Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Microbial metabolism of abietane diterpenoids by Pseudomonas abietaniphila BKME-9 and Burkholderia xenovorans… Smith, Daryl James 2006

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2006-200933.pdf [ 10.96MB ]
Metadata
JSON: 831-1.0093012.json
JSON-LD: 831-1.0093012-ld.json
RDF/XML (Pretty): 831-1.0093012-rdf.xml
RDF/JSON: 831-1.0093012-rdf.json
Turtle: 831-1.0093012-turtle.txt
N-Triples: 831-1.0093012-rdf-ntriples.txt
Original Record: 831-1.0093012-source.json
Full Text
831-1.0093012-fulltext.txt
Citation
831-1.0093012.ris

Full Text

MICROBIAL M E T A B O L I S M OF A B I E T A N E DITERPENOEDS B Y PSEUDOMONAS ABIETANIPHILA B K M E - 9 A N D BURKHOLDERIA XENOVORANS LB400 by Daryl James Smith B.Sc., Dalhousie University, 1989 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE F A C U L T Y OF G R A D U A T E STUDIES (Microbiology and Immunology) UNIVERSITY OF BRITISH C O L U M B I A August 2006 © Daryl James Smith, 2006 / ABSTRACT This dissertation has elucidated initial steps in the degradation of abietane diterpenoids by Pseudomonas abietaniphila B K M E - 9 and Burkholderia xenovorans LB400. A 10.4-kbp extension of the dit cluster in B K M E - 9 containing genes involved in abietane diterpenoid degradation has been sequenced. The ditQ gene was found to encode a cytochrome P450 monooxygenase. Knocking out ditQ had little effect on growth of B K M E - 9 on abietic acid (AbA) but impaired growth on dehydroabietic acid (DhA) and palustric acid (PaA). A xylE transcriptional fusion showed that a range of diterpenoids induced transcription of ditQ. Substrate binding assays of DitQ revealed that DhA binds to the enzyme (Kd = 0.43 + 0.03 uM). These results indicate that DitQ is involved in the metabolism of DhA and PaA and are consistent with its putative role in converting DhA to 7-hydroxy-DhA. The genome of LB400 was found to contain a large cluster of genes with high similarity to the BKME-9-dit cluster. Microarray transcriptional analysis revealed that of the 72 genes encoded by an 80.5-kb cluster, 43 are up-regulated at least 2-fold in expression during growth on DhA versus on succinate. This cluster has been named the LB400 dit cluster. Through 2D gel proteomic analysis, we have determined that a key difference in the catabolism of the abietane diterpenoids, DhA and A b A lies in the differential expression of a cytochrome P450, DitU (CYP226A2) encoded by the dit cluster. DitQ was expressed during growth on both DhA and AbA, whereas DitU expression was only detectable during growth on AbA. Phenotypic studies of knockout mutants in LB400 containing insertion mutations of ditQ or ditU showed that ditQ was required for growth on DhA, whereas ditU was required for growth on AbA. In C e l l suspension assays, patterns of metabolite accumulation confirmed the role of DitU in AbA transformation and DitQ in DhA transformation. Substrate binding assays revealed that DitQ binds both DhA (Kd = 0.98 + 0.01 uM) and PaA (X d =1.6 + 0.1 uM). An in vitro P450 assay confirmed that DitQ transforms DhA to 7-hydroxy-DhA. These results demonstrate distinct roles ii of DitQ and DitU in the transformation of DhA and AbA to the central intermediate, 7-oxo-DhA, in a convergent degradation pathway. 111 TABLE OF CONTENTS Abstract 1 1 Table of Contents , iv List of Tables viii List of Figures i x List of Abbreviations xi Acknowledgements xiii Contribution of Others xiv 1. Introduction 1 1.1 Preface '. 1 1.2 Terpenoid Synthesis 2 1.2.1 IPP/DMAPP Synthesis 2 1.2.2 Mevalonate pathway 4 1.2.3 Mevalonate-independent pathway 5 1.2.4 The isoprene rule 5 1.2.5 Prenyltransferases 6 1.2.6 Terpene synthase 7 1.2.7 Diterpenoid cyclization 8 1.2.8 Oxidative formation of acidic abietane diterpenoids 10 1.3 Terpenoid significance •11 1.3.1 Plant defense and growth 11 1.3.2 Acidic diterpenoids as pollutants in pulp and paper production 12 1.3.3 Medicinal significance 12 1.3.3.1 Monoterpenoids - limonene and perillyl alcohol 12 1.3.3.2 Sesquiterpenoids - artemisinin 14 1.3.3.3. Diterpenoids - Taxol and others 14 1.3.3.4. Triterpenoids - betulinic acid 15 1.3.3.5. Obstacles in the production of terpene pharmaceuticals 15 1.3.3.6. Metabolic Engineering... 15 1.4. Cytochromes P450 in terpenoid metabolism 16 1.4.1. P450 heme-thiolate proteins 16 1.4.2 Classification of P450s 16 iv 1.4.3 General mechanism IV 1.4.4 P450s in diterpenoid metabolism 19 1.5 Biotransformation of terpenoids 21 1.5.1. Isosteviol 22 1.5.2. Kaurene/pimerane diterpenoids 22 1.5.3 Abietane diterpenoids 26 1.6 Summary • 30 1.7 Conclusions • 31 1.8 Thesis Objectives. 32 1.9 References • •' 33 2. A cytochrome P450 involved in the metabolism of abietane diterpenoids by Pseudomonas abietaniphila B K M E - 9 40 2.1 Introduction 40 2.2 Methods and Materials 41 2.3 Results 50 2.3.1 Sequencing of 10.4 kbp region containing a putative P450 gene 50 2.3.2 Sequence analysis of 10.4-kbp dit cluster extension. 53 2.3.3 Growth of ditQ mutant on abietanes 54 2.3.4 Abietane removal by P450KO 56 2.3.5 Specific induction of P450dit by abietane diterpenoids 57 2.3.6 P450dit reduced carbon monoxide and substrate binding spectra 58 2.4 Discussion 60 2.6 References 65 3. The LB400 dit Cluster 67 3.1 Introduction 67 3.2 Methods and Materials 68 3.3 Results 77 3.3.1 LB400 growth on abietane diterpenoids 77 3.3.2 Metabolic analysis of LB400 and Di tAIKO cell suspensions 79 3.3.3 Transcriptomic analysis 81 3.3.3.1 Dit cluster 82 3.3.3.2 Global analysis - COG distribution 85 3.3.3.2.1. Up regulated genes 85 3.3.3.2.2 Down- regulated genes 87 3.4 Discussion • 88 3.4.1 Competitiveness of LB400 89 3.4.2. The dit clusters 90 3.4.3 Catabolic transposon 91 3.4.4. Uptake of diterpenoids. 92 3.4.5. Diterpenoid metabolism 93 3.4.6. A putative oxygenase-driven electron transport system 95 3.4.7. Role of lipid metabolism genes 97 3.5 References 103 4. DitQand DitU 106 4.1 Introduction .....106 4.2 Materials and Methods 107 4.3 Results 114 4.3.1 Succinate, DhA and AbA proteomes 114 4.3.2 Growth of mutant strains 117 4.3.3 Cell Suspension assays 117 4.3.4 Binding assays 121 4.3.5 P450 in vitro activity assay 121 4.4 Discussion 124 4.4.1 Consistency of Proteome and Transcriptome 124 4.4.2 Cytochromes P450 DitQ and DitU 124 4.4.3 Binding properties of DitQ 126 4.4.4 Demonstration of DitQ activity 127 4.4.5 Proposed initial steps of the diterpenoid pathway 127 4.4.6. Electron transport ferredoxins 130 4.4.7. CYP226A family 131 4.5 Conclusion..'. 132 4.6. References 133 vi 5. Conclusions • • 5.3. Comments on future research 1 3 9 140 5.4 References Appendix ^ L I S T O F T A B L E S Table 1.1 Biotransformation of selected diterpenoids 23 Table 2.1 Strains and plasmids used in the study of Chapter 2 41 Table 2.2. Amino acid sequence comparison of deduced DitQ (P450dit) to proteins in the non-redundant database obtained by BLASTP a search 50 Table 2.3 Conserved domain search and COG comparison 54 Table 3.1 Strains and plasmids used in the study of Chapter 3 68 . Table 3.2. Growth characteristics of LB400 on four abietane diterpenoids 77 Table 3.3 Dit cluster annotation and gene expression on DhA. 84 Table 3.4. Summary of transcriptional analysis based on COG protein classification... 86 Supplementary Table 3.1 Up regulated genes outside of the dit cluster. 99 Supplementary Table 3.2 Down regulated genes 102 Table 4.1 Bacterial strains and plasmids used in this study .107 Table 4.2 Oligonucleotide primers used in PCR 108 Table 4.3 Proteins involved in abietane diterpenoid catabolism identified by MASCOT-based analysis of MALDI-TOF spectra 115 Table 4.4 CYP226A family 132 LIST OF FIGURES Figure 1.1 Chemical structures of abietane diterpenoids .* 1 Figure 1.2 Consecutive condensation of isopentyl pyrophosphate in the prenyl transferase reaction 3 Figure 1.3 The mevalonate and D X P pathways of EPP production 4 Figure 1.4 General scheme - prenyltransferase reaction 7 Figure 1.5 Abietane diterpenoid cyclization reactions 9 Figure 1.6 Consecutive oxidation of abietadiene to abietic acid ..10 Figure 1.7 Medically significant terpenoids 13 Figure 1.8 General mechanism of P450 catalytic cycle 18 Figure 1.9 Main route of gibberellin biosynthesis of gibberellins in Gibberella fujikuroi 20 Figure 1.10 Initial steps in the proposed degradation pathway of DhA by A. Arthrobacter sp., B F. resinovorum and C. Alcaligenes sp. and Pseudomonas sp ".27 Figure 1.11 Proposed convergent pathway for abietane diterpenoid degradation pathway by Pseudomonas abietaniphila B K M E - 9 29 Figure 2.1. Proposed pathway for abietane degradation by P. abietaniphila BKME-9...51 Figure 2.2 Physical map of the dit gene cluster of B K M E - 9 , the tdt gene cluster of A19-6a and putative homologues from LB400 52 Figure 2.3 Growth and substrate removal of B K M E - 9 and P450KO on DhA and AbA 57 Figure 2.4 Expression of ditQ-xylE gene fusion product in response to various diterpenoids and aromatic compounds ...58 Figure 2.5 Binding spectrum for P450dit with DhA 59 Figure 3.1 Growth curves of LB400 on four abietane diterpenoids 78 Figure 3.2 Cell suspension of LB400 and Di tAIKO with AbA DhA, and PaA 79 Figure 3.3 Plots of M versus A of combined genomic chip set after Lowess intensity dependent normalization 81 Figure 3.4 A . Plot shows M values (LOG2(DhA/Succinate) for signal intensities for genes of the LB400 dit cluster B. Physical map of the LB400 dit cluster of genes..: 83 Figure 3.5 Proposed convergent pathway for abietane diterpenoid degradation by Burkholderia xenovorans LB400 94 Figure 4.1 Global analysis of 3 LB400 proteomes 114 Figure 4.2 Differential expression of DitU 116 Figure 4.3 Growth characteristics of LB400 and two mutant strains, DitUKO andDitQKO 118 Figure 4.4 Cell suspension of LB400, DitQKO and DitUKO with AbA, DhA, and PaA 120 Figure 4.5 Binding spectra for DhA or PaA to DitQ. 122 Figure 4.6 In vitro DitQ activity assay 123 Figure 4.7 Proposed convergent pathway for abietane diterpenoid degradation byLB400. . . . '. 129 x L I S T O F A B B R E V I A T I O N S A b A abietic acid Asp aspartate ATPase adenosine triphosphate synthase B C A bicinchoninic acid Bcc Burkholderia cepacia complex B L A S T Basic Local Alignment Search Tool BLASTP protein-protein B L A S T C230 catechol 2,3-dioxygenase CDS coding sequences C F U colony forming unit CO carbon monoxide CoA Coenzyme A COGs clusters of orthologous groups cpm counts per minute CTP cytidine triphosphate dATP deoxyadenosine triphosphate dCTP deoxycytidine triphosphate dGTP deoxyguanosine triphosphate DhA dehydroabietic acid D M A P P dimethylallyl diphosphate D M D H A dimethyl heptanedioic acid DMSO dimethylsulfoxide D N A deoxy ribose nucleic acid D X P deoxyxylulose pathway dNTP deoxyribonucleotide triphosphate DTT dithiothreitol dUTP , deoxyuridine triphosphate EB elution buffer EDTA ethylenediaminetetraacetic acid EI electron impact EMP Embden-Meyerhof-Parnas F A D flavin adenine dinucleotide FID flame ionization detector F M N flavin adenine mononucleotide FPP farnesyl diphosphate GC gas chromotography GCMS gas chromatography-mass spectroscopy GFPP geranylfarnesyl disphosphate GGPP geranylgeranyl diphosphate Gm gentamicin GPP geranyl diphosphate h hour HIV human immunodeficiency virus H M M hidden Markov model IPG immobilized pH gradient rpp isopentyl diphosphate IPTG isopropyl-beta-D-thiogalactopyranoside L B Luria-Bertani N A D H reduced nicotinamide adenine dinucleotide N A D P H reduced nicotinamide adenine dinucleotide phosphate NCBI National Center for Biotechnology Information NIST National Institute of Standards and Technologies NPLC1L1 Niemann-Pick CI-like 1 MFS Major Facilitator Superfamily min minute MS mass spectroscopy OD optical density OPP pyrophosphate P450 cytochrome P450 PaA palustric acid P A G E polyacrylamide gel electrophoresis PCR polymerase chain reaction pi Isoelectric point PMSF phenylmethylsulphonylfluoride psi pounds per square inch T C A tricarboxylic acid TE tr isEDTA TMS transmembrane subunits U V ultra violet UV-Vis ultra violet-visible X-gal 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside xii A C K N O W L E D G E M E N T S First, I would like to thank my supervisor, Dr. William Mohn, for giving me the opportunity and for his understanding, patience, guidance and support. I would also like to thank my supervisory committee, Drs. Tom Beatty, Lindsay Eltis and Michael Murphy for their guidance. Lindsay was instrumental in work on the P450s by providing helpful suggestions on both experimentation and data interpretation. He also provided purified DitA3, BphF and BphG for in vitro P450 assays. I would like to thank Leticia Gomez for assistance with the in vitro P450 assays. I would like to thank Gord Stewart for everything from un-jamming the printer to help with operation of the GCMS. I would like to thank all members, past and present, of the Mohn Lab group for support and camaraderie. I am also grateful to all members of the "RHA1 group" for support and guidance in annotation and interpretation of genomic data. In particular, I would thank Dr. Marianna Patrauchan and Christine Florizone for valuable assistance on all aspects of the 2D gel work. I would like to thank Nancy Smith and Dr. Dominic Frigon for proofreading parts of this dissertation. I also acknowledge Dr. J. Tiedje for the use of his laboratory at Michigan State University to conduct microarray experiments and Dr. Joonhong Park for helpful discussion and assistance with the microarray work. I also thank Dr. R.C. Wyndham for tdt cluster sequence data prior to publication, cloned tdtD, and helpful discussions. This work was supported by the Natural Science and Engineering Research Council of Canada. To Kristen, my appreciation for accepting me, and all that goes along with that, including grad school. To Simon, Regan, and Talia, my appreciation for putting a smile on my face on those days when nothing was going right. xiii C O N T R I B U T I O N S O F O T H E R S Chapter 2 Vince Martin conducted Southern hybridization reactions. Lindsay Eltis suggested P450 substrate binding assays at a Committee meeting. Lindsay Eltis also helped interpret difference spectra. Chapter 3. Summer student Ryan Farnsworth contributed to growth assay of LB400. Xeotron microarray hybridization and scanning were conducted by Joonhong Park at Michigan State University. Chapter 4. Summer student, Angle Yu , provided help with preparation of knockouts. A portion of 2D gel analysis was conducted by Chirstine Florizone, including running and scanning gels. Marianna Patrauchan provided valuable assistance with Progenesis Workstation software. Excision of protein from gels, in-gel digests of selected protein spots and MALDI-TOF were conducted by Christine Florizone. Lindsay Eltis suggested the P450 in vitro assay and kindly provided the both BphG and Dit A3. xiv 1. I n t r o d u c t i o n 1.1 P r e f a c e Resin is produced by most species of coniferous trees including grand fir (Abies grandis) (28) and Norway spruce (Picea abies) (56). Resin consists of a volatile turpentine fraction, 5 containing mostly monoterpenes and sesquiterpenes, and a non-volatile rosin fraction. The rosin fraction, which consists mostly of diterpenoids, has been reported to constitute up to 0.8% ofthe total dry weight of wood (21). Considering the total biomass of conifers on the planet and their continual growth and decay, these compounds contribute significantly to the global carbon cycle. The most abundant diterpenoids in the rosin fraction are acidic abietanes and pimeranes (56). 