAAGGTCG TAGGGGTCAC CCAGTTCATC GGCCGTCACC AGGCAGGGGC CCATGATATT 5641 CGCTGTGTCG AAGTCCTTGC TCTTGGCCGG GCCAAGCATG CCCGGCATCT CGGCGGCCTG 5701 TGCGTCACGG GCGCTCATGT CATTGAAGAT GGTGAATCCG ACGATGTGAT CGCGAGCGTT 57 61 GTCCCGGGAA ATGTCCTTGC CTTTCTTGCC GATGTAGCAG CCAAACTCCA GCTCGAAGTC 5821 CAGCGCTTTA CTGAACGCGG GCCAGAGGAA TTCATCATCG GCTCCGATCA CGGCGAAGCG 5881 ATTGCATTTG TAGTAGATGG GCTGTCGGTT GAAGGTCTCG ATCACCCGCT CGTCGGCGCG 5941 GGTATTCATC GCTTTGAGCG TGGCCTCCGG GTCTTCGGTG CGAAGGGCCC GGGCGCGTCT 6001 GGCCGCAGCA AAAGCCTGAC GCAGATGCAG CTCGAAACAG GAGCAGTCGC GCATCTGTGG 6061 CGGCGGCTGG ATCGGCGCAC GCAGCCGGAC CTCGTTGCGC GCAATGACGC TGGCTGTCGG 6121 GGATTTACGC AACAGCGTGC GCGCCAGTTC CAGCGCCTGC TCGCCCCCTT CCACCAGCTT 6181 CAGCACGCTG CTCAGCGCGG CGCTTTCGCC ACCCTGCACC TGACGGTGGG CGGCCTGCAG 6241 GTCAACCACC ACCTGATCGT TGTCGATCAG CGCGCCGGCA CGTTCGATGC CATCGTCCTG 6301 AGTGAAGGTT ACCAGCCTCA TACCCGGCCA CTCACTGGCA GGCAGGCGCC GGTCACGGCG 6361 TCTGCGTCGT CGCAGAGCAG AAAGGCGATG ACAGACGCCA GTTGCGTCGG CCTCACCCAG 6421 CGGCTGAAGT CGGCATCAGG CATGTCGGTG CGGTTGGTCG GGGTGTCGAT GATGCTGGGC 64 81 AATACGGCGT TAACCGTGAT GCCGCGATCC TTGAGTTCTT CGGCCAACGC TTCGGTGAGC 6541 CGCGAGACAC CGGCCTTGGA GGCCGCATAA GCGCCCATGC CCATTCCTGC CTTGAGGCTG 6601 GCCTGAGCGC CGATATTGAC GATCCGCCCG CCATCATGGG CCAGCAGATG CTCAAGCACC 6661 GCCTGCGAAG CGATGACGGC GGTACGCAGG TTCATTTGGT AAAGGTGGTC CCAGGTCGCC 6721 AGGCTTCCGC TCTCCAGCGT TTCCCAGGCG AAACCGCCGG CGACATTCAC TAGGCCGTCG 6781 ATCCGCCCGA AGTGTTCGGC CACTTGCGCA AACGCCGAGC GGGCCGATTC GACAGAAGTC 6841 AGGTCGACAC CTCCCAGCGC AAGCACGTTG GCGTCGTCGC TGACCCTGGT CTGAGACGTC 6901 TCGGCCCGAT CCAGCAGCGC GACACGGGCA CCGCGGCCAC TTAAATACTG ACCGAGCGCT 6961 GTCCCGAGAA CCCCGAATCC ACCGGTTACA ACGATCACTC TGGCTTGCAA GGGTGAGTCT 7021 TCAAGCGCCT GGTCCTGGCG AGAACTGGAC ATCGCGATTT CCTCTTATTG TTGTTGGGCG 7081 CACCCTGGAA GGGTGAGCTA GGCATGCACA CAATATAAGA CACTTTTTAA AAATATGTAC 7141 AGATGATACT GTGCATTGGT TGACTGGAAT CAATAAAACC TCATCCGGAC TTCCACGCCC 72 01 ATGCCCAGAA AACTTCCTGC ACTGAACGCC GATAACCGTG CCTTCTGGCA AGGTGGCAAA 7261 GACGGTCAGT TGCTCATTCA CCACTGCGAT GCCTGCCGGC GGTTCTTCCA TCCGCCCTCG 7321 CCCATCTGTC CGCGCTGCGC CAGCTTCGAC GTCAAGCCGC GCGGCGTTTC CGGCAAGGGC 7381 TCCGTCGCCA GCTTCACCGT GAATCATCAG CAGTGGACGC CTGAACTCGA CACCCCCTTC 7441 GTGGTGGCGA TCATCGAGCT GATCGAACAG CCAGGATTGC GCTTCCTCAG CAACGTCATC 7501 GGTTGTGACC CACTTTCGGT CAGCATCGGC ATGCGCGTGC ATGTCACATT CGACAACGTC 7561 GAGGATGTCT GGCTGCCCCT GTTCGAGAAG GATCAATGAT GTTCGATCAT CGTTACGAGA 7621 AAGACGTCGC CATTACCGGT ATCGGTCAGT CGGAGGTGGG CCGCCCCTCC AGCAAGTCGG 114 7681 CCATGCGTCT GACACTGGAG GCGTGTCTTG AAGCCATCGC CGATGCCGGC CTGACCCGTG 7741 AGGACATCGA CGGTGTCGCC TGCTGGCCCG GTGACAACAA CAATGGCGAT CCCTTTTCCC 7801 CGGTGGGGCC CAGTGCGCTG AAGAGTGCGC TGGGGCTGAA CGTCAACTGG TTTGGCGCCG 7 861 GCTACGAAGG GCCTGGCCCG TTGGCGGGAG TCATCAACGG AGCGATGGCC ATCGCCGCAG 7921 GCTTGTGTCG ACATGTGCTG GTGTTCCGCA CCATTACCGA AGCCTCGGCC CGCCAGCACA 7981 ACAAGCAGGC CGGGGCCTTG AGTGCAAAAA CCCAGGGGCG GGACAGCAGT CATGCCTGGC 8041 AATGGTTTAC ACCGTTTAAC GTCCATTCGG GCATCAACCT GATGGCGCTG TATGCGCAAC 8101 GCCACTTCCA CGAATATGGC ACCCGGCCGG AACAACTGGC ACAGATCGCG TTGACCTGTC 8161 GAGCCAACGC TCAGCGTAAT CCCAAGGCCA TCTATCGCAC GCCCATGACC ATGGACGACT 8221 ACATGGCATC GAGGATGATC TCCTCGCCGC TGCGCATGTT CGACTGTGAC GTTCACTGCG 8281 ATGCCTCCAC CGCCATTGTG CTGTCTCGTC GGGACGTCGC CAAAGATACG CGGCATCAGC 8341 CGATCCGTAT CGAGGCAATG GGGGCGGCGC TCGATCAGCC TTGGTCGTGG GACCAGATCT 8401 CGTTGACACA AATGGCGGCG TTCGACGTGG GCCGCATGAT GTGGGCGCGC ACCGACTACA 8461 CGCCGTCTGA TGTCGGTTCG GCACAGCTTT ACGATGGGTT TTCGATCCTG ACGATGATCT 8521 GGCTGGAAGC CTTGGGCCTG TGCTCGACCG GGCAAAGTGG TGCATTCGTC GAAGGTGGGG 8581 AGCGTATTGC ATTGACCGGC CAGTTACCGA TCAACACCAA TGGCGGGCAA TTGTCCGGCG 8641 GCCGGACCCA CGGGCTGGGT TATGTGCATG AAGCGTGTCT GCAGCTTTGG GGGCGCGCCG 8701 AGGGTCGTCA GACCCGGCCT CACACCGTCG CCGCCGTTGC CGCAGGAGGG GGCCCGTTGG 8761 GAGGCAGCCT CCTGCTGGCC AGGGACTGAG CAACAGGCTG GGCAGAGTGC TTAAAACAGC 8 821 ACCTTGCTCA GCTCTGCGCA GAACGTCACG AGGGTCCGGC CGATTTCCTC GATTCCTTCT 8881 TCATCGCGCT GGCCTCTGAA CAGCACAGCC GATACCGTAA AGTCTATGTT GCCGGACGGG 8941 GAAAACACTG GCGCCGCAAT GGCCAAGATG CCGATGGAAA AGTAGCCATC ATCCACCGCC 9001 CAGCCCCGCT CGGCCGCAAG CGCCACATCC TGGCGATAGA CCTTGAGCGG CAAAGGGCGC 9061 GCCCACCGAA TGGCCTCGAA ATCGTCCCTG ATTTTTTCCT CATCCAGATC CATCCGGGTG 9121 GCGAACAGCC GCCCGCTTGC CCCCATCAGA ATCGGCAAGC GTTGCCCTTC GGCCATGTCG 9181 ATGCGTACGT CGGTGGGGCT CGCGGCAGAG CTGACAATCA CAATGCGATC AGGGCCCATG 9241 CGGCGCCACA GGGTCACCGT GACGCGCAAA CGGGCAGCCA GTTCCTGCAG