10 Acidic abietanes are tricyclic C-20 carboxylic acid-containing compounds with an isopropyl group at C13 (Fig. 1.1), whereas the pimeranes have a vinyl and methyl substituents at this position. They are both produced in conifers through the cyclization of geranylgeranyl . diphosphate by diterpene synthase and subsequent oxidations involving cytochromes P450 (27, 71). 17 D e h y d r o a b i e t i c a c i d 7 - O x o - d e h y d r o a b i e t i c a c i d F i g u r e 1.1 C h e m i c a l s t r u c t u r e s o f a b i e t a n e d i t e r p e n o i d s . 1 15 This dissertation will focus on the microbial metabolism of abietic acid (AbA), dehydroabietic acid (DhA), palustric acid (PaA), and 7-oxo-dehydroabietic acid (7-oxo-DhA) (Fig. 1.1). One of the first documented applications of these tree derived natural products was in shipbuilding (12). Rosin from conifers in the form of pitch or tar was used as a sealant for wooden ships. In the biblical account of Noah and the ark, Noah is commanded by God in 20 Genesis 6:14 ".. .So make yourself an ark of cypress wood and coat it with pitch inside and out." Rosin was also used as a sealant for wine vessels in ancient Rome (55) leaving the contents with a unique terpene flavour. This taste sensation can still be found today in the Retsina wines of Greece. To gain a better understanding of the metabolism of these compounds this introduction 25 will first look at their biosynthesis and significance. This will be followed by a brief overview of cytochromes P450 (P450), key enzymes involved in both the synthesis and degradation of several terpenes, and conclude by reviewing what was known about the microbial degradation of diterpenoids prior to the work presented in the following chapters. 30 1.2 T e r p e n o i d Synthesis 1.2.1 IPP/DMAPP synthesis Terpenoids constitute the largest single family of natural products with estimates of the number of characterized terpenoids ranging from 25,000 to 50,000. Isopentyl pyrophosphate (IPP) and its allylic isomer, dimethyl allyl pyrophosphate (DMAPP) are the precursors for a vast 35 array of biologically important compounds (Fig. 1.2). These two 5-C isomers are the structural 2 OPP Dimethylallyldiphosphate(DMAPP) OPP IPP Cytokinin Steroids Cholesterol Sesquiterpenes Farnesylated proteins (RAS, lamin, etc) Heme a Dolichols Natural rubber ^OPP Geranyl diphosphate IPP ^OPP Farnesyl diphosphate (FPP) IPP OPP Geranylgeranyl diphosphate (GGPP) IPP OPP Monoterpenes Carotenoids Diterpenes Geranylgeranylated proteins (rab, rho y-subunit of G-proteins, etc) Chlorophylls Archaebacterial ether linked lipids Geranylfarnesyl diphosphate (GFPP) IPP F i g u r e 1. 2. Consecu t i ve condensa t ion o f i sopen ty l p y r o p h o s p h a t e i n the p r e n y l t rans fe rase r e a c t i o n . Adapted from (68) 3 subunits from which all terpenes are synthesized. Two pathways have been identified in the synthesis of IPP and DMAPP, the mevalonate pathway and the mevalonate-independent or deoxyxylulose (DXP) pathway (Fig. 1.3). (17, 19, 73). Until the early nineties the mevalonate pathway was considered the only route of IPP production, however recent work has 40 demonstrated a major reliance on the D X P pathway in both plants and many bacteria. 1.2.2 Mevalonate pathway A l l Archaea and Eukarya, and some Bacteria, utilize the mevalonate pathway. Mevalonate is formed from 3 acetyl Coenzyme A (acetyl-CoA) subunits via acetoacetyl-CoA 45 and 3-hydroxy-3-methylglutaryl-CoA (Fig. 1.3 A). Mevalonate is then reduced, phosphorylated and decarboxylated to yield IPP which undergoes isomerization to D M A P P through the activity of IPP isomerase. A. Mevalonate Pathway o 3 X Isopentenyl diphosphate (IPP) acetyl CoA mevalonic acid B. Deoxyxylulose Pathway multiple steps o pyruvic acid o OH 0 .OP OH dimethylallyl diphosphate (DMAPP) OH 1-deoxy-D-xylulose 5-phosphate glyceraldehyde 3-phosphate Figure 1.3 The mevalonate and DXP pathways of IPP production. 4 1.2.3 Mevalonate-independent pathway 50 This route of IPP and D M A P P production is essential in plant plastids and many Bacteria, but not in animals or Archaea (Fig. 1.3 B). The absence of this pathway in animals has made it an attractive area of research in antimicrobial therapies (19). Observations of unexpected isotope patterns in radiolabeled terpenoids in eubacteria growing on [13C]glucose, which were not compatible with the classic mevalonate pathway predictions, led to further investigation of 55 an alternate pathway (as reviewed in (19)). In one report, the fermentative ethanologenic bacterium Zymomonas mobilis was grown on radiolabeled glucose. The 2-megabase genome of Z. mobilis has recently been sequenced (78). Gene annotation confirmed the absence of essential genes for the Embden-Meyerhof-Parnas (EMP) and tricarboxylic acid (TCA) cycle pathways. Z. mobilis exclusively uses the Entner-Doudoroff pathway for the metabolism of glucose. Growth 60 on [13C]glucose showed the incorporation of 3 contiguous carbons from glucose into IPP which cannot be accounted for in the mevalonate pathway utilizing 2-C Acetyl CoA (74). In the D X P pathway IPP is synthesized via the condensation of pyruvate and glyceraldehyde 3-phosphate to form deoxy-D-xylose-5-phosphate through decarboxylation and rearrangement. This condensation of two 3-C subunits accounts for the isotope pattern observed in Z. mobilis 65 terpenoid production. 1.2.4 The isoprene rule After production of IPP, biosynthesis of the terpenoids follows the "biogenic isoprene rule" which was originally proposed by Nobel laureate Leopold Ruzicka in 1953 using the limited structural and enzymatic data available at the time (77). Ruzicka proposed that the 70 structural precursors of the various classes of terpenoids were composed from linear chains of specific lengths which were synthesized from "head to tail" joining of 5 carbon isoprene units. It is now known that the parents of the various classes include, 10-C geranyl pyrophosphate (GPP), 5 15-C farnesyl pyrophosphate (FPP), 20-C geranylgeranyl pyrophosphate (GGPP), 25-C geranylfarnesyl pyrophosphate (GFPP), 30-C squalene and 40-C phytoene (Fig. 1.2). 75 1.2.5 Prenyltransferases Prenyltransferases catalyze the synthesis of linear prenyl pyrophosphate in a 1,4 head to tail condensation reaction via a positively charged carbon ion (carbocation) intermediate from IPP and DMAPP. This is the first committed step in the synthesis of terpenoids (52). Structural analysis of prenyltransferases show two juxtaposed conserved aspartate-rich regions, which 80 coordinate substrate binding and catalysis via divalent cations in these alpha helical proteins. D M A P P is the electrophilic allylic isomer of IPP, produced by IPP isomerase, which facilitates the 1 '-4 condensation of these two 5 carbon units (16). Chain elongation involves the consecutive addition of an IPP to D M A P P or its growing allylic pyrophosphate counterpart (85) (Fig. 1.4). The reaction is initiated by formation of a carbocation at CI of the allylic 85 pyrophosphate generated by elimination of pyrophosphate via C-0 bond cleavage. Condensation occurs through the nucleophilic attack of the terminal double bond of IPP on the carbocation at CI of the allylic pyrophosphate leading to a new C-C bond. This forms a tertiary carbocation, which is eliminated through deprotonation at C2 of the now condensed IPP, leading to double bond formation between C2 and C3 of the growing chain. The reaction may be repeated with the 90 addition of more IPPs. Chain length is determined by the transferase catalyzing the elongation. For example, farnesyl pyrophosphate synthase only produces 15-C isoprenes and chain elongation is completed after the condensation of geranyl pyrophosphate with IPP, whereas geranylgeranyl pyrophosphate synthase produces a 20-C isoprene from the condensation of farnesyl 95 pyrophosphate with an IPP. Chain length is governed by the presence of a "large" amino acid residue, such as phenylalanine, located near the aspartate rich motif. The larger residue blocks 6 further increases in chain length and leads to termination (68). Substitution of these large amino acids with smaller amino acids, such as alanine, allows for increases in chain length. IPP GPP Figure 1.4 General scheme - prenyltransferase reaction. The reaction is initiated by elimination of the pyrophosphate group from D M A P P yielding a carbocation at CI . Nucleophilic attack of IPP on the carbocation yields a new C-C bond. Stereospecific deprotonation at C2 yields a new allylic pyrophosphate that is available for addition of another IPP. 1.2.6 Terpene synthase After production of linear universal precursor prenyl pyrophosphates (GPP, FPP, GGPP, 100 and GFPP), terpene production reaches a second flux point catalyzed by the terpene synthases. These enzymes determine the subclass of terpene formed. The reaction mechanism of both chain elongation and cyclization involve similar steps, which has led to speculation that both enzymes share a common ancestor (52). Both share conserved catalytic residues in primary sequence analysis as well as overall structural similarity. However, the diversity in the type and 105 mechanistic complexity of the synthases far outweighs the relatively conserved transferases. In the cyclization of C-10 monoterpenes and C-15 sesquiterpenes, catalysis is similar to the prenyltransferase reaction in that it begins with a carbocation formation through cleavage of the pyrophosphate. This is followed by intramolecular attack of the prenyl chain at CI to yield a cyclic carbocation intermediate. After cyclization, rearrangement and modification of the ring 7. 110 structure may occur, including internal additions involving the remaining double bonds to generate additional rings, hydride shifts, methyl migrations and Wagner-Meerwein rearrangements before water capture or deprotonation (as reviewed in (13)). In some cases a single synthase may produce more than one product, as seen below with abietadiene synthase. 1.2.7 Diterpene cyclization 115 The major routes of diterpene cyclization are, macrocyclic leading to the taxane (see Fig. 1.7 pg. 13) and casbene skeletal structures, or cyclization via a copalyl diphosphate intermediate (see Fig. 1.5) leading to tricyclic kaurene (see Fig. 1.9 pg. 20) and abietadiene structures (Fig. 1.5). The latter cyclization reaction has two key intermediates, the A / B ring closure to form the copalyl diphosphate intermediate and C ring closure to yield a pimaradiene intermediate. A /B 120 ring closure is initiated by protonation of the terminal C20 double bond, followed by internal additions and proton eliminations to (-)-copalyl pyrophosphate in the case of kaurene and (+)-copalyl pyrophosphate with abietadiene. A common mechanism of A/B ring closure in tricyclic diterpenoid formation leads to a common A/B ring structure. C ring closure is similar to the cyclization reaction of the monoterpene and sesquiterpenes in that it is initiated by carbocation 125 formation through pyrophosphate cleavage. Intramolecular proton shift, a 1,2 methyl migration and deprotonation complete the reaction and lead to the final product. It has been determined that this second step in catalysis may result in multiple product formations, as seen in abietadiene cyclization. In kaurene synthesis, two enzymes catalyze the two steps, whereas in abietadiene synthesis a single enzyme catalyzes both steps (70). There is evidence that the single abietadiene 130 synthase has two active sites, one for formation of the copalyl intermediates and the second for C ring closure and other structural modifications (70). 8 Figure 1.5 Abietane diterpenoid cyclization reactions. Protonation of GGPP's terminal double 135 bond yields a carbocation at CI5. Internal addition of CI 5 carbocation to CIO yields the A ring and a CI 1 carbocation. Internal addition of CI 1 carbocation to C6 yields the B ring and a C8 carbocation. Deprotonation at the C8 carbocation yields (+)-copalyl diphosphate. Elimination of pyrophosphate via C-0 bond cleavage yields a charged delocalized carbocation at C13-C15. Internal addition of C13 carbocation to C17 yields the C ring and a carbocation at C8. An 140 intramolecular proton shift from C15 to CI7 yields the pimaradienyl intermediate. A 1,2 methyl shift from C13 to C14 yields a carbocation at CI 3. Finally, deprotonation yields one of several tricyclic products. Adapted from (13) 145 The enzyme catalyzing the cyclization of geranylgeranyl pyrophosphate to abietadiene, the precursor of abietic acid in A. grandis, has been purified and the gene encoding it has been cloned and expressed in Escherichia coli (47, 84). The proposed pathway for abietadiene formation proceeds via (+)-copalyl pyrophosphate and pimaradiene intermediates (70) (Fig. 1.5). An abietadiene synthase in vitro activity assay yielded 3 major products (abietadiene, 150 levopimaradiene, and neoabietadiene) and 3 minor products (pimaradiene, sandaracopimaradiene 9 and palustradiene). These 6 products constitute the skeletal precursors of all resin acids produced in A. grandis (70). 1.2.8 Oxidative formation of acidic abietane diterpenoids Sequential oxidation of the C18 methyl group by two cytochromes P450 and an aldehyde 155 dehydrogenase are proposed to lead to the carboxylic functional group on C18 of diterpenes in A. grandis (27) (Fig. 1.6). Recently, a methyl jasmonate-induced cytochrome P450 (CYP720B1) abietadiene abietadienol abietadienal abietic acid Figure 1.6 . Consecutive oxidation of abietadiene to abietic acid. Oxidation of abietadiene at 160 C18 yields abietadienol. Oxidation of abietadienol at C18 yields abietadienal. Oxidation of abietadienal at C18 yields abietic acid. from Loblolly pine (Pinus taeda) was cloned, sequenced and characterized (71). In vitro, CYP720B1 was found to catalyze consecutive C18 oxidations of diterpene alcohols and 165 aldehydes. When expressed in yeast, this single P450 could completely oxidize C18 abietadiene to abietic acid but with lower activity than seen for transformations of the alcohol or aldehyde (71). The authors suggested that an additional P450 might be required for the first oxidative step. In summary, production of a wide array of acidic diterpenoids from IPP and D M A P P subunits in several conifers requires a total of only five enzymes. GGPP prenyltransferase 170 synthesizes a 20-C linear diterpenoid, and abietadiene synthase catalyzes cyclization and skeletal rearrangements to produce of a variety of diterpene structures. Finally, 1 or 2 cytochromes P450 and in some conifers, an aldehyde dehydrogenase catalyze oxidations to yield the carboxylic functional group. 10 1.3 Terpenoid significance 175 1.3.1 Plant defense and growth A number of plant and fungal terpene products are used for plant defense or stimulation of plant growth. It has been proposed that the large number of terpenes may be the result of evolutionary development of plant defense against insect attack, or signaling to enhance growth (38). Gibberellins are a family of tetracyclic diterpenoid carboxylic acids produced in plants and 180 fungi by transformations of ent-kaurene that act to stimulate plant growth (81) (see Fig. 1.9). The genes required for synthesis of various gibberellins from e«/-kaurene have been sequenced and characterized. The microbial metabolism of e«?