CAATGGTTTG 9301 GCGAACTCGA CTCGCTGGCC CTGAGTCACC AATTGTTCGA CCAATCGCGC AAGGCCGAGC 9361 CCTGCGGAAT ACCGTTTGGA CAGTGGATTG AAATCCACCA CGTCTTCCAT GACCAGGGTG 9421 CGCAAGATGT TGAAGCAGGT GCTGGGATTG ATACCCAGAA CTCTCGCCAG ATCCACCGAG 9481 CGCTCCGGAG TGCCGGCCTG GCTCAGATGA CGAAGGATGC GGATAGCATT CGAAACCGGC 9541 TTGACGCTGA CCGCGCCTTT CTGGACGTCG GTGGTGGTTT CAGCCGTGTC AGGACGTTGT 9601 GGAGCATTGC TGTCGGTCTG GGTACGAGGC ATCTTGAATA CCGGGTGATC GGGCGATGCG 9661 GCATTCTAGC CTGAGAACGA CAGCGTATTA AAAGTTTAAT ACTGAAACCA CTCAGTTATT 9721 TTCCGGGGCA CCGGCGAGTG GCTGATGAAT GTAGCGCCTC GATATCTGCG CGTCACAACG. 97 81 CATGCCACTC AAGCGGGCCT CCAGAAGCTG ATCTGCCTGC GTGGTTCGCG ACTGCTGACG 9841 TAGGTAGTCC AGCCACGACT CGACGATGAA ACGCTCTACA TAATGGCCGT CTTCGTCGAG 9901 GTCGTGATAA AGCCGCCAAT TCTTCGCTCC GTTGCGACGC CTGGACTGGC CAACCGCGTA 9961 GGCAACCGCG ATGAAGTTGG CGCGGTCTTC CGGGGCGACC CGGTACGCCA GTTCAATTGA 10021 GACCGGGCCG GGGCCAGGGT TCAACTGCTC GGGTACCAAT AATCGGTCTG TCGGCTGAAT 10081 CGCGCTGGCG TAATCGGCCT CTTGCCCCAG TTCCAGGCGA CCGCTGCGAG TCAGAATCAG 10141 ACCAACCCCA AGTGTCACCG CTGCCAGCAC CAGGCTGTTC TGCAAGCCCA GGTACTCGGC 10201 GAGTGTTCCC CAGATAGCGC CGCCGATAGC CATTGCCCCC ATCAGCATCA GCAGGTAGAC 10261 CGACGCGACC CGGGCGCGTA CCCAGTTCGC CGCGCAGGTT TGTACCACTG TTGAAATCGT 10321 CGCGTTGACG GCCATCCAGG ACATCCCGGC GATCATCAAC GTTCCACAGA CCGCCCACCG 10381 GCTGTGGGAC AGCGCGGCTA CCAGCGTGGC AAGCGCGAAT CCCGCAGTGC CTGCGATGAT 10441 CAGAACACGC AGGGGAAAGC GGCGGTACAT GGCCGTGAGA TTCAAAGCCC CGGCCACCGC 10501 ACCGGCGCCC AGAAAACCCA TCAACAGGCC GTAGCCTCCG GCGTCCATTC CCAACTCGTG 10561 CTTGGCCACC AATGGCAGCA GCGCCCACAG CGCGCTGGCA CTCAGCGTGA AGATGAAGAC 10621 CTGTTTCAAT GCGCTGACCA GCACCGCGGA ATGCCGTATG TAACGCACGC CACTGCGCAG 10681 GCCTGCGGCA AAATTTTCCG CTGGCAGGGC GTGAGGCGTC TGACGAGGGG TGAATGCGGC 10741 GACCAGCACG ATGACCAGCG CCAGGCACAC CGTGATGATC ACGAACACCG AGGCGGCATT 10801 CCAATAGGCG ATCAACCCGC CAGCCAGCGC CGGGCCCAGC GCGCGGGCGG CATTGGTGGA 10861 AATACCGTTC AGGGAAATCG CCGCCGGAAC CTGATCCCGC GGCAACACCG AGACGATCGT 10921 GACCATCCAG GCTGACATGC TGAGCGCACT GCCGGTGCCC AGCAAAAACG TGAACGCCAG 10981 CAATGACCAG TCGCTGAGCA TGTCCAGCGA CGACAAGACG CTCAGGGCTG CCGCCGCGAT 11041 CAGCATGACG CCCTGGGTCA TCATCAGCCA TTTGCGACGG TCGACCAGAT CGGCAATCAC 11101 GCCACCGGGC AATCCGAACA GAAATGCTGG CAGGGAGATT GCCGTCTGCA CCAGCGAGAC 11161 CAT CAG CGGT GAGGTCGACA GCAAGGTCAT GATCCAGGCG GCACCGACGG TCTGCATCCA 11221 GATCGCCAGA TTGACCATGG CGCCACCGCA CCACAACCAG CGGAAACTGC GATTGCGTAT 11281 TGGCGCCCAG GCTGAGGCCG GCTGGGCGCG GGGTACCGCT GGCTCGTCCG GGCTTAGCCC 11341 CGAAAGATCG CTCATGGGCT GACAGGCTGG CCAAGCCTGC CCACCGAACG TGCTGTAGTG 115 11401 AGAACCCGTG GCTGGCTGAC GCGGTTGCTC 114 61 ACCCGATCGC CCGGCTGCAG AAAACGGCCA 11521 GTGAACACCA GATCGCCGGG CTGCAGCGTT 11581 TCGATTGGGA AAAGCATCTG TCGGGTGTTG 11641 AATTCGATGT CCAGGTGCGG TTGTCCGACG 117 01 CCGACCGGCG TCGTGCGATC CATGGCCTTC 11761 TTGCCCGCAT CCCGGGCGCT GATGTCATTG 11821 GCGCAAGGCT CATCACCCAG GGCACGAGCG 11881 AGTTGCGCGG TCAGCGCGGG GTACTGGATG 11941 GGTTTGATAT AGCCCAGCGG ATGCTCCGGG 12001 AGGTAATTCA GGCCCACGCC AAAAACCCGG 12061 CATTGCGCCA ACGCTATACC GACCTTGTCC 12121 TATTCGCCAG CCCATTCGGC AAATGGTCCA 12181 TCGAGCAAGG CCCAGAAGCA TTCTGCGCGA 12241 CTTGCTCGCC AGGTATTCCG GGTCCAGGAC 12301 AATCGGACCG ACCATTTCAT GCCCCCACAG 12 361 GTCGCCCTGC CACGGTTCGA TTTCAGCGAT 12421 GTGATAGAAC GAAAGATGCG GATCCTGGGT 124 81 TTCGCGCTTC TTGACGATAT CGTAGGTTTC 12541 GCCGACGTGC TGGACACCCA TGCGCCCGAC 12601 GTGGTGATTG AGTCTGGAAC GGAAGAAACC 12 661 CCAGTGAAAA CCCATGATCC GCGTCAGAAA 12721 GATGACGACA ACGTGCCCTG CCCCGCGCTC 12781 CGGCATGAAT GAACGGGGCG TCCACTTCTG 12841 CGGGTCGCGA AAGCGAATCA GCTCGCGAAC 12901 GCGGGTGATC TCGACGTGAT TGGCTTCGAA 12961 GCCCATGGCC TCCCAGCCGA TGTAGGCCAG 13021 GCGGTGCCGC CGGTCGTCTA TCTTGAAGTA 13081 CGCGATCCCA AAACCCATGA CCTGCGGGCC 13141 TTCAAAACCC AGATAAC CAA TACCGATGAT 13201 AGGTGGGTGT CAGTCAGATT GCCAGCAAAC 13261 TGAAGGAGGC CTCGGCAGAG GCAACGAACA 13321 CCGGTCGATC CATCAGCACG CCATCGAGCA 13381 ACGCAGTAGT GCCTGGCGTG GCGATGAATC 13441 CCGGAGCGCC TTCGACGGCA AGTTGCCGGG 13501 CATGTGCGCT GATGCCCGCG ACTTTTGAAC 13561 TGATCACGCC GCGCTGTTCA GCCAGATGAT 13621 AATCGATTTC GTTGCGCATG GTGAATTGCC 13681 CGAATTTCGC CGCCGAAGCG TTGTTGTACA 13741 CTGCTTCGAT CCAAGCCTTG GCTTGCTCAG 138 01 GTAATTTCAG CCCCTCTGCA CGCATCAGCG 13861 TATCACACCC CACCACGACT GCGCCTTCAC 13921 GTCCACCACC GGTTCCGGTG ATCAAAACCA 13981 CCTTCCCCTG TTCATGCGCA CACGGTTGTT 14041 AAAGACTCAG GCAGCGTTGT CGGGAGTATC 14101 GCAGGCTGCG GCGCCTTCGC GGGCTTCGTC 14161 AAGATAGACC AGTCCATCGT CGTCAAGCTT 14221 GCCATAGCCG