-kaurene is discussed below. Both monoterpenes and diterpenes play a role in plant defence against attacks from herbivores and pathogens (50). Fungal inoculation or wounding resulted in the production of 185 terpenoids in Maritime pine (Pinus pinaster) (10). Mechanical wounding or insect attack of A. grandis stems leads to an induction of enzymes required for synthesis of resin components (28). In addition to the induced terpene synthesis some plant species performed terpenes, which are stored in specialized structures such as resin ducts in Pinus contorta (Lodgepole pine) or resin blisters in A. grandis. 190 Terpene production is induced by methyl jasmonate in Norway spruce stems (56). Both monoterpene and diterpene synthases are induced following wounding with a peak in production at 10 days post wounding. The simultaneous production of both monoterpenes and diterpenes is consistent with their combined role in plant defence. The volatile monoterpenes solubilize and facilitate mobilization of the lipophilic diterpenes for transfer to the point of attack. Here, 195 following volatilization of the monoterpenes, the diterpenes can then crystallize to form a protective barrier that seals the plant off from further damage. Meanwhile the volatile monoterpenes are potentially toxic to invading plant pathogens. 11 1.3.2 Acidic diterpenoids as pollutants in pulp and paper production Usually when one thinks of natural products from trees, the first thought that comes to 200 mind is not pollution. However, during pulp and paper making, resin acids including abietane diterpenes are extracted from wood and discharged in wastewater at concentrations far above those occurring naturally (2, 54). Research on the microbial removal of these compounds from wastewater treatment systems has been the driving force behind investigations of the degradation of resin acids. Removal of resin acids from the wastewater is necessary before it is discharged 205 into receiving water because of the acute toxicity of resin acids to fish (2). Additionally, resin acids are a major component of pitch. Pitch is formed during pulp and paper making by lipophilic wood extracts which coalesce to form droplets. Pitch deposits lower pulp quality and interfere with the operation of papermaking equipment. Accumulation of pitch in the papermaking water limits re-use of that water, which otherwise would reduce the environmental 210 impact of papermaking (32). The resin acids from pulp and paper production are usually removed in biotreatment systems. Several resin acid degrading microorganisms isolated from pulp and paper mill biological treatment systems have been characterized (6, 54, 60, 62-65). 1.3.3 Medicinal significance Greater than half of chemotherapeutic drugs for cancer treatment and infectious disease 215 are of natural origin (67). Terpenoids are the largest class of natural products and comprise a significant portion of treatments in use today. 1.3.3.1 Monoterpenoids - limonene and perillyl alcohol Limonene is one of the most abundant monoterpenes found in nature (Fig. 1.7). It is the major component of peel oil from oranges, citrus, and lemons, and is the oil of caraway. 220 Monoterpenoids are C-10 isoprene compounds that differ from other classes of terpenoids in 12 Figure 1.7 Medically significant terpenoids. D-limonene and perillyl alcohol are 225 monoterpenoids currently being studied for their potential use as chemotherapeutic agents. Artemisinin is a sesquiterpenoid with antimalarial activity. Taxol is a taxane diterpenoid with potent chemotherapeutic activity (Ph, phenyl; Ac, acetate; Bz, benzoate). Betulinic acid is a triterpenoid currently under investigation for use in prevention and treatment of HIV infection and cancer. 230 their volatility and aromaticity. Much of the work on monoterpenoids in the past has been focused on the development of flavours and fragrances related to the food and cosmetic industry. Recently, additional interest has centred on these compounds as chemotherapy agents against tumors (88). Limonene and its hydroxylated derivative perillyl alcohol, respectively, are 235 undergoing Phase 1 and Phase II clinical testing of their chemotherapeutic activity (88). The proposed mechanisms of action against tumors are (a) the interference of protein prenylation of key regulatory proteins including RAS (29) or (b) induction of apoptosis (3). 13 1.3.3.2 Sesquiterpenoids - artemisinin Multiple drug resistance of Plasmodium falciparum has led to the necessity for 240 development of alternative forms of treatment against malaria (87, 88). Artemisinin is a potent antimalarial extract of sweet wormwood (Artemisia annua), which has been used in traditional Chinese medicine for centuries under the name Qinghaosu (Fig. 1.7). It is a sesquiterpene lactone with an endoperoxide trioxane ring structure, which is required for medicinal activity. It is postulated to act through the proliferation of free radicals (61), or more likely through 245 inhibition of the parasite's calcium ATPase (18). 1.3.3.3 Diterpenoids - Taxol and others AbA and its derivatives have recently been evaluated for their ability to function as inhibitors of fungi (20), tumors, mutagenesis, viruses, nitric oxide production (30, 44), inflammation, (22) and lipoxygenase activity (82). A recent increase in the interest in natural 250 products isolated from marine organisms has led to the discovery of a wide range of novel diterpenoids. These novel products could potentially lead to the development of new medicinal applications (4, 33). One of the most widely recognized diterpenoids in pharmaceutical applications today is Taxol (Fig. 1.7). This taxane diterpenoid extracted from the bark of Pacific Yew (Taxus 255 brevifolia) has proven to be a potent chemotherapeutic agent. Taxol acts by stabilizing microtubules, which in turn hinders the rearrangement of microtubule networks required for mitosis and cell proliferation (88). Extraction of Taxol from its natural source is not practical in quantities required for widespread clinical use. At present, research is underway to better understand the synthesis of Taxol in Yew to enhance cell culture and semisynthetic means of 260 production (41, 42). 14 1.3.3.4 Triterpenoids - betulinic acid Betulinic acid is a pentacyclic lupane-type triterpenoid found in several plant species, including the birch tree (Betula spp. Betulaceae) (1.1). Recently, much of the focus on betulinic acid and its derivatives has centered on prevention and treatment of HIV infection and cancer. In 265 HIV infection treatment a betulinic acid derivative was found to disrupt the cellular entry of HJV-1, while in cancer treatment it was found to induce apoptosis (11). 1.3.3.5 Obstacles in the production of terpene pharmaceuticals Significant obstacles exist in our ability to extract large quantities of potentially beneficial terpenoids from their natural sources. Complications include multiple biosynthetic 270 pathways in plants resulting in a mixture of products, and low product concentrations and product yields (88). For example, extraction of 1 kg of Taxol required 6.7 t of Taxus bark, which is roughly equivalent to 2000-3000 trees (as reviewed in (40)). Cell culture, aquaculture, semi, and total synthesis are being investigated as means to circumvent some of the issues related to production of terpenoids. Metabolic engineering is also a viable option. 275 1.3.3.6 Metabolic engineering Recently, Martin et al. (59) reported the synthesis of amorphadiene in E. coli. Amorphadiene is a cyclic sesquiterpene that is a precursor in the synthesis of artemisinin. As mentioned above, artemisinins are potent antimalarial drugs produced naturally in sweet wormwood. Extraction of this natural product is costly and therefore prohibitive to treatment of 280 large human populations. Therefore a cost-effective means of production is currently under investigation. A significant advancement toward this goal was achieved by the expression of the mevalonate pathway for IPP production in E. coli (59). By using the mevalonate pathway for IPP production as opposed to E. colVs DXP pathway, it was possible to circumvent regulatory controls of IPP production in E. coli and produce large quantities of the terpenoid precursor. 15 285 Another example of metabolic engineering for terpene production is perillyl alcohol. Van Beilen et al. (83) screened 1800 bacterial strains for the ability to transform limonene to perillyl alcohol. Mycobacterium sp. HXB-1500 expressing cytochrome P450 (CYP 153 family) had the most promising transformation activity. The P450 system from HXB-1500, including a ferredoxin and ferredoxin reductase, was expressed in Pseudomonas putida (83). This strain was 290 able to convert 3 umol of limonene to perillyl alcohol per minute per gram of cells (dry weight). 1.4 Cytochromes P450 in terpenoid metabolism 1.4.1 P450 heme-thiolate proteins Cytochromes P450 are heme-thiolate proteins present in all domains of life, which 295 catalyze a wide variety of oxygenation reactions. During terpene synthesis, many of the post cyclization reactions required for production of active terpenoid compounds are catalyzed by P450s, as seen above in the synthesis of diterpene resin acids (71). P450s produce regiospecifically and stereospecifically modified terpene products (36). Understanding the role of these enzymes is of interest in the metabolic engineering of pharmaceuticals and the 300 metabolism of xenobiotics. As cytochromes P450 form a significant portion of the findings of this dissertation, the following section will briefly consider this extremely interesting enzyme group. 1.4.2 Classification of P450s P450-dependent oxygenation systems can be classified into four groups depending on the 305 number of proteins involved in electron transfer from reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) (26). Type I systems have 3 proteins including a reductase - containing either flavin adenine dinucleotide' (FAD) or flavin adenine mononucleotide (FMN), a ferredoxin, and the catalytic P450 subunit. This is the most common scheme found in prokaryotes and mitochondria. Type II systems 16 310 involve 2 proteins; an NAD(P)H reductase and a catalytic subunit. The reductase component contains both an F A D and F M N . Electrons are transferred from F A D to F M N and then to the catalytic P450 component. Type II enzymes are found in the endoplasmic reticulum of higher animals and most other eukaryotic cells. Type III systems are composed of a single protein, which contains both a reductase and a catalytic domain. This protein contains an FAD, F M N and 315 heme group associated with a P450 and requires N A D P H as an electron donor. Type III enzymes were first reported in Bacillus megaterium (P450 BM-3) (66) and then later found in Fusarium oxysporum (P450foxy) (45). Recently Roberts et al. (72) identified a new class of cytochrome P450 from the genus Rhodococcus. In these type IV systems, electron transport is mediated through a dioxygenase-320 reductase-like activity. Electrons are transferred from N A D H to the P450 active site via an F M N centre and a 2Fe2S ferredoxin-like component. As in Type III P450s, the electron transport proteins are linked to the Type IV P450 in one polypeptide. Heterologous expression of Type IV P450, P450Rhf, in E. coli produced the expected 85-kilodalton protein. Although the natural function of this P450 is unknown, it was able to mediate the O-dealkylation of 7-325 ethoxycoumarin. Additional Type IV cytochromes P450 have.been identified in the recently sequenced genome of Rhodococcus sp. RHA1 (86), however the proteins and not fused suggesting a type V classification. A Basic Local Alignment Search Tool (BLAST) search of the National Center for Biotechnology Information (NCBI) non-redundant database using the P450RhF sequence identified several putative Type IV P450s from the genus Burkholderia. 330 1.4.3 General mechanism Much of our knowledge of the structure and mechanism of P450s has been determined by work on P450 c a m(CYP101). This enzyme, isolated from P. putida, stereospecifically hydroxylates the monoterpene camphor to 5-exo-hydroxy camphor. The general mechanism of 17 oxygen activation is described as follows (15, 31,51) (Fig. 1.8). In the resting state, the heme 335 ferric iron is hexacoordinated by the conserved cysteine residue of the heme-binding pocket, the Figure 1.8 General mechanism of P450 catalytic cycle. R H - substrate, ROH - hydroxylated product, LS-low spin iron, HS- high spin iron. Adapted from (51) and (15). 18 4 nitrogens of the heme pyrrole rings, and a distal water molecule. Binding of the substrate, in 340 most cases, leads to the displacement of the iron-bound water which in turn changes the spin state of the heme iron. This change of spin increases the reduction potential of the heme iron, facilitating the first electron transfer from NAD(P)H leading to ferrous iron. The production of ferrous iron facilitates dioxygen binding generating an Fen-C>2 complex followed by a second electron transfer generating Fe n-0 2". Protonation of the distal oxygen with heterolytic cleavage 345 of 0 -0 bond leads to water formation and Fe I V=0. This high valent complex drives most of the P450 reactions (31). In a typical hydroxylation reaction, the high valent complex abstracts a hydrogen atom from an unactivated C atom of the substrate followed by rebound and transfer of oxygen to bound substrate and dissociation of product regenerating the ferric heme iron. 1.4.4 P450s in diterpenoid metabolism 350 As mentioned above, P450s are required for post-synthase modification of terpenes to functional compounds. This is well documented in the production of gibberellins from the diterpenoid e«/-kaurene (81) (Fig. 1.9). A cluster of genes in Gibberlla fujikuroi encodes 4 cytochromes P450 designated P450-1 through P450-4. P450-4 is required for carboxylic acid formation. Three consecutive oxidations by P450-4 of the diterpenoid enf-kaurene at C19 355 proceed from an alcohol, to an aldehyde, and finally to e«/-kaurenoic acid (Fig. 1.7). This is similar to the transformation of abietadiene to abietic acid in Loblolly pine discussed above. P450-1 is required for several oxidation steps in the production of gibberellins in G. fujikuroi, including a 7-alpha-OH (Fig. 1.9). Rojas et al. (75) characterized the C7 a hydroxylation reaction of e«£-kaurenoic acid by P450-1 through gene knockout and expression 360 of P450-1 in a fungal strain lacking several gibberellin genes. P450-1 mutant strains failed to transform e«£-kaurenoic acid to e«/-7a-hydroxykaurenoic acid. The e«/-7-a-hydroxykaurenoic 19 P450-4 P450-4 P450-4 e n f - k a u r e n e e r r t - k a u r e n o i c a c i d e n f - 7 a - h y d r o x y -k a u r e n o i c a c i d G A 1 2 - a l d e h y d e I P450-1 'COOH P450-1 G A 1 4 - a l d e h y d e 'COoH C ° 2 H G A 14 P450-2 G A 7 C 0 2 H Figure 1.9 Main route of gibberellin biosynthesis of gibberellins in Gibberlla fujikuroi. Des - desaturase, G A - gibberellin, Adapted from (81) 20 acid product is a precursor of B ring contraction, an essential step in gibberellin synthesis. The enzyme was also found to catalyze 3C P hydroxylation. In fact, this single P450 was found to be involved in oxidations at four different carbon atoms on the tetracyclic compounds. (C3, C6, C7, 365 C18). This allows for the production of several types of gibberellins, which in turn increases the range of plants these compounds can affect during fungal infection. Given that the enzyme contains a single heme and substrate binding requires a specific orientation of substrate proximal to the heme iron, subsequent hydroxylations must require flexibility in substrate binding to accommodate variable substrates. In their discussion, the. authors give some possibilities 370 including shifts in substrate binding brought about by subsequent hydroxylation reactions (75). 