CAGCAGCGTG AACGGTCGGC 14281 CATTTCTTGT CTCCGTTGTA GGTTCAGTTG 14341 TAAATATTTC ATCCACATCC TGTGCTCTTG 14401 TGAGCATCAG CGAGTTTTTC AGCGGACAGC 144 61 CTTTTTGGCC TATTTGATGA ACATATATTC 14521 CGGTCAGACG GCTAGACAGA CATAACCAGC 14581 TCAGGGAGAA CGACCATAGC GTCGCTGTTG 14641 AACCTATAAA GGAAGCCAAT ACATGACCTC 14701 CGAATTTCGA AGCGCCTGGA AGATCCTCAT 14761 CAATGCTGCG CTGCTTTATG GCTTTGGAAC 14821 CTGGACCAGG CCGGAATTGC AGGCGTGCAT 14881 CCTGCAATTG GTCGGCTGGT TCAACCTTCG 14941 ATTGCTGTTG CTGGTGCTCG GCTACCTGGC 15001 GATGTATCTG GCATTCGCAC TGCTGCCCAT 15061 GACCCAATTG TTGAGCCTCT GGTACGAACG ATGCTGCCAA TCCCCTCGAT ACGAACCTCC TCCTCAAGGC CCACACCGTG GGTTGAACCC ATCCGAGCGT CGACGTAGTT GAGCAACTCA TCGAACTGGC GACATTCATC GTTGACCGAC CCGATTTCAT CGAGGGTGAC GACCCATGGG TGGGTCAGCA GATCGAGGCG GCCGATCCGT CCGCAGGTGT AGCCGAGCAG GCAGGCACTG ATGACCGCGA CAAGCTCCAC TTCGTAGTCG TCGTCTGAAG CGCCCACCAG CGCAGATTCC GCGGTACTCC CCAGATGCTG CAGGTGGCTA GCGCCGGGCT CCAGTGGCGG CAGGATCCGG AGCGCCAGCG CGGCAATCCC CCGGGATGCG CGAATACGCT GCAGGTAGGC TGCGTCAGCA TACTCGACCC TTAACAGGCG CATCTCAGCG CTCTTCGGGC GTCTTGACGC TCGGGCCAAG ACTCAGGCAC TCAGGATTGA GCTCGAAAGG GGTTTCGAAG GCGAAACCGG AGGGCGTGAA GTGTTGCCCC AGCGTCATCA TCATCGGCAG ACCGACGTCG CGAATGCTGT TAAC GAACAG ACCATGACCA TAGGCGATGT CATGGCTGGT GGTACGCCCC TTGCCTGCTC CGGCGCCGTA GTCTTCCAGT TCGTCCGTGT ACTCGGGGGT ATTGGCAAGA AAGCCACCAT GGGGACGGCC GCCGTAGAAC AATTCGTGTT GATAGCCGAC ACCGCGCTTC TCGCAGAGGG CAGCGTCGCC GGTCTTGATC GCCGCATCGA ACGCCTGTCT GCGGTCAACT TTGCCGGGGT GGAAGGCAAA CAGCGATTCT TTGTCGCTGG CAGGCGACGG GTATTCGCGC CACGCGTCCA GGTCTGGCGT GTCCATGTGA ATGCACCTTT TGTTCTTGTC CGCCATCGAT GACGATGTCG GAGCCTGTGA GTGCCATGGC AACGACCTCT TCCGGCTCTC GCGCTGCACG AACCTTCGGG TTTTCCATGA CAGGGCTGAT GCTCACCGCC CTTATACCCA TGAAGGCTAT GACCGCGCCT TTGGCGGCGG CGCCCCAGCC TGCCGTCGAG GAAATATTGA TCCAGGCATA TTTGGTGGTG TAGAACAGCA AGTCCTCGAT GGACAGCTCG CTCACCGGCC ACACATCGAT GCGGCCATGT TCCTGCACGG GGTCTCCAAG ATCGACGGGT GCCGATCCGT CGGCAGTTTC CTTGTTCGCG CCGGCGTTGG GAGCGAATTT CAGCGCCGCT ACGCGACCCT CTTTTTCCTG CAAACGACCT GCCATCCTTT CGATTGCTGA CGGCAAAAGC GTCGTCAGGC GAGGGGTTCG AGCCAGATCG CTTCGGCCGG TTCAAGCTCG GGAGGCACTC GGTCATCATC GTAGATGCTG GGGCAAACGG CGGCGCACAG CACCACTTTG AATTGCGGTT TTTCTGAACT CGCCAATAAT GAACACTTTC TGAGCATGTG GGTGCAATGC