1.5 Biotransformation of terpenoids Cytochromes P450 play a key role in the metabolism of xenobiotics in mammals. Much of the research on microbial metabolism of terpenes focuses on creating mammalian models of metabolism (76). This research aims to predict metabolites that are produced in mammalian 375 systems and then produce large enough quantities of these metabolites for further study. Fungal biotransformation experiments are most often used because of the similarities between cytochrome P450 systems in fungi and mammals. Typically these fungal transformations are limited and the organisms do not mineralize the substrates. Such partial transformation suggests that the reactions may be fortuitous or cometabolic. In these types of investigations little 380 attention is given to the genetics or enzymology behind the transformations occurring in the microbes. These reports therefore are not very informative regarding the mineralization of growth substrates by bacterial systems. However, by considering the results of this type of work it is possible to gain insight into reactions that may initiate metabolism and identify susceptible carbon positions on the substrate. Table 1.1 contains a selection of the microbial metabolism of 385 cyclic diterpene compounds illustrating the general range of activities reported. For a review of these transformations see (34). 21 1.5.1 Isosteviol Stevioside, a natural non-caloric sweetener extracted from the leaves of Stevia rebaudiana, is reported to be 250-300x sweeter than sucrose (35). It is presently used in many 390 parts of Asia and South America as a substitute sweetener. Isosteviol (Table 1.1) is a beyerane diterpene produced from the acid hydrolysis of the 3 glucose side chains of stevioside. Isosteviol and its derivatives have several reported bioactive properties including antibacterial activity (53), inhibitory effects on Epstein Barr Virus activation (1) and D N A polymerase and D N A topoisomerase inhibitors. In an effort to increase the bioactivity of isosteviol compounds, de 395 Oliveira et al. (14) examined transformation of isosteviol by Aspergillus niger, Penicillium chrysogenum and Rhizopus arrhizus. Transformation of this beyerane diterpenoid resulted in the production of 3 identified products, two monohydroxylated products (at C7 and CI7) and one dihydroxylated product (CI and C7). 1.5.2 Kaurene/pimerane diterpenoids 400 9-epi-ent Pimaradiene diterpenes resemble AbA's B ring structure, with a C7,8 double bond (24) (Table 1.1). Biotransformation of 2a, 19-dihydroxy-9-ep/-en?-pimara-7,15-dieneby G. fujikuroi resulted in the accumulation of more than 7 compounds. Three compounds contained a 7,8 epoxide, 2 were C7-keto and 2 were C7-hydroxylated. The main reaction with this substrate was the alpha epoxidation of the 7,8 double bond. It was suggested that the epoxide 405 was subsequently converted to the 7-keto compound. Similar results were found for biotransformation of 18-hydroxy-9-epz'-e«^-pimara -7,15-diene, and 18-hydroxy-9,13-epi-e«^-pimara-7-15-diene by G. fujikuroi (23, 25). Fraga et al. suggested that the 7,8 epoxide rearranged to the 7-ketone or the allylic alcohol 7-hydroxy by opening of the oxirane ring. 22 410 Name & Structure Table 1 . 1 Biotransformation of selected diterpenoids Microbe(s) Transformation(s) 18-hydroxy-9,13-ep/-enf-pimara-7,15-diene Gibberella fujikuroi -Epoxidation of 7,8 double bond ( main product) -Rearrangement of 7,8 epoxy to 7-hydroxy or 7-keto derivative -Other hydroxylations Reference (23) Gibberella fujikuroi - -Epoxidation of 7,8 double bond (main product) -Rearrangement of 7,8 epoxy to 7-hydroxy or 7-keto derivative -Other hydroxylations (24) 2a,19-dihydroxy-9-ep/'-enf-pimara-7,15-diene Gibberella fujikuroi -Epoxidation of 7,8 double bond (main product) -Rearrangement of 7,8 epoxy to 7-hydroxy or 7-keto derivative -Other hydroxylations (25) CHZDH"' 7 " 6 18-hydroxy-9-ep/'-enf-pimara-7,15-diene Name & Structure Microbe(s) Transformation(s) V /codH 19 isosteviol Aspergillus niger Penicillium chrysogenum Rhizopus arrhizus C12, C17, C1, C7 hydroxylation Reference (14) Gibberella fujikuroi C7 hydroxylation £OOH 15a-hydroxy-enf-kaurenoic acid (5) Rhizopus arrhizus C7, C14, C16, C17, C18, C19 hydroxylation 19 18 . s t e m o d i n (39) to 4^  Name and Structure Microbe(s) Transformations Reference 15 12 1 16 2 01 1 p > J 3 / C 0 0 H 10 8 5 7 s-" \ 6 19 ^ * 1 8 grindelic acid Aspergillus niger Penicillium brevi-compactum Major product was 3p-hydroxy derivative Minor products include 3-ketone, 18-alcohol, and 7cc,8a-epoxide (37),(69) 12 OH 1 2 0 J 1 7 > s 1 6 32 r j ^ l o ^ 1 5 19* 18 sclareol Septomyxa affinis Cunningham-ella sp. 38-alcohol, 3-ketone, 2a-alcohol, 18-alcohol (34, 46) 1.5.3 Abietane diterpenoids Several strains capable of growth on abietane or pimerane diterpenoids have been described as mentioned above. Few studies, however, have focused on the mechanism of 415 mineralization of these compounds. What is known regarding the catabolism of abietane diterpenoids was recently reviewed (54, 60). Early reports of the microbial degradation of abietane diterpenoids (as reviewed in (43)) focused on the identification of metabolites, which accumulated during growth in typical salt media, in cation deficient media, or in the presence of the metabolic inhibitor, bipyridyl. Flavobacterium resinovorum, isolated from the soil of a Pinus 420 maritima forest, was found to grow on the non-volatile components of pine oleoresin as a sole source of carbon and electrons. During growth on DhA, Biellmann and Wennig (9) identified a single metabolite extracted from the growth medium, the 3-oxo, decarboxylated derivative of DhA (Fig. 1.10, III). This compound was not expected as degradation was thought to begin at the aromatic ring. Growth of F. resinovorum on DhA using metabolic inhibitors (alpha, alpha'-425 dipyridyl) or in cation deficient media resulted in slower growth allowing for identification of additional metabolites including, VI the diphenol, diketone, decarboxylated derivative of DhA (7). From the identification of additional metabolites they proposed a degradation pathway for DhA by F. resinovorum (Fig. 1.10 B). The same group used similar methods to investigate the degradation of DhA by 430 Pseudomonas sp. and Alcaligenes sp. (8). Both strains were isolated by aerobic enrichment on DhA. Unlike metabolites found with F. resinovorum, only 7-oxo-DhA was isolated and not the 3-oxo derivatives. As before, based on isolated metabolites they proposed a degradation pathway, which differs from the F. resinovorum in that no C3 oxidation or decarboxylation occurs in the initial steps (Fig. 1.10 C). 26 Figure 1.10 Initial steps in the proposed degradation pathway of DhA by A. Arthrobacter sp., B. F. resinovorum and C. Alcaligenes sp. and Pseudomonas sp. I, dehydroabietic acid; II, 3-oxo-dehydroabietic acid III, 2-oxo-dehydroabietin; IV, 7-oxo-dehydroabietic acid; V , 7-440 hydroxy-dehydroabietic acid; VI, 5,6-dihydroxy-2,9-dioxo-dehydroabietin; VII, 11,12-dihydroxy-7-oxo-dehydroabietic acid. Additionally, Levinson and Carter (49) reported an Arthrobacter sp. isolated from Lodgepole pine which when incubated with methyl dehydroabietate produced 3-oxo-DhA 445 without identification of any 7-oxo-DhA. They also tentatively reported an A ring degradation product of DhA with an intact B and C ring. This collection of early work indicated 3 possible degradation pathways for DhA degradation, one via 3-oxo-DhA, a second via 7-oxo-DhA and a third combining both 3 and 7-oxo-DhA derivatives. As mentioned above, biodegradation of abietane diterpenoids in pulp and paper mill 450 effluent has been a driving force behind research into the microbial metabolism of terpenes. Recently, molecular investigations have focused on a bacterium isolated from bleach kraft pulp 27 mill effluent, Pseudomonas abietaniphila BKME-9 , that mineralizes and grows on the abietane diterpenoids, AbA, DhA, PaA, and 7-oxo-DhA as its sole organic substrates (6). The dit gene cluster of B K M E - 9 encodes enzymes required for the catabolism of these compounds (57, 58). 455 Several genes of the dit cluster were sequenced and characterized, and a convergent pathway for abietane diterpenoid metabolism was proposed (Fig. 1.11). Evidence for a convergent pathway for AbA, DhA and PaA catabolism came from studies of a ditAl knockout mutant (57). DitA ring hydroxylating dioxygenase catalyzes the formation of a catecholic intermediate in the proposed pathway (58). 7-Oxo-DhA accumulated in cell suspensions of the ditAl mutant strain incubated 460 with AbA or DhA, while DhA and 7-oxo-DhA accumulated in cell suspensions incubated with PaA. The aromatisation of the C ring of both AbA and PaA suggested a convergent pathway with DhA serving as an intermediate. The initial steps in the biodegradation pathway have not been elucidated, but some evidence suggests that a P450 monooxygenase is involved. A putative P450, encoded by tdtD 465 that may function in abietane diterpenoid degradation, was recently identified in Pseudomonas diterpeniphila A19-6a, another resin acid-degrading bacterium closely related to B K M E - 9 (64, 65). Morgan and Wyndham (64) reported that a tdtD knockout mutant of A19-6a was retarded in its removal of DhA or A b A from its growth medium when compared to that of the wild type. The mutant retained the ability to grow on DhA and AbA as sole organic substrates; however, 470 any effects of the mutation on growth rates were not reported. These results suggest involvement of the tdtD gene in diterpenoid metabolism but give no conclusive evidence for a functional P450 gene product or the role of such an enzyme in resin acid metabolism. Morgan and Wyndham also provided evidence for a homologue of the tdtD gene in B K M E - 9 but were unable to conclude whether this gene was linked to the previously described dit cluster. 28 F i g u r e 1.11 P roposed convergen t p a t h w a y f o r ab ie tane d i t e r p e n o i d d e g r a d a t i o n p a t h w a y b y Pseudomonas abietaniphila B K M E - 9 . Adapted from (57) Chemical Designations AbA, abietic acid; PaA, palustric acid; DhA, dehydroabietic acid, IV, 7-hydroxy-dehydroabietic acid; V , 7-oxo palustric acid; 7-oxo-DhA, 7-oxo-dehydroabietic acid; VII, 7-oxo-ll, 12-dihydroxy-480 8,13-abietadien acid; VII, 7-oxo-11,12-dihydroxydehydroabietic acid. 29 Little is known about the anaerobic degradation of abietane diterpenoids (60). A pathway for the anaerobic metabolism of DhA has been proposed using results obtained from experiments with deuterium labelled DhA. Deuterium labelled DhA was incubated in sediment collected downstream of a pulp and paper mill and compared to DhA transformation in autoclaved 485 sediment under the same conditions (79, 80). The main intermediates in the pathway are decarboxylated and aromatized products with intact tricyclic abietane structures. After 264 days of incubation, d-tetrahydroretene (10-d-1 -methyl-7- methylethyl-1,2,3,4-tetrahydrophenanthrene) was found to be the major product. A small amount of retene (7-isopropyl-l-methylphenantrene) was also observed. Retene has also been identified in sediment 490 particles in lakes receiving pulp and paper mill effluent (48). 1.6 Summary Terpenoids are ubiquitous and play a variety of critical roles in biology. Not only do they constitute an important part of the carbon cycle, they also raise concerns in pollution control, and 495 have potential benefits as pharmaceutical agents. This introduction has presented several elements of the abietane diterpenoid carbon cycle. Synthesis of a wide variety of compounds from a simple 5-carbon "building block", utilizing relatively few enzymes, leads to common structural intermediates while additional enzymes are used for modification to produce functional compounds. 500 While much attention has been given to the synthesis of these compounds, little is known about their catabolism. A survey of biotransformation of terpenes above and in Table 1.1 shows the prevalence of the oxidation of these hydrophobic compounds to more polar products. Most aerobic biotransformations of terpenes are initiated by hydroxylations or epoxidations. Carbons 7 and 3 of diterpenes synthesized via copalyl diphosphate (Fig. 1.5) are most often hydroxylated. 505 Diterpenes containing a C7-C8 single bond are susceptible to alpha or beta hydroxylation at C7, 30 which may be followed by further oxidation to a ketone. In the case of a C7-C8 double bond there is support for C7-C8 epoxidation also leading to a C7 hydroxyl or ketone. The transformations presented in Table 1.1 and studies involving strains capable of growth on diterpenoids, emphasize the importance of C7 and C3 oxidations in their metabolism. To date, 510 however, no genes or enzymes catalyzing these transformations leading to substrate degradation have been reported. We hypothesize that a cytochrome P450 is required for this transformation and represents the first required step in aerobic abietane diterpenoid catabolism following substrate uptake. 1.7 Conclusions 515 A review of both the synthesis and the limited data that is available concerning the degradation of tricyclic diterpenoids has shed some light on the unknowns regarding their metabolism. It is interesting to note that the three proposed pathways for abietane diterpenoid degradation are initiated by oxidation of the A ring at C3 or the B ring at C7 (Fig 1.10). This same type of initial reaction is often observed in biotransformations of other terpenoid 520 compounds (Table 1.1). This raises the possibility that there are common catabolic pathways for degradation. Alternatively, this suggests that these carbons are simply most susceptible to hydroxylation. Looking again at the synthesis of these compounds it is worth noting that the formation of the A and B ring in all known tricyclic diterpenoids is similar, while the variability between the classes of tricyclic diterpenoids is found mainly in the formation of the C ring (Fig. 525 1.5, Table 1.1). If the C ring is cleaved and removed, then resulting intermediates would have similar structures resembling a decalin derivative. Little is known about the microbial catabolism of this fused two-ring structure, however, this compound could be a common intermediate in tricyclic diterpenoid degradation. At this point there is little evidence to support this hypothesis. These possibilities raise further issues regarding the enzymes involved in catabolism. 530 Degradation may rely on relatively few enzymes, perhaps with broad specificities or, possibly 31 there is a proliferation of divergent degradative enzymes specific for the broad range of terpenoids. Similar to a divergent synthetic pathway utilizing relatively few enzymes to produce a variety of compounds (Fig. 1.5), the same may be true for their catabolism, that is relatively few enzymes are used to transform the broad range of diterpenoids into a common intermediate 535 in a convergent degradation pathway (Fig. 1.1). This thesis will focus on the microbial metabolism of abietane diterpenoids and attempt to shed more light on some of the above issues. 1.8 Thesis Objectives The overall objective of this doctoral research is to characterize the initial steps in the aerobic degradation pathway of abietane diterpenoid catabolism by Pseudomonas abietaniphda 540 B K M E - 9 and Burkholderia xenovorans LB400. This overall objective was pursued through 3 specific aims that focused on genes of the B K M E - 9 and LB400 dit clusters, including 3 cytochrome P450 genes: (l)to identify, clone and sequence a P450 contiguous with the dit gene cluster of B K M E - 9 and determine its involvement in the initial steps of abietane diterpenoid degradation; (2) to characterize the growth of LB400 on abietane diterpenoids, particularly 545 looking at the induction of genes of the LB400 dit cluster using microarray transcriptomic analysis and using a knockout of ditAl encoding a ring hydroxylating dioxygenase, an enzyme critical in the degradation pathway; and, (3) to investigate the roles of two cytochromes P450 in the degradation of abietane diterpenoids by LB400. The three cytochromes P450 that were studied were DitQ-BKME-9, DitQ-LB400 (CYP226A1), and DitU-LB400 (CYP226A2). 550 Expression of the enzymes during growth on abietane diterpenoids was assessed using a fused reporter construct in the case of DitQ-BKME-9 and both microarray transcriptomic analysis and 2D gel proteomic analysis in the cases of CYP226A1 and CYP226A2. The functions of these P450s in the catabolism of abietane diterpenoids were determined by gene knockout phenotypes, substrate-binding assays, and in the case of CYP226A1, an in vitro activity assay. The results are 555 discussed and the physiological relevance of the observed activities is addressed. 32-1.9 References 1. Akihisa, T., Y. Hamasaki, H. Tokuda, M. Ukiya, Y. Kimura, and H. Nishino. 2004. Microbial transformation of isosteviol and inhibitory effects on Epstein-Barr virus activation of the transformation products. J Nat Prod 67:407-10. 2. Ali, M., and T. R. Sreekrishnan. 2001. Aquatic toxicity from pulp and paper mill effluents: a review. Advances in Environmental Research 5:175-196. 3. Ariazi, E. A., Y. Sato mi, M. J. Ellis, J. D. Haag, W. Shi, C. A. Sattler, and M. N. Gould. 1999, Activation of the transforming growth factor p signaling pathway and induction of cytostasis and apoptosis in mammary carcinomas treated with the anticancer agent perillyl alcohol. Cancer Res 59:1917-1928. 4. Baker, D. D., and K. A. Alvi. 2004. Small-molecule natural products: new structures, new activities. Curr Opin Biotechnol 15:576-83. 5. Barrero, A. F., J. E. Oltra, E. Cerda-Olmedo, J. Avalos, and J. Justicia. 2001. Microbial transformation of e«?-kaurenoic acid and its 15-hydroxy derivatives by the SGI 38 mutant of Gibberella fujikuroi. J. Nat. Prod. 64:222-225. 6. Bicho, P. A., V. Martin, and J. N. Saddler. 1995. Growth, induction, and substrate specificity of dehydroabietic acid-degrading bacteria isolated from a kraft mill effluent enrichment. Appl Environ Microbiol 61:3245-50. 7. Biellmann, J. F., G. Branlant, M. Gero-Robert, and M. Poiret. 1973. Degradation bacterienne de l'acide dehydroabietique par Flavobacterium resinovorum. Tetrahedron 29:1227-1236. 8. Biellmann, J. F., G. Branlant, M. Gero-Robert, and M. Poiret. 1973. Degradation bacterienne de l'acide dehydroabietique par un Pseudomonas et une Alcaligenes. Tetrahedron 29:1237-1241. 9. Biellmann, J. F., and R. Wennig. 1970. Microbial degradation of dehydroabietic acid. Chemical Communication 6:346. 10. Cheniclet, C. 1987. Effects of wounding and fungus inoculation on terpene producing systems of maritime pine. Journal of Experimental Botany 38:1557-1572. 11. Cichewicz, R. H., and S. A. Kouzi. 2004. Chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection. Med Res Rev 24:90-114. . 12. Coppen, J. J. W., G. A. Hone, Food and Agriculture Organization of the United Nations., and Natural Resources Institute (Great Britain). 1995. Gum naval stores : turpentine and rosin from pine resin. Food and Agriculture Organization of the United Nations, Rome. 33 13. Davis, E. M., and R. Croteau. 2000. Cyclization enzymes in the biosynthesis of monoterpenes, sesquiterpenes, and diterpenes. Topics in Current Chemistry 209:53-95. 14. de Oliveira, B. H., M. C. dos Santos, and P. C. Leal. 1999. Biotransformation of the diperpenoid, isosteviol, by Aspergillus niger, Penicillium chrysogenum and Rhizopus arrhizus. Phytochemistry 51:737-41. 15. Denisov, I. G., T. M. Makris, S. G. Sligar, and I. Schlichting. 2005. Structure and chemistry of cytochrome P450. Chem Rev 105:2253-77. 16. Dewick, P. M. 2002. The biosynthesis of C5-C25 terpenoid compounds. Nat Prod Rep 19:181-222. 17. Dubey, V. S., R. Bhalla, and R. Luthra. 2003. An overview of the non-mevalonate pathway for terpenoid biosynthesis in plants. J Biosci 28:637-46. 18. Eckstein-Ludwig, U . , R. J . Webb, I. D. A. van Goethem, J . M. East, A. G. Lee, M. Kimura, P. M. O'Neill, P. G. Bray, S. A. Ward, and S. Krishna. 2003. Artemisinins target the SERCA of Plasmodium falciparum. Nature 424:957-961. 19. Eisenreich, W., A. Bacher, D. Arigoni, and F. Rohdich. 2004. Biosynthesis of isoprenoids via the non-mevalonate pathway. Cell Mol Life Sci 61:1401-26. 20. Feio, S. S., S. Franca, A. M. Silva, B. Gigante, J. C. Roseiro, and M. J. Marcelo Curto. 2002. Antimicrobial activity of methyl cis-7-oxo deisopropyldehydroabietate on Botrytis cinerea and Lophodermium seditiosum: ultrastructural observations by transmission electron microscopy. J Appl Microbiol 93:765-71. 21. Fengel, D., and G. Wegener. 1984. Wood : chemistry, ultrastructure, reactions. W. de Gruyter, Berlin ; New York. 22. Fernandez, M. A., M. P. Tornos, M. D. Garcia, B. de las Heras, A. M. Villar, and M. T. Saenz. 2001. Anti-inflammatory activity of abietic acid, a diterpene isolated from Pimenta racemosa var. grissea. J Pharm Pharmacol 53:867-72. 23. Fraga, B. M., P. Gonzalez, M. G. Hernandez, M. C. Chamy, and J. A. Garbarino. 2003. Microbial transformation of 18-hydroxy-9,13-epi-ent-pimara-7,15-diene by Gibberella fujikuroi. J Nat Prod 66:392-7. 24. Fraga, B. M., P. Gonzalez, M. G. Hernandez, M. C. Chamy, and J. A. Garbarino. 1998. The microbiological transformation of a 9-epi-ent-pimaradiene diterpene by Gibberella fujikuroi. Phytochemistry 47:211-215. 25. Fraga, B. M., M. G. Hernandez, P. Gonzalez, M. C. Chamy, and J. A. Garbarino. 2000. The biotransformation of 18-hydroxy-9-epi-ent-pimara-7,15-diene by Gibberella fujikuroi. Phytochemistry 53:395-399. 26. Fulco, A. J. 1991. P450BM-3 and other inducible bacterial P450 cytochromes: biochemistry and regulation. Annu Rev Pharmacol Toxicol 31:177-203. 34 27. Funk, C , and R. Croteau. 1994. Diterpenoid resin acid biosynthesis in conifers: characterization of two cytochrome P450-dependent monooxygenases and an aldehyde dehydrogenase involved in abietic acid biosynthesis. Arch Biochem Biophys 308:258-66. 28. Funk, C , E. Lewinsohn, B. S. Vogel, C. L. Steele, and R. Croteau. 1994. Regulation of oleoresinosis in Grand Fir (Abies grandis) (coordinate induction of monoterpene and diterpene cyclases and two cytochrome P450-dependent diterpenoid hydroxylases by Stem Wounding). Plant Physiol 106:999-1005. 29. Gelb, M. H., F. Tamanoi, K. Yokoyama, F. Ghomashchi, K. Esson, and M. N. Gould. 1995. The inhibition of protein prenyltransferases by oxygenated metabolites of limonene and perillyl alcohol. Cancer Letters 91:169-175. 30. Gigante, B., C. Santos, A. M. Silva, M. J. Curto, M. S. Nascimento, E. Pinto, M. Pedro, F. Cerqueira, M. M. Pinto, M. P. Duarte, A. Laires, J. Rueff, J. Goncalves, M. I. Pegado, and M. L. Valdeira. 2003. Catechols from abietic acid, synthesis and evaluation as bioactive compounds. Bioorg Med Chem 11:1631-8. 31. Guengerich, F. P. 2001. Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem Res Toxicol 14:611-50. 32. Gutierrez, A., J. C. del Rio, M. J. Martinez, and A. T. Martinez. 2001. The biotechnological control of pitch in paper pulp manufacturing. Trends Biotechnol 19:340-8. 33. Hanson, J. R. 2004. Diterpenoids. Nat Prod Rep 21:785-93. 34. Hanson, J. R. 1992. The microbiological transformation of diterpenoids. Nat Prod Rep 9:139-51. 35. Hanson, J. R., and B. H. De Oliveira. 1993. Stevioside and related sweet diterpenoid glycosides. Nat Prod Rep 10:301-9. 36. Hefner, J., S. M. Rubenstein, R. E. Ketchum, D. M. Gibson, R. M. Williams, and R. Croteau. 1996. Cytochrome P450-catalyzed hydroxylation of taxa-4(5),l l(12)-diene to taxa-4(20),ll(12)-dien-5alpha-ol: the first oxygenation step in taxol biosynthesis. Chem Biol 3:479-89. 37. Hoffmann, J. J., Punnapayak, H. Jolad, S.D., Bate, R.B., Camou, F.A. 1988. Bioconversion of Grindelic Acid into 3-alphahydroxygrindelic acid. J Nat Prod 51:125-128. 38. Huber, D. P. W., S Ralph, and J.R. Bohlmann. 2004. Genomic hardwiring and phenotypic plasticity of terpenoid-based defenses in confiers. Journal of Chemical Ecology 30:2399-2418. 39. Hufford, C. D., F. A. Badria, M. Abou-Karam, W. T. Shier, and R. D. Rogers. 1991. Preparation, characterization, and antiviral activity of microbial metabolites of stemodin. J Nat Prod 54:1543-52. 35 40. Jennewein, S., and R. Croteau. 2001. Taxol: biosynthesis, molecular genetics, and biotechnological applications. Appl Microbiol Biotechnol 57:13-9. 41. Jennewein, S., R. M. Long, R. M. Williams, and R. Croteau. 2004. Cytochrome p450 taxadiene 5alpha-hydroxylase, a mechanistically unusual monooxygenase catalyzing the first oxygenation step of taxol biosynthesis. Chem Biol 11:379-87. 42. Jennewein, S., C. D. Rithner, R. M. Williams, and R. B. Croteau. 2001. Taxol biosynthesis: taxane 13 alpha-hydroxylase is a cytochrome P450-dependent monooxygenase. Proc Natl Acad Sci U S A 98:13595-600. 43. Kieslich, K. 1976. Microbial transformations of non-steroid cyclic compounds. J. Wiley, [Chichester, Eng.]. 44. Kinouchi, Y., H. Ohtsu, H. Tokuda, H. Nishino, S. Matsunaga, and R. Tanaka. 2000. Potential antitumor-promoting diterpenoids from the stem bark of Picea glehni. J Nat Prod 63:817-20. 45. Kitazume, T., N. Takaya, N. Nakayama, and H. Shoun. 2000. Fusarium oxysporum fatty-acid subterminal hydroxylase (CYP505) is a membrane-bound eukaryotic counterpart of Bacillus megaterium cytochrome P450BM3. J. Biol. Chem. 275:39734-39740. 46. Kouzi, S. A., and J. D. McChesney. 1991. Microbial models of mammalian metabolism: fungal metabolism of the diterpene sclareol by Cunninghamella species. J Nat Prod 54:483-90. 47. LaFever, R. E., B. S. Vogel, and R. Croteau. 1994. Diterpenoid resin acid biosynthesis in conifers: enzymatic cyclization of geranylgeranyl pyrophosphate to abietadiene, the precursor of abietic acid. Arch Biochem Biophys 313:139-49. 48. Leppanen, H., J. V. K. Kukkonen, and A. O. J. Oikari. 2000. Concentration of retene and resin acids in sedimenting particles collected from a bleached kraft mill effluent receiving lake. Water Research 34:1604-1610. 49. Levinson, A. S., B. C. Carter, and M. L. Taylor. 1968. Microbial degradation of methyl dehydrobietate. Chem. Commun.: 1344. 50. Lewinsohn, E., M. Gijzen, T. J. Savage, and R. Croteau. 1991. Defense mechanisms of conifers: Relationship of monoterpene cyclase activity to anatomical specialization and oleoresin monoterpene content. Plant Physiol. 96:38-43. 51. Lewis, D. F. V. 2001. Guide to cytochromes P450 : structure and function. Taylor & Francis, London ; New York. 52. Liang, P.-H., T.-P. Ko, and A. H.-J. Wang. 2002. Structure, mechanism and function of prenyltransferases. Eur J Biochem 269:3339-3354. 36 53. Lin, L. H., L. W. Lee, S. Y. Sheu, and P. Y. Lin. 2004. Study on the stevioside analogues of steviolbioside, steviol, and isosteviol 19-alkyl amide dimers: synthesis and cytotoxic and antibacterial activity. Chem Pharm Bull (Tokyo) 52:1117-22. 54. Liss, S. N., P. A . Bicho, and J. N. Saddler. 1997. Microbiology and biodegradation of resin acids in pulp mill effluents: a minireview. Can J Microbiol 43:599-611. 55. Maria Perla Colombini, F. M., E. Ribechini,. 2005. Direct exposure electron ionization mass spectrometry and gas chromatography/mass spectrometry techniques to study organic coatings on archaeological amphorae. Journal of Mass Spectrometry 40:675-687. 56. Martin, D., D. Tholl, J. Gershenzon, and J. Bohlmann. 2002. Methyl jasmonate induces traumatic resin ducts, terpenoid resin biosynthesis, and-terpenoid accumulation in developing xylem of Norway Spruce stems. Plant Physiol. 129:1003-1018. 57. Martin, V. J., and W. W. Mohn. 2000. Genetic investigation of the catabolic pathway for degradation of abietane diterpenoids by Pseudomonas abietaniphila BKME-9 . J Bacteriol 182:3784-93. 58. Martin, V. J., and W. W. Mohn. 1999. A novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium Pseudomonas abietaniphila B K M E - 9 . J Bacteriol 181:2675-82. 59. Martin, V. J., D. J. Pitera, S. T. Withers, J. D. Newman, and J. D. Keasling. 2003. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21:796-802. 60. Martin, V. J., Z. Yu, and W. W. Mohn. 1999. Recent advances in understanding resin acid biodegradation: microbial diversity and metabolism. Arch Microbiol 172:131-8. 61. Meshnick, S. R., A . Thomas, A . Ranz, C. M. Xu, and H. Z. Pan. 1991. Artemisinin (qinghaosu): the role of intracellular hemin in its mechanism of antimalarial action. Mol Biochem Parasitol 49:181-9. 62. Mohn, W. W. 1995. Bacteria obtained from a sequencing batch reactor that are capable of growth on dehydroabietic acid. Appl Environ Microbiol 61:2145-50. 63. Mohn, W. W., A . E. Wilson, P. Bicho, and E. R. Moore. 1999. Physiological and phylogenetic diversity of bacteria growing on resin acids. Syst Appl Microbiol 22:68-78. 64. Morgan, C. A . , and R. C. Wyndham. 2002. Characterization of tdt genes for the degradation of tricyclic diterpenes by Pseudomonas diterpeniphila A19-6a. Can J Microbiol 48:49-59. 65. Morgan, C. A . , and R. C. Wyndham. 1996. Isolation and characterization of resin acid degrading bacteria found in effluent from a bleached kraft pulp mill. Can J Microbiol 42:423-30. 37 66. Narhi, L., and A. Fulco. 1986. Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium. J. Biol. Chem. 261:7160-7169. 67. Newman, D . J., G. M. Cragg, and K. M. Snader. 2003. Natural products as sources of new drugs over the period 1981-2002. J Nat Prod 66:1022-37. 68. Ohnuma, S.-i., K. Hirooka, N. Tsuruoka, M. Yano, C. Ohto, H. Nakane, and T. Nishino. 1998. A pathway where polyprenyl diphosphate elongates in prenyltransferase. Insight into a common mechanism of chain length determination of prenyltransferases. J. Biol. Chem. 273:26705-26713. 69. Orden, A. A., D . A. Cifuente, E. J. Borkowski, C. E. Tonn, and M. K. Sanz. 2005. Stereo- and regioselective hydroxylation of grindelic acid derivatives by Aspergillus niger. Nat Prod Res 19:625-31. 70. Peters, R. J., J. E. Flory, R. Jetter, M. M. Ravn, H.-J. Lee, R. M. Coates, and R. B. Croteau. 2000. Abietadiene synthase from Grand Fir (Abies grandis): Characterization and mechanism of action of the "pseudomature" recombinant enzyme. Biochemistry 39:15592-15602. 71. Ro, D.-K., G.-I. Arimura, S. Y. W. Lau, E. Piers, and J. Bohlmann. 2005. Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP720B1) is a multifunctional, multisubstrate cytochrome P450 monooxygenase. PNAS 102:8060-8065. 72. Roberts, G. A., G. Grogan, A. Greter, S. L. Flitsch, and N. J. Turner. 2002. Identification of a new class of cytochrome P450 from a Rhodococcus sp. J. Bacterid. 184:3898-3908. 73. Rohdich, F., K. Kis, A. Bacher, and W. Eisenreich. 2001. The non-mevalonate pathway of isoprenoids: genes, enzymes and intermediates. Curr Opin Chem Biol 5:535-40. 