AGCTCCATGA CCCCCGATTT CATGGATTTT TCGGCCTTCT GCTATGACGA GATTTATGTC TATTATTTTG TCGAGACGCG TGCCATATTC TCGATCCTGC ACGCGATTCT CTTGCACGGC TCCAACACCT GGCTCGACAA TTCGAAAAGC GAAGCGGCGA TCGATTTCAC CCTGTCCGTG GCCGGCGTTG CGATCAGCAT CCTGGTCGTT CCATTGAACC AGGCGTTCGG CACTTTCCTG TTCGGCGGCG CCGTCATTTC CTACGGCATC AAGCGCGTGA CGGTGATCTC AACCACCCAA CTCACTCATT CGATCTGGTC CGTCGGCATG GGCACGCTTG CGGTCACCTG CAATCGCGGG CTGGCGCTGG CAATCGGCTT 116 15121 GTCGGGCACT GGGCTGACAG CAGCCATCGT CCCGCGCCTG ATGAGCTGGG GTATCGAACA 15181 GTGGGACTGG CGGGCGGCGT TCGTCATGCT CGCAATCCTG AACCTTGTGG TGCTTCTGCC 15241 GCTGACCCTG CTGTGGTTCC GCTTGCCGGG TGTCGAAGTC AAACGCTCCG AGCTAGCCGA 15301 GAAAGCCGCT GACCATCTGT TGCTCACCCT CCCCGGCGTG AGCTTTCGCG AGGGTATGCG 15361 CTCCGGCAAG TTCTGGATCT GTAATCTGGC CCTGTCGCTG GTGGTGTCCT CGGTGGTCGG 15421 CATGGTCACC AGCACCATTC CGTTGTTGCA GTCCAAAGGG CTGAGTGCCG CCGATGCCGG 15481 ACTGATCTTC AGTGGGTTCG GGATCTCCCT GATCGTCGGC CGGATGTTGA TCGGCTATCT 15541 GCTCGATCGG CTCTGGCCGC CCGCCGTGGC AGCGGCCAGC CTGATGATGC CAGCCGTCGG 15601 CTGCATGATT TACCTCAGTG GCACCACGGA TTTCCAGATG CTGCTGATGG CCGCCATGCT 15661 GGTGGGTTTC GGGGCCGGTG CCGAATTCGA TATCGCGGCC TTTCTGGTCG CACGTTACTT 15721 CGGCCTGCGC GAGTACGGCC GCCTGTTTGG CGTGCACCAG GGCCTGAATA CCGTGGCCTC 15781 TGCCCTCGCT CCGTTGCTGT TCGCCTTCAT GCTCAGCCGC AGTGGCGACT ACTCGGCAAT 15841 GCTGGTGTAT TGCATGGCCT GCTGCCTGAT CGGGCCGCTG TTGTTACTGA TGCTGGGACG 15901 CGCCCCCCGA TTCCAGGGCG CTGCACTGGC GGCGTCCTCC TGAAGACTCG TCGCCCACGC 15961 CAACGCTCTT GACTTCCGAC AATAAG 117 APPENDIX m S p e c t r u n P l o t F i l e : O : \UM3 D a t e : 09 Sep 1998 1 7 : 3 3 : 4 4 C o n n e n t : S c a n : 1753 S e g : 2 G r o u p : 8 R e t e n t i o n : 2 3 . 3 5 R I C : 5027G M a s s e s : 9 8 - 5 5 0 tt P k s : 330 Base P k : 253 I n t : 4238 100 .00X = 4230 1BBZ 253 G C - M S of7-oxoDhA p-p- |T | 11 i | i | > 1 i p 1' I ' p i 1 1 • 1 ' 1 ' I ' I ' I "i-p ' TI ' 1 '1 P P | ' P P P P ; ' P I 1 I 1 P P I 1 I 1 P P | 1 P P P P P I 100 150 200 250 380 350 400 450 508 550 S p e c t r u n P l o t F i l e : O: \UM3 D a t e : 09 Sep 1998 1 7 : 3 3 : 4 4 C o n n e n t : S c a n : 1847 S e g : 2 G r o u p : 0 R e t e n t i o n : 2 4 . G I R I C : 384785 M a s s e s : 90 -550 tt P k s : 29G Base Pk : 284 I n t : 22389 1 0 0 . 