74. Rohmer, M., M. Knani, P. Simonin, B. Sutter, and H. Sahm. 1993. Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem J 295 ( Pt 2):517-24. 75. Rojas, M. C , P. Hedden, P. Gaskin, and B. Tudzynski. 2001. The P450-1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis. Proc Natl Acad Sci U S A 98:5838-43. 76. Rosazza, J. P., and R. V. Smith. 1979. Microbial models for drug metabolism. Adv Appl Microbiol 25:169-208. 77. Ruzicka, L. 1953. The isoprene rule and the biogenesis of terpenic compounds. Experientia 9:357-67. 78. Seo, J.-S., H. Chong, H. S. Park, K.-0. Yoon, C. Jung, J. J. Kim, J. H. Hong, H. Kim, J.-H. Kim, J.-I. Kil, C. J. Park, H.-M. Oh, J.-S. Lee, S.-J. Jin, H.-W. Um, H.-J. 38 Lee, S.-J. Oh, J. Y. Kim, H. L. Kang, S. Y. Lee, K. J. Lee, and H. S. Kang. 2005. The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4. 23:63-68. 79. Tavendale, M. H., P. N. McFarlane, K. L. Mackie, A . L. Wilkins, and A . G. Langdon. 1997. The fate of resin acids-1. The biotransformation and degradation of deuterium labelled dehydroabietic acid in anaerobic sediments. Chemosphere 35:2137-2151. 80. Tavendale, M. H., P. N. McFarlane, K. L. Mackie, A . L. Wilkins, and A . G. Langdon. 1997. The fate of resin acids-2. The fate of resin acids and resin acid derived neutral compounds in anaerobic sediments. Chemosphere 35:2153-2166. 81. Tudzynski, B. 2005. Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology. Appl Microbiol Biotechnol 66:597-611. 82. Ulusu, N. N., D. Ercil, M. K. Sakar, and E. F. Tezcan. 2002. Abietic acid inhibits lipoxygenase activity. Phytother Res 16:88-90. 83. van Beilen, J. B., R. Holtackers, D. Luscher, U. Bauer, B. Witholt, and W. A . Duetz. 2005. Biocatalytic production of perillyl alcohol from limonene by using a novel Mycobacterium sp. cytochrome P450 alkane hydroxylase expressed in Pseudomonas ' putida. Appl Environ Microbiol 71:1737-44. 84. Vogel, B. S., M. R. Wildung, G. Vogel, and R. Croteau. 1996. Abietadiene Synthase from Grand Fir (Abies grandis). cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase involved in resin acid biosynthesis. J. Biol. Chem. 271:23262-23268. 85. Wang, K. C , and S.-i. Ohnuma. 2000. Isoprenyl diphosphate synthases. Biochimica et Biophysica Acta (BB A) - Molecular and Cell Biology of Lipids 1529:33-48. 86. Warren, R., W. W. Hsiao, H. Kudo, M. Myhre, M. Dosanjh, A . Petrescu, H. Kobayashi, S. Shimizu, K. Miyauchi, E. Masai, G. Yang, J. M. Stott, J. E. Schein, H. Shin, J. Khattra, D. Smailus, Y. S. Butterfield, A . Siddiqui, R. Holt, M. A . Marra, S. J. Jones, W. W. Mohn, F. S. Brinkman, M. Fukuda, J. Davies, and L. D. Eltis. 2004. Functional characterization of a catabolic plasmid from polychlorinated- biphenyl-degrading Rhodococcus sp. strain RHA1. J Bacteriol 186:7783-95. 87. Woodrow, C. J., R. K. Haynes, and S. Krishna. 2005. Artemisinins. Postgrad Med J 81:71-78. 88. Zhang, L., and A . L. Demain. 2005. Natural products : drug discovery and therapeutic medicine. Humana Press, Totowa, N J . 39 2. A cytochrome P450 involved in the metabolism of abietane diterpenoids by Pseudomonas abietaniphila BKME-9 1 2.1 Introduction 5 Pseudomonas abietaniphila B K M E - 9 contains a cluster of genes required for the catabolism of abietane diterpenoids (10, 11). Several genes of the B K M E - 9 diterpenoid degradation (dit) cluster were sequenced and characterized, and a convergent pathway for abietane diterpenoid metabolism was proposed (see Fig. 1.11)(10). As presented in the Introduction, some evidence from research on the abietane diterpenoid degrading bacterium 10 Pseudomonas diterpeniphila A19-6a suggests that a P450 monooxygenase is involved in the catabolism of these compounds (13). No candidate P450 encoding genes, however, have been identified in the characterized B K M E - 9 dit cluster. Based on the above evidence we investigated the possibility of a cytochrome P450 involvement in the initial steps in the abietane diterpenoids degradation pathway of BKME-9 . To 15 accomplish this a genomic D N A fragment contiguous to the dit cluster was cloned and characterized. Here we provide previously missing evidence for the existence of a P450 enzyme in the dit cluster and demonstrate that it is involved in diterpenoid metabolism. We also show an apparent difference in strains B K M E - 9 and A19-6a, as the former does not require the P450 for normal metabolism of abietic acid. We characterized the P450 using a knockout mutant, a gene-20 fusion transcriptional reporter, and by determining carbon monoxide (CO)- and substrate-binding spectra of the protein expressed in E. coli. We also provide additional evidence for the homology of diterpenoid degradation genes in both B K M E - 9 and A19-6a. 1 A version of this chapter has been published as: Smith, D.J., V.J.J. Martin, and W.W. Mohn 2004. A Cytochrome P450 Involved in the Metabolism of Abietane Diterpenoids by Pseudomonas abietaniphila BKME-9 . J Bacterid 186:3631-9. Copyright © 2006, the American Society for Microbiology. 40 2.2 Methods and Materials 25 30 Bacterial Strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 2.1. Escherichia coli was cultured on Luria-Bertani medium and P. abietaniphila strains was cultured on tryptic soy broth, or mineral medium supplemented with diterpenoids as previously described (12). Mutants of strain B K M E - 9 were cultured with 4 u,g of gentamicin. Diterpenoids were supplied by Helix Biotechnologies, Richmond, Canada. Table 2.1 Strains and plasmids used in this study Genotype or Description Reference or Source Strains P. abietaniphila BKME-9 P450KO P. diterpeniphila A19-6a E. coli DH5a S17-1 Plasmids pUC19 pEXIOOT pX1918G pDS1 pEXP450 pEXP450KO pLC48 pLC162 Wild type; grows on abietane diterpenoids ditQ:: xylE-accC1; Gm r Wild type; grows on DhA (3) This study (13) endA1 hsdRU (rk" mk") supE44 thi-1 recA1 gyrA (Naf) relA1 Gibco BRL A(laclZYA-argF) U169 deoR (<S>80dlacA(lacZ)M15) recA pro thi hsdR with integrated RP4-2-TcMu::Kna::Tn7; (17) Tra+ Trr Sm r Cloning vector, Ap r (22) sacB conjugable plasmid for gene replacement; Ap r (16) xylE-accC7 fusion cassette-containing plasmid; Ap r Gm r (16) 5.1-kb EcoR I fragment of pLC162 cloned into the EcoRI site This study ofpUC19 1482-bp EcoRV-Sma\ of pDS1 cloned into the Smal site of This study pEXIOOT Pst\ xylE-accC1 cassette of pX1918G cloned into the Pst\ This study site of pEXP450 SuperCosI cosmid library clone containing DhA degradation (11) genes SuperCosI cosmid library clone containing DhA degradation (11) genes 35 The AbA used was approximately 96% pure, with another diterpenoid, most likely DhA, comprising the remainder. The PaA used was approximately 90% PaA, 7% DhA, and 3% AbA. The 7-oxo-DhA used was greater than 97% pure with trace amounts of several undetermined diterpenoids. DhA, and isopimaric acid (IpA) used in the study were greater than 99% pure. 41 Southern hybridization. Standard techniques of Southern hybridization analysis were followed as previously described. Briefly, SS Maximum Strength Nytran Plus was used for blotting (Schleicher and Schuell, Keene, New Hampshire). The immobilized D N A was hybridized to tdtD labeled with [a 3 2-P] dCTP (NEN, Boston, Mass.) using the Nick Translation System from 40 Gibco B R L (Gaithersburg, Md.). Dr. Cam Wyndham, Institute of Biology, Carleton University Ottawa, Canada, kindly provided tdtD from P. diterpeniphila A19-6a. Labeling efficiency was recorded at 6.7 x 10 7 cpm/ul of probe solution. After blotting the membrane was washed in 6X SSC (0.9 M NaCl, 90 m M sodium citrate, pH 7.0) for 5 min and then nucleic acids were immobilized by heating at 80° C for 90 45 min. Hybridization solution was prepared by combining 5 ml formamide, 3 ml 20X SSPE (3.6 m M NaCl, 200 m M NaP0 4 (pH 7.7), 1 m M EDTA), 0.5 ml 100X Denhart's (0.2% ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin), 0.5 ml 2 ug/ul salmon sperm DNA. Prior to adding the probe the membrane was incubated in 5 ml of hybridization solution at 42°C in a preheated tube roller for 2 hours and then drained. The probe was boiled for 3 min and then the 50 entire 50 ul was added to 5 ml of hybridization solution in the tube containing the membrane. After overnight hybridization at 42°C the membrane was briefly washed with 20 ml 7X SSPE/1% sodium dodecyl sulfate (SDS) followed by a second wash with 150 ml 7X SSPE/1% SDS at 42° for 1 hour. Hybridization was analyzed by using standard phosphorimager scanning and autoradiography techniques. 55 DNA manipulation. Plasmid D N A was isolated by the standard alkali lysis (2) or by QIAprep Spin Miniprep Kit (Qiagen, Santa Clarita, California). Restriction endonuclease (New England Biolabs [NEB], Beverly, Mass., or Gibco BRL) digestions were performed by standard procedures. D N A fragments were purified from agarose gels with QIAquick gel extraction kit (Qiagen). Plasmid pUC19 and E. coli DH5oc were used to clone the 5.1 kbp EcoRl digested 42 60 fragment from pLC 162 to generate pDS 1. Prior to ligation EcoRl digested pUC 19 was treated with alkaline phosphatase (Gibco) as per manufacture's instructions. Nucleic acids were then ethanol precipitated and suspended in 5 ul of ddFkO. Ligation reactions were conducted using T4 D N A ligase (NEB) as per manufacture's instructions. The ligase reaction mixture was incubated at 15° C over night and used to 65 transform E. coli DH5cx by electroporation according to the protocol supplied with the Bio-Rad (Hercules, Calif.) gene pulser. Positive transformations were selected by blue/white colony growth after over night incubation on L B agar plates supplemented with ampicillin (50 pg/ml) and 40 ul of X-gal (40 mg/ml in dimethyl formamide). Two dilutions of transformed cells were used for plating: 10 pi and 990 ul. White colonies were picked from the plates and grown 70 overnight in 5 ml of L B broth supplemented with 50 p.g/ml ampicillin. Plasmid D N A was then extracted with QIAprep Spin Miniprep columns as above, digested with EcoRl, and run on a 0.7 % agarose gel to identify insert and vector. The successful ligation was designated pDSl . S u c c e s s i v e u n i d i r e c t i o n a l d e l e t i o n s . Successive unidirectional deletions of pDSl D N A were prepared using the double-stranded nested deletion system from Pharmacia Biotech (Uppsala, 75 Sweden). Support protocol provided with the system was followed as per manufacturer's recommendations. In general, pDSl was prepared for nested deletions by extracting plasmid D N A from overnight L B grown cultures supplemented with 100 pg/ml ampicillin using QIAprep Spin Miniprep Kit eluted with 50 pi of ddH 2 0. Plasmids were linearized by digesting with Sail (NEB) at 37°C for 2 hours. D N A was then ethanol precipitated and suspended in 80 ddH20. Sail digested samples were then backfilled with thionucleotides using Klenow fragment provided with the system. The reaction mixtures were incubated in a water bath at 37°C for 30 min followed by 20-min incubation at 65°C to inactivate the Klenow fragment. Backfilled D N A samples were purified using QIAquick PCR Purification Kit (Qiagen) and suspended in 30 pi of 43 ddFkO. The concentration of backfilled D N A for this sample was 380 ng/pl as determined by 85 agarose gel electrophoresis analysis. 5'- overhanging ends susceptible for Exonuclease III digestion were prepared by Xbal (Gibco) digest incubated in a 37°C water bath for 90 min. Exonuclease III deletion reactions were carried out at 37°C under the following conditions; 20 pi Xbal digested D N A (~2 pg), 20 pi 2X Exo III buffer solution (8 pi Exo III buffer, 6pi 0.3 M NaCl and lOpl ddH 2 0, and 1 pi Exonuclease III (90,000-130,000 units/ml). Twenty-four 1.6 pi 90 aliquot, were removed from the reaction mixture at 4 min intervals and combined with 3 pi of S1 nuclease solution as described in the support protocol. After a 30 min incubation at room temperature 1 pi of SI Stop Solution was added to each sample followed by 10-min incubation at 65°C. Deletions were analyzed by electrophoresis on a 1% agarose gel of 2.6 pi from each 95 aliquot. The remaining 3 pi of each aliquot were used for circularization of the linearized plasmids as outline in the protocol provided with the system. Based on the extent of deletion, circularized plasmids were selected for transformation into chemically competent DH5a E. coli cells prepared essentially as described by Hanahan (6). Transformed DH5cx cells were screened on L B agar plates using blue/white screening as described above. White colonies from each plate 100 were used to inoculate 5 ml of L B broth supplemented with 100 pg/ml of ampicillin. Alkaline lysis mini-preps, as described above, were used to isolate plasmid DNA. Following EcoRl digestion plasmid sizes were analyzed by gel electrophoresis on 0.7% agarose gel. Clones of the appropriate sizes were then used to inoculate 5 ml L B broth with 100 pg/ml of ampicillin. Plasmid D N A was isolated with Qiagen Miniprep kit as described above and suspended in 50 pi 105 of ddH 2 0. 1 pi of each sample was then linearized by an EcoRl digest and analyzed by electrophoresis to confirm fragment size and determine D N A concentration. 44 Sequencing and sequence analysis. Standard M l 3 primers for sequencing of successive unidirectional deletion clones were supplied by the Nucleic Acid and Protein Service (NAPS) at the University of British Columbia. The forward primer used was -21 M l 2, (5 ' -TGT-AAA-110 ACG-ACG-GCC-AGT-3 ' ) and the reverse primer used was M13R, ( 5'- C A G - G A A - A C A -GCT-ATG-ACC-3 ' ) . Primers used for "primer walking" of cosmid library clones were supplied by AlphaDNA (http://www.alphadna.com). AlphaDNA also supplied primers used for colony PCR of P450KO and sequencing of the PCR product. D N A sequences were determined at NAPS at the University of British Columbia using the AmpliTaq dye terminator cycle sequencing 115 (Applied Biosystems, Forest City, CA). Conditions of the sequencing reaction were as follows; 4 pi terminator premix (NAPS), 0.25-0.5 pg of plasmid template or 1 pg of cosmid template, 3.2 pmol primer, and ddH 2 0 as required to 20 pi. Primer extension products were prepared by PTC-150 Minicycler with a Hot Bonnet (MJ Research, Waltham, M A ) using the following program for 25 cycles; rapid thermal ramp to 96°C, 96°C for 30 seconds, rapid thermal ramp to 50°C, 120 50°C for 15 seconds, rapid thermal ramp to 60°C, 60°C for 4 min. Extension products were purified using Centri-Sep columns (Princeton Separation, Adelphia, NJ). A consensus nucleic acid sequence was prepared using Bioedit (Version 5.0.0), available at http://vvww.mbio.ncsu.edu/RNaseP/info/programs/BIOEDIT/bioedit.html). ORF finder software at http://www.ncbi.nlm.nih.gov/gorf/gorf.html was used to determine open reading frames and to 125 conduct sequence similarity searches using BLASTP software (2.2.6) from the National Center for Biotechnology Information website. The ClustalW Multiple Alignment program included with the Bioedit software was used to align and analyze protein sequences using the default setting. Knockout of the putative P450 by gene replacement. A knockout of ditQ was generated by 130 gene replacement to yield strain P450KO. Plasmid pEXP450 was constructed by ligating a 1482 bp EcoRV Smal blunt end fragment of pDSl into the dephosphorylated unique Smal site of 45 pEXIOOT (8) containing the SacB counterselectable maker and transforming Dh5a. Next, a i n -digested xylE-accCl transcriptional fusion antibiotic cassette of pX1918G (8) was ligated into the dephosphorylated unique Pstl site of pEXP450, which disrupted the P450 gene and the 135 product was used to transform E. coli S17-1 to create pEXP450KO. Successful transformants were selected by growth on L B plates containing 10 u,g/ml of gentamycin and the ability to cleave catechol producing a yellow colour after spraying colonies with 0.1 M potassium phosphate (pH 7.5) containing 100 m M catechol as in (8). Ligation and transformation were confirmed by I-Sce digest of alkaline lysed extracts of overnight culture yielding two bands on 140 an agarose gel representing the vector and the insert. Homologous recombination of the mutated allele into strain B K M E - 9 was accomplished by diparental conjugation as in (5) followed by a two step selection method as previously described (11). Successful gene replacement was monitored by colony PCR (23) with primers targeted to the P450 gene (P450-4041eft, 5'-GCG G A C CTT G A A GGT A G C GA-3', and P450-3567right, 5'-GCA A C T T C A TGG C A G GCC 145 TT-3') at an annealing temperature of 61°C and a 4-min extension time. In order to confirm insertion into the gene of interest the 3163 bp amplicon from the above PCR was then used in two sequencing reactions with P450-4041eft and P450-3567right as primers. G r o w t h assays a n d cel l suspensions. Cultures of B K M E - 9 and P450KO were grown overnight at 28°C on mineral medium supplemented with 90 mg of DhA per litre, or with 1 g of sodium 150 pyruvate per litre supplemented with 4 mg of gentamycin per litre. These overnight cultures were then transferred to mineral medium supplemented with 1 g sodium pyruvate per litre. After overnight growth, cells were collected by centrifugation, washed and suspended in sterile saline at an optical density at 600 nm (ODeoo) of 0.6. These cell suspension was then used to inoculate (0.1%) 2-ml cultures in solvent-washed tubes of mineral medium supplemented with either 1 g 155 of sodium pyruvate, 90 mg of AbA, 90 mg of DhA, 90 mg of PaA, or 95 mg of 7-oxo-DhA per litre. A l l cultures were then incubated on a rotary shaker at 28°C. At selected time intervals 2 to 46 4 replicates of 2-ml cultures of each strain were removed from the incubator. Half of the cultures were acidified with 2 drops of 1 M HC1 and immediately frozen at -20°C for later analysis of abietanes by gas chromatography using a flame ionization detector (GC-FID), as previously 160 described (12). The other half of the cultures (1 ml) was centrifuged, the pellet washed with 0.9% sterile saline, the suspension centrifuged again and the pellet frozen at -20°C. These samples were later used to determine protein concentration using the micro-bicinchoninic acid (BCA) protein assay kit (Sigma) and bovine serum albumin as the standard (18). B C A protein quantification was used to monitor growth as opposed to optical density, because resin acids 165 precipitating in the medium prevented accurately measuring OD. Cell suspension assays were conducted as previously described (11). GC electron impact (EI) mass spectrometry (MS) of methyl ester derivatives was conducted as previously described (12) using an Agilent Technologies 6890N Network GC system equipped with an Agilent 5973 Mass Selective Detector. National Institute of Standards and Technology MS Search (2.0) was used to analyze 170 mass spectral data. Catechol 2,3-dioxygenase (C230) assays. Previously it was reported that BKME-9 shows no endogenous C230 activity and that activity served as an adequate reporter for gene induction studies (10). For C230 assays, strain P450KO was grown on mineral medium supplemented with l g per litre sodium pyruvate to an OD 6 0o between 0.15 and 0.3 and then spiked with a 175 potential inducer, 150 mg per litre DhA, 150 mg per litre AbA, 158 mg per litre 7-oxo-DhA, 150 mg per litre isopimaric acid, 37 mg per litre 12,14-dichlorodehydroabietic acid, 15.4 mg per litre biphenyl, 12.0 mg per litre naphthalene, or 17.8 mg per litre phenanthrene. These cultures were incubated until they reached an OD 6oo between 0.6 and 0.7. Cultures were then harvested, washed in 10 m M K P 0 4 buffer (pH 7.5) at 4°C and suspended in the buffer at an OD 6 0o of 6.0. 180 Triplicate enzyme assays were performed on whole cells suspended at an O D e o o of 0.1 in 1 ml of 47 the buffer containing 500 (iM catechol. C230 activity was assayed spectrophotometrically at 30°C as the formation of 2-hydroxy semialdehyde at 375 nM (s = 44 mM" 1 cm"1) for 3.5 min. S p e c t r o p h o t o m e t r i c assays. A 2-litre flask containing 1 litre of L B with 50 pg/ml of ampicillin was inoculated with 5 ml of an overnight culture of E. coli harbouring pEXP450 or pEXlOOT. 185 The culture was incubated with shaking until the ODeoo reached approximately 0.6. Expression of ditQ was induced by addition of 1 m M IPTG and further incubation for 18 to 24 hours. Cells were harvested by eentrifugation at 8275 x g in a Sorvall SLA 3000 rotor for 15 min. The pellet was washed with 1 litre of TrisCl pH 7.4 and centrifuged as above. The pellet was then suspended in 5 ml of the buffer plus 1 m M DTT and 1 m M PMSF. The suspension was passed 190 through a French pressure cell 2 times, and the crude lysate was centrifuged for 30 min at 25000 x g in a Sorvall SS-34 rotor. The supernatant was removed and the crude extract was used for spectrophotometric analysis, using a Cary IE spectrophotometer and Cary U V W i n Scan Application Version 2.00 software. The reduced CO binding spectrum was obtained with 200 pi of crude extract added to 195 1.80 ml of the same buffer as above plus a few crystals of sodium dithionite to reduce the sample. The sample was then equally divided in two 1-ml, optically matched cuvettes. One sample was treated by bubbling carbon monoxide through the cuvette slowly for 30 sec. The second sample was used as the reference in difference spectroscopy with the carbon monoxide treated sample. 200 Substrate binding assays were performed in two optically matched 3-ml cuvettes, each with 300 pi of the above crude extract ofE. coli harbouring pEXlOOT or pEXP450 plus 2.70 ml of buffer used above. Increasing concentrations of substrate in buffer were added to the sample cuvette and equal volumes of buffer were added to the reference cuvette. The difference spectra were determined from 350 nm to 500 nm. The binding constant, was determined using the 205 following non-linear fitting equation: 48 AA' = A A M (( [Lj] + [ E T ] + IQ - (([L T ] + [ E T ] + KAf - 4 [ L T ] [ E T ] ) ° 5)/ (2 [ E ] T ) where A A is the difference in absorbance between 387 and 425, A A M is the maximum change in absorbance, [L]T , is the total ligand concentration, and [E]T is the total enzyme concentration (4). P450 levels were calculated from the absorbance at 450 nm of the ferrous form complexed with 210 CO using the cytochrome P450 extinction coefficient, 91 mlVr1 cm - 1 . Nuc leo t i de sequence accession n u m b e r . The nucleotide sequences reported in this study were submitted to GenBank under accession no. AF119621. 49 2.3 Results 2.3.1 Sequencing of 10.4 kbp region containing a putative P450 gene 215 To determine i f a gene cluster corresponding to the tdt cluster of P. diterpeniphila A19-6a is located adjacent to the dit cluster of BKME-9 , we analyzed by Southern blot nine EcoRI-digested B K M E - 9 cosmid library clones containing ditAl, using the tdtD gene as a probe. The lanes containing fragments from cosmids pLC48 and pLC162 had single bands with high intensity of approximately 6 kbp and 5.1 kbp respectively (Fig 2.2). The 5.1 kbp fragment of 220 pLC162 hybridizing to the tdtD probe was cloned and sequenced. This 5.1 kbp fragment is located approximately 2.8 kbp downstream from ORF1 of the previously characterized dit cluster (Fig. 2.2). The 2.8 kbp gap was sequenced from cosmid library clone pLC162 by primer walking. We also used primer walking to sequence 2.5 kbp beyond the end of the 5.1 kbp fragment opposite to the gap, using the pLC48 cosmid as a template. Thus, a total region 10.4 225 kbp adjacent to the dit cluster was sequenced. Table 2.2 Amino acid sequence comparison of deduced DitQ (P450dit) to proteins in the non-redundant database obtained by BLASTP search Proteins with similar sequence (protein function) E-values % identity (no. of residues) Organism Reference Accesion no. TdtD (cytochrome P450) e-178 84 (424) Pseudomonas diterpeniphila (13) AAK95585 ERYK Cytochrome P450 113A1 (erythromycin B/D C-12 hydroxylase) 7e-22 26 (397) Saccharopolyspora erythraea (19) P48635 CYP108 cytochrome P450 t erp (a terpineol oxidation) 1e-18 23 (428) Pseudomonas sp. (15) P33006 CamC P450cam (cytochrome P450) (camphor 5-monooxygenase) 0.023 17 (415) Pseudomonas putida (20) P00183 230 The region of the above 5.1-kbp fragment, presumably hybridizing to the tdtD probe, corresponds to an ORF designated ditQ. A similarity search of the non-redundant GenBank database using BLASTP (1) with the deduced amino acid sequence of ditQ indicates that ditQ codes for a putative P450 (Table 2.2). Comparison of the inferred amino acid sequence of ditQ 50 Figure. 2.1 Proposed pathway for abietane degradation by P. abietaniphila BKME-9. Chemical designations: I, palustric acid; II, dehydroabietic acid; III, 7-hydroxydehydroabietic acid; IV, 7-oxodehydroabietic acid;, V , 7-oxo-1 l,12-dihydroxy-8-13-abietadien acid; VI , 7-oxo-11,12-dihydroxydehydroabietic acid; VII, abietic acid pLC48 pLC162 E c o R l ~7~ 6.0 kbp "7 E c o R l Cos 1 EcoR I site 5.1 kbp 2.8 kbp E c o R l E c o R l Dit Ouster E c o R \ E c o R \ E c o R \ 7" P. abietaniphila BKME-9 ^ # 6^  6^ ° 6^ 6^ 6^ 6^ / # P. diterpeniphila A19-6a ^ # # ^  # / # 7*A Burkholderia sp. L B 4 0 0 74 82 81 82 74 72 83 Jp <<S 75 81 <?T <2T <??" <$*• <?r qjr c? c? <P e-^  ^ j i * ' jsr j « r ,er ,«r 40 r # ^ 71 60 70 74 70 84 52 75 69 60 74 74 59 76 63 56 73 63 57 52 / - Dehydrogenase • - CoA Ligase < ^ • - Hypothetical • - (i subunit dioxygenase 62 • -P450 • - a subunit dioxygenase m -ACR • - Sterol Carrier-like protein • - Thiolase • - Regulator • - Hydrolase • - Ferredoxin • - Ring cleavage dioxygenase • - Permease Figure 2.2 Physical map of the dit gene cluster of BKME-9, the tdt gene cluster of A19-6a and homologues from LB400. Genes are represented by arrows with patterns and colours corresponding to putative functions. Numbers below each gene represent the percent amino acid identity corresponding to the deduced protein sequence of the B K M E - 9 gene above. Double vertical lines indicate gaps in the genome sequence of unspecified length, which may contain additional ORFs. Horizontal lines refer to cosmid library clones used for subcloning and sequencing of BKME-9 D N A and the dit cluster. IO to the Cluster of Orthologous Groups of protein (COGS) and Protein families database of alignments and H M M S showed similarity to P450s (Table 2.3). Alignment of the P450dit 235 deduced protein with the well-characterized P450 c a m showed conservation of functional residues including the highly conserved heme-binding region with consensus sequence FG(F/H)G(P/S)H(M/L)C. 2.3.2 Sequence analysis of 10.4-kbp dit cluster extension 240 Sequence analysis of the 10.4-kbp fragment revealed nine complete ORFs and one partial ORF which are homologues of genes encoding 2 dehydrogenases, a thiolase, a hydrolase, an ancient uncharacterized conserved region, a cytochrome P450, a transcriptional regulator, a CoA ligase and 2 conserved hypothetical proteins (Fig. 2.2). The CoA ligase sequence is contiguous with the previously identified ORF1 (10) of the dit cluster and completes this gene sequence, 245 now designated ditJ (Fig. 2.2, Tables 2.2 and 2.3). A region of the 10.4-kbp extension corresponds very closely to the tdt cluster (13), having the same ORF arrangement and greater than 72% identity of deduced amino acid sequences. The gene encoding DitP was identified by COG analysis as an ancient conserved region common to two or more phylogenetic branches (Table 2.3). Morgan and Wyndham (13) did not identify this coding region on the tdt cluster. 250 Further analysis of the tdt sequence did not reveal an ORF corresponding to ditP. 53 Table 2.3 Conserved domain search and COG comparison Gene Deduced Functional assignment based on Conserved Domains no. of COG comparison Pfam Data base Search residues Domain E-value Residues ORF3 398 COG1960 Acyl-CoA dehydrogenases 1. Acyl-CoA dehydrogenase, N-terminal domain (pfam02771) 2. Acyl-CoA dehydrogenase, middle domain. (pfam02770) 2.4e-6 3.4e-16 27-145 147-248 ORF4 435 No Hits Amidohydrolase family (pfam04909) 3.4e-08 41-373 ditQ 424 COG2124 Cytochrome P450 Cytochrome P450 (pfam00067) 2.3e-24 39-421 ditP 140 COG2128 Uncharacterized ancient conserved region No Hits ditO 391 COG0183 Acetyl-CoA acetyl transferase 1. Thiolase, N-terminal domain (pfam00108) 2. Thiolase, C-terminal domain. (pfam0280) 1.6e-86 1.6e-63 1-260 265-389 ditN 301 COG1250 3-Hyd roxyacyl-CoA dehydrogenase 1. 3-hydroxyacyl-CoA dehydrogenase, NAD binding domain (pfam02737) 2. 3-hydroxyacyl-CoA dehydrogenase, C-terminal domain (pfam00725) 2.3e-53 5.8e-41 3-188 190-285 ditM 289 COG0179 2-keto-4-pentenoate hydra tase/2-oxohepta-3-ene-1,7-dioic acid hydratase (catechol pathway) Fumarylacetoacetate (FAA) hydrolase (pfam01557) 1e-45 86 254 ditL 359 No Hits Amidohydrolase (pfam04909) 3.6e-32 1-344 ditK 249 No Hits Bacterial regulatory proteins, tetR family (pfam00440) 2e-12 67-113 ditJ 546 COG0318 Acyl-CoA synthetases (AMP-forming)/AMP-acid ligases II AMP-binding enzyme (pfam00501) 7.5e-105 43-459 2.3.3 Growth of ditQ mutant on abietanes 255 A ditQ mutant strain, P450KO, was used to investigate the function of ditQ in abietane diterpenoid metabolism. Characterization of the growth of P450KO on abietane diterpenoids revealed that ditQ is required for a growth phenotype similar to the wild type on DhA and PaA but not on AbA or 7-oxo-DhA. B K M E - 9 growing on AbA had a doubling time (+ standard error) of 12 + 2.0 h and reached a protein concentration of 10 + 0.4 u.g of protein/ml for a 260 growth yield of 0.1 + 0.4 x 10"2_g of protein per g of AbA, while P450KO had a doubling time of 54 10 + 4.0 h and reached a protein concentration of 11 + 0.5 ug /ml for a growth yield of 0.1 + 0.6 x 10"3 g of protein per g of AbA (Fig 2.3 A) Similarly growth of B K M E - 9 on 7-oxo-DhA had a doubling time of 4.5 + 0.6 h and reached a protein concentration of 16 + 1.0 ug/ml for a growth yield of 0.16 + 0.01 g of protein per g of 7-oxo-DhA, while P450KO had a doubling time of 7.7 265 + 0.7 h and reached a protein concentration of 12 + 4.0 x 10"2 ug/ml for a growth yield of 0.13 + 0.01 g of protein per g of 7-oxo-DhA. The growth rates and yields of P450KO were substantially lower than those of B K M E - 9 on DhA or PaA. Using DhA as a carbon and energy source, B K M E - 9 had a doubling time of 3.6 + 0.2 h and reached a protein concentration of 22 + 0.5 ug/ml for a growth yield of 0.24 + 0.88 x 270 10"2 g of protein per g of DhA; whereas, P450KO had a doubling time of 10 + 1.7 h and a reached a protein concentration of only 10 + 0.6 ug/ml for a growth yield of 0.11 + 0.40 x 10" g of protein per g of DhA (Fig. 2.3B). Similarly, on PaA, B K M E - 9 had a doubling time of 5.6 + 3.0 x 10"2h and reached a protein concentration of -27 + 2.2 ug/ml for a growth yield of 0.3 + 0.1 x 10"2 g of protein per g of PaA; whereas P450KO had a doubling time of 23 + 2.8 h and 275 reached a final protein concentration of 18 + 1 ug/ml for a growth yield of 0.2 + 0.8 x 10" g of protein per g of PaA. These results suggest that a P450 encoded by ditQ plays an important role in metabolism of DhA and PaA but not metabolism of A b A or 7-oxo-DhA. We cannot exclude the possibility that the xylE-accCl insertion cassette used to create P450KO may have a polar effect on transcription of ORFs downstream of ditQ. But, this is . 