0 0 * = 22389 108* 284 GC-MS of major metabolite i | • i i i i p i i i i p i i i i p i i i • i i i i i i i i i i i , i i i i p i i i i p i i i i p i 180 158 280 250 300 3S& 400 450 500 550 1 1 8 9 <Ni <-i ^ d d ^ S K ^ S d 3 3 3 I I I O O O z z z "to T- 3 S SI i- f~J o 3 £ 8 S K ™ o) ]J jj |J Jj n irt to o en oi to •* c> <o d i ~ * d d in «* v> T- oi r> n rJn <nui in o *r S 2 r r q q ooo q q q £ c CO O ^ TO O CU iiO 119 GC-MS chromarogram and spectra of intermediates found in the supernatant of ditC mutant strain BKME-93 cells suspension C h r o n a t o g r a n P l o t C o n n e n t : S c a n : 1T50 S e g : 2 G r o u p : P l o t t e d : 15B0 t o 2888 C : \S f lTURN\DATf i \MUTZl D a t e : 8 4 / 8 1 / 9 8 8 5 : 8 6 : 4 8 R e t e n t i o n : 2 3 . 3 1 R I C : Z57G58 M a s s e s : 188-644 R a n g e : 1 t o Z8Z6 188* - 16228132 6 . 2 5 * TOT 1E1G08 115994188 441822692 1584| 514923| 1GG8759 431816 18191291 1544 15927G 407138 1722 484511 1232847J 1768 1638 164113 555388 87234 1211967 296797 1866 441832 f£\ 1708572^2/ | 1886 [290053 1218559 1500 1 9 . 9 8 1608 2 1 . 3 1 1700 2 2 . 6 5 1888 2 3 . 9 8 1980 2 5 . 3 2 C : \SATURN\DfiTH\MUT21 Date : 0 4 / 0 1 / 9 8 05 :06 :40 S p e c t r u n P l o t C o n n e n t : . S c a n : 1722 S e g : 2 G r o u p : 0 R e t e n t i o n : 2 2 . 9 4 R I C : 624411 Mass | H P k s : 251 Base P k : 237 I n t : 95141 1 0 0 . 0 0 * = 95141 188* 237 90 - 408 INTJ 0 147 115 131 195 1G2 177 lll|M|lll 287 312 253 265 2 8 1 2 9 7 330 355 3 7 1 I 1 i '" I 1 f 1 I 1 i 1 T 100 128 148 168 180 200 220 240 260 280 300 320 340 360 388 488 120 C:xSflTURNsDATAsMUT21 D a t e : 04/81/98 85:88:48 90 - 480 S p e c t r u n P l o t Comnent : D=+=«+ir.Yi- 23 27 R I C : 351533 Mass S c a n : 1747 S e g : 2 G r o u p : 0 R e t e n t . o n . 2 3 K l ^ M x _ 4 7 G 0 B tt P k s : 281 Base P k : 147 * 100/. 147 IMT-J ® 115 131 311 177 207 298 1G5 1 OQ7 255 221 CV . 269 4 326 341355 3 8 5 'I i f i I I " | 1 'I ' I 1 I ' 'I C:VSATURNSDATASMUT21 D a t e : 04/01/98 05=06:40 S p e c t r u n P l o t £TU S e , : 2 G r o u p : 0 R e t e n t i o n : 23^ 98 RIC : asxm ^ B L tt P k s : 386 Base P k : 147 1 < 1 X 100X 147 INT 131 115 297 177 207 165 195 Ijllyljllljl 223 255 269 284 237 312 II 327 3 4 1 3 5 5 371 387 f\ ! I"i • | ' l 1"| 'V ' I ' ' ' I 121 C :\SATURN\DATA\MUI21 D a t e : 8 4 / 8 1 / 9 8 0 5 : 8 6 : 4 8 S p e c t r u n P l o t C o n n e n t : , S c a n : 1866 S e g : 2 G r o u p : 8 R e t e n t i o n : 2 4 . 8 6 R I C : 688823 Mass | « P k s : 312 Base P k : 313 I n t : 187528 1 8 8 . 8 8 * = 187528 188* 313 98 - 450 INT 147 115131 287 177 jllaJli •-•1-i"-f"r-|-'i-|'4IJfr'^'j*<''if'tl**|t^r''f*r''Jj*'*'l"i>*Jr'*'*'V1»"'f'*fr'l*'^-1'"*"-! '"'I' |"n ''i ' 1"' | -'"I ' ) ' 100 120 148 160 180 200 220 240 260 280 300 320 340 360 380 480 420 440 247 2 7 2 8 1 i|¥f'if'i't ll 4 'T'T'> l i 299 388 356 328 373 415 C : \SAIURN\DATA\MUT21 Date : 0 4 / 0 1 / 9 8 05 :06 :40 90 . -^450 S p e c t r u n P l o t C o n n e n t : S c a n : 1886 S e g : 2 G r o u p : 8 R e t e n t i o n : 2 5 . 1 3 R I C : 564124 Mass it P k s : 316 Base P k : 147 I n t : 48231 1 8 8 . 8 8 * = 48231 188* 147 INT 183 131 l l U U l l 311 269 287 177 165 191 255 2 2 1 2 3 8 JiiLi 286 299 386 327 371 341 415 I I ''I ' ' I r [ 1 T ' 1 | '"I ' | | 328 340 360 380 400 420 440 100 120 148 168 188 200 220 240 260 280 300 1 2 2 C : \ S A TURNSDATASMUT21 D a t e : 0 4 / 8 1 / 9 8 8 5 : 8 6 : 4 8 98 - 450 S p e c t r u n P l o t C o n n e n t : S c a n : 19Z6 S e g : Z G r o u p : 0 R e t e n t i o n : 2 5 . 6 6 R I C : 48969Z Mass L tt P k s : 345 Base P k : 147 I n t : 44845 1 0 0 . 0 0 * = 44845 180* 147 299 INT 131 115 IdJlllJ 287 177 133 221 Lllilllll.lllllllll.llllil.IlL 281 269 257 K-r1 374 327342 314 ^ 4 415 | ' I ' | ' I ' | ' I ' | '''I ' l L | i 7 • | i I 188 128 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 Date : 0 4 / 0 1 / 9 8 05 :06 :40 98 - 450 S p e c t r u n P l o t C : \SATURN\DATA\MUT21 C o n n e n t : S c a n : 1947 S e g : 2 G r o u p : 0 R e t e n t i o n : 2 5 . 9 4 R I C : 385590 M a s s | tt P k s : 312 Base P k : 147 I n t : 47158 1 0 0 . 0 0 * = 47158 188* 147 (9 I N T J 131 115 luiill 207 177 165 191 297 281 257 223 372 313 I I | I | I | I | I | I |T"| i p | !"| i ]" i | i | i | 1 | 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 341 357 415 ,1. 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