280 unlikely given that the cassette does not contain a transcription terminator and so should allow transcription of downstream sequences. Additionally, there is a classic r/zo-independent terminator sequence located 30 bp downstream of ditQ. The terminator mRNA sequence forming the stem-loop is 5' A C C C G U G C C U - G A G A - A G G C G C G G G U U U U U U - 3 ' (with underlined bases indicating the stems and hyphens indicating the loop). The 3' end of the mRNA 55 285 has poly(U) tail that is required for termination. The hairpin structure has a free energy of-20.5 kcal/mol as predicted by Kinefold (http://kinefold.u-strasbg.fr/) or -19.53 kcal/mol as predicted by RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). 2.3.4 A b i e t a n e r e m o v a l b y P 4 5 0 K O Abietane diterpenoid removal coincided with increases in protein concentration during 290 growth by both B K M E - 9 and P450KO (Fig 2.3). Kinetics of removal of AbA and 7-oxo-DhA by P450KO were similar to removal by BKME-9 . In contrast, removal of DhA and PaA by P450KO required longer incubation periods than those observed for B K M E - 9 (Fig. 2.3). By 24 h B K M E - 9 had removed 100% of the initial DhA, whereas P450KO had only removed -12%. Similarly, B K M E - 9 had removed 98% of the initial PaA by 48 hours, whereas P450KO had only 295 removed ~5% by the same time. After 90 hours P450KO had only removed 65% of the original PaA. Along with the removal of PaA by P450KO, accumulation of DhA was also observed (data not shown), suggesting that PaA is transformed to DhA by the strain. The PaA reagent is 90% pure, containing also 7% DhA and 3% AbA. In cultures of P450KO on PaA, the DhA concentration increased to a maximum at 80 hours and was reduced to an undetectable level by 300 100 hours. The AbA concentration did not increase and was also undetectable by TOO hours. The increase in DhA was not observed when B K M E - 9 grew on PaA, and in those cultures, the trace amounts of both DhA and AbA associated with the PaA reagent were removed by 50 hours. When P450KO grew on DhA, 2 putative metabolites accumulated. The same two metabolites were also found at lower concentrations when P450KO was grown on PaA. These metabolites 305 did not accumulate when B K M E - 9 was grown on any resin acid tested. Mass spectral analysis by GC-MS was not sufficient to determine the structure of the metabolites.. 56 14 12 10 1 1 8 <D l 6 O O ) 4 0 25 20 1 ' E 1 5 B o 5 - i o o Wildtype growth P450KO growth Wildtype AbA removal P450KO AbA removal Wildtype growth P450KO growth . Wildtype DhA removal \ y ^-jf P450KO DhA removal #^  20 40 60 80 100 Time (hours) A A 80 O) 1 _ ' c ' r o 60 | rx "D 40 '£ o 20 5 B - 1 0 0 100 80 ^ tr 60 -g < o 4 40 I ro o 420 I-o 120 Figure 2.3 Growth and substrate removal of BKME-9 and P450KO on DhA and AbA. A. Protein growth curves of B K M E - 9 and ditQ mutant strain, P450KO, on AbA showing AbA removal. B. Protein growth curves of BKME-9 and the ditQ mutant strain on DhA showing DhA removal. Error bars indicate standard deviation of the average of 3 samples, strain. The highest rates of removal occurred during highest rates of growth in all cases. 2.3.5 Specific induction of P450dit by abietane diterpenoids The xylE transcriptional fusion of the ditQ knockout strain encodes catechol 2,3-dioxygenase (C230), which allowed for analysis of induction of ditQ transcription by spectrophotometrically monitoring cleavage of catechol in suspended cells incubated with various inducers. The ditQ gene was induced by all 4 abietane diterpenoids tested (Fig. 2.4). In fact, a pimerane diterpenoid, isopimaric acid, and a chlorinated diterpenoid, 12,14-dichlorodehydroabietic acid, also induced ditQ, despite these two compounds not being growth substrates for BKME-9 . However, this is not surprising considering that the same inducers were 57 identified for ditAl and ditA3, the genes encoding the a subunit and ferredoxin of the ring hydroxylating dioxygenase, respectively (10). As previously seen with the dioxygenase components, non-diterpenoid compounds did not induce expression of ditQ above the level of the pyruvate control. c •5 o a. at £ c I i o < CM O 2 6 0 - , 240 220 200 H 180 160-1 140 H 120 100 8 0 - I 60 ^| 40 20 H 0 X •9 •3" <2T - / Figure 2.4 Expression of ditQ-xylE gene fusion product in response to various diterpenoids and aromatic compounds. C230 activity was assayed spectrophotometrically at 30°C as the formation the yellow catechol cleavage product, 2-hydroxy semialdehyde, at 375 nM (s = 44 mM" 1 cm"1) for 3.5 min. Activity values are means + SD (n=3) of enzyme assays. 2.3.6 P450dit reduced carbon monoxide and substrate binding spectra P450s are identified by a characteristic Soret maximum at 450 nm in the CO-bound form of the reduced enzyme. The difference spectrum of reduced P450dit expressed in E. coli with and without carbon monoxide produced a Soret maximum at 450 nm (not shown). This result confirms that ditQ codes for a cytochrome P450. 58 DhA substrate binding experiments with the crude lysate of E. coli expressing P450dit yielded a type I substrate binding spectrum, which is a strong indicator that DhA is a substrate for P450dit. Titration of P450d,t with DhA yielded a type I substrate binding spectrum with a minimum at 387 and a maximum at 425 nm (Fig. 2.5). Type I curves result from the conversion of low spin hexacoordinated ferric heme with a Soret peak at around 417 nm to a high spin pentacoordinated ferric heme with the displacement of the distal water ligand after substrate binding (9). This results in a decrease in the Soret peak at 417 and an increase of a Soret peak at 387 nm. A plot of the difference in absorbance between 387 nm and 425 nm versus substrate concentration fitted to the binding curve equation gave an estimated of 0.43 uM with a standard deviation of + 0.03. OJ o c CD XI o (0 J3 0.05 h 0.04 h 0.03 \-< 0.02 V < 0.01 h 0.00 360 380 400 420 440 460 480 500 Wavelength (nm) _L 5 10 15 Dehydroabietic Acid Concentration (\M) 20 Figure 2.5 Binding spectrum for P450 d i t with DhA. Data points represent the difference in absorbance between 387 nm and 425 nm caused by increasing DhA concentration. The curve represents a best fit of the data to the binding equation in which KA = 0.43 + 0.03 uM and AA, = 0.05 + 6.81 x 10"4. Inset: UV/Visible difference spectra of P450dit with increasing concentration of DhA. 59 Neither AbA nor PaA produced typical P450 substrate binding spectra, and so, are likely not substrates for P450d,t (data not shown). Although both seemed to cause a perturbation of the heme environment, resulting in a shift of the Soret maximum, the curves produced were ambiguous, and further study is required for definitive analysis of binding using these compounds. 7-Oxo-DhA is also a growth substrate for B K M E - 9 but clearly did not bind to P450d,t and is therefore not likely a substrate for P450dit- Isopimaric acid, which is not a growth substrate for BKME-9 , may bind to P450dit weakly but did not yield a typical binding spectrum, and therefore, is not likely a substrate of P450 d l t. 2.4 Discussion In this study we demonstrated the involvement of a newly identified cytochrome P450 in the metabolism of abietane diterpenoids by BKME-9 . A gene knockout of ditQ, coding for P450djt, indicates that this gene is involved in the degradation of DhA and PaA. The knockout increased the doubling times, and lowered the protein yields of the mutant growing on either DhA or PaA, in comparison to B K M E - 9 (Fig. 2.3). The P450 d i t mutant retained the ability to grow slowly on DhA relative to BKME-9 . This is an indication that an alternate degradation pathway, not involving P450dit, is able to metabolize DhA and PaA. Perhaps it is this same alternate pathway that is responsible for the metabolism of A b A and 7-oxo-DhA. In addition, growth of strain P450KO on PaA or DhA produced the same putative metabolites, suggesting disruption in the metabolism of these compounds at the same point in a convergent pathway. Induction analysis indicates that ditQ expression is inducible by a range diterpenoids (Fig. 2.4), while a substrate binding assay showed that DhA is a likely substrate for this enzyme with a relatively low Kd of 0.4' u M (Fig. 2.5). The results of this study are not in complete agreement with those of Morgan and Wyndham (13), who reported that a tdtD mutant of Pseudomonas sp. A19-6a retained the ability to grow on DhA and A b A and exhibited similar decreases in removal rates for both substrates. 60 However, since abietanes were not extracted from the cells in that study, abietanes sorbed to cells but not necessarily degraded would have been considered removed. It is also possible that A19-6a differs from B K M E - 9 in its complement of diterpenoid degradation enzymes in a way that does not allow for metabolism of AbA in the A19-6a tdtD mutant. We hypothesize that the function of P450dit is to hydroxylate DhA at C-1 (Fig. 2.1). In a previous study on abietane degradation by BKME-9 , Martin and Mohn (10) showed that a ring-hydroxylating dioxygenase mutant, BKME-41, accumulated 7-oxo-DhA in cell suspension assays on DhA, PaA or AbA. They also showed that the substrate for the ring hydroxylating dioxygenase, DitA, required a ketone group at C-1, as DhA was not a substrate for the dioxygenase. The bacterial degradation of several natural plant products involves P450 monooxygenases that catalyze ring hydroxylation followed by oxidation of the hydroxyl group to a carbonyl. Some examples of this mechanism include the degradation of camphor involving P450 c a m(14), the degradation of limonene involving the P450, limonene-6-hydroxylase, (21) and the recently reported degradation of cineole involving P450cjn (7). The metabolism of abietane diterpenoids appears to follow the same pattern, with P450dit catalyzing the hydroxylation of DhA to 7-hydroxy-DhA before a further oxidation to 7-oxo-DhA. Since 7-oxo-DhA is a metabolic intermediate of AbA, and substrate binding assays indicate that AbA is not a substrate of P450dit,how then is AbA transformed to 7-oxo-DhA? Possibly another pathway is used for AbA metabolism, involving another P450 which functions to hydroxylate A b A or one of its derivatives. The existence of an additional P450 that can partially complement P450dit could also explain how the P450di t mutant strain was able to grow, albeit slowly, on DhA and PaA. Further, the possibility of a second P450 is also consistent with sequence analysis of Burkholderia sp. LB400 (see below). The results of this study and a previous one (10) are consistent with a mechanism of PaA degradation involving DhA as an intermediate that is subsequently hydroxylated at C-1 by 61 P450dit (Fig. 2.1). Martin and Mohn (10) showed an accumulation of DhA along with 7-oxo-DhA from PaA in a cell suspension assay of the ditAl mutant. In this study, we observed an increase in DhA concentration during growth of the P450dit mutant on PaA. The dit cluster contains several putative dehydrogenase genes, which could function in the formation of DhA from PaA. Substrate binding data strongly suggest that DhA is the better substrate for P450dit; while, PaA did not produce a typical substrate binding spectrum and does not appear to be a good substrate for this enzyme. Additional work, using a more pure PaA reagent would lead to greater insight regarding this potential substrate-. This study confirms the relationship between the newly described 10.4 kbp extension of the dit cluster in B K M E - 9 and the tdt cluster oi Pseudomonas diterpeniphila A19-6a. These two sequences encode highly similar proteins and share the same gene arrangement (Fig. 2.2). Sequence alignment of the deduced amino acid sequences from the tdt cluster with the corresponding putative homologues in the dit cluster showed 72% or greater amino acid identity. We hypothesize that the P450 and the putative thiolase, dehydrogenase, isomerase, hypothetical, regulator and CoA-ligase genes of the two organisms are functional homologues. Based on deduced amino acid sequence identity between TdtD and P450dj t,the latter would constitute a second member of the new P450 family proposed by Morgan and Wyndham (13). (Recently named CYP226A.) Sequence comparison of the dit cluster with the recently sequenced Burkholderia sp. LB400 genome suggests that LB400 also contains homologues of dit cluster genes. With the exception of ditE, coding for a putative permease of the major facilitator superfamily, every protein encoded by the dit cluster (including the 10.4 kbp extension) has a putative homologue in a 60 kbp region of the LB400 genome (Fig. 2.2). Further, most of the genes are in small groups that have the same gene order as their putative homologues in B K M E - 9 . An alignment of the deduced amino acid sequence shows high sequence identity between these deduced proteins of 62 B K M E - 9 and LB400. Preliminary results indicate that LB400 can grow on DhA as a sole organic substrate (unpublished data). We are currently testing the hypothesis that this 60-kbp region of the LB400 genome codes for proteins that are required for diterpenoid degradation. Interestingly, the genome sequence of LB400 provides additional evidence for the involvement of two P450s in diterpenoid metabolism. The above 60 kbp region in the LB400 genome includes two genes coding for putative cytochromes P450, BxeC0599 and BxeC0631 (Fig. 2.2), whose deduced protein products both have a high percent identity to P450djt, relative to other P450 homologues in the data bases (Fig. 2.2). Possibly one of the two genes codes for a P450dit homologue responsible for DhA/PaA degradation while a second codes for a second P450 responsible for A b A degradation. Other genes of interest in the 60-kbp region include (i) BxeC0579 with high sequence identity to ferredoxin reductase genes, (ii) BxeC0601, a ferredoxin gene homologue with similarity to those of P450 ferredoxins, and (iii) BxeC0612, a gene putatively coding for a methyl accepting chemotaxis protein. Additionally, the most highly conserved genes shared between the dit cluster and the 60kbp region of LB400 are the 2 encoding hypothetical proteins. High sequence conservation suggests that the gene products may perform an essential unknown function. Mutations are currently being generated in LB400 to investigate the functions of selected genes. Figure 2.1 shows a proposed pathway for abietane diterpenoid metabolism in BKME-9 . In this convergent scheme, PaA is transformed to DhA followed by hydroxylation at C-1 and further oxidation to form 7-oxo-DhA. This agrees with a previous reports on resin acid degradation (10, 11), which showed the requirement of a carbonyl group at C-7 for DitA dioxygenase activity and showed the accumulation of 7-oxo-DhA during growth of a ditAl knockout mutant on AbA, DhA or PaA. In accordance with the results of this study, AbA is transformed to 7-oxo-DhA without the formation of DhA. Possibly a P450, other than P450dj t, is involved in this transformation, as suggested by the LB400 genome analysis. We are confident 63 that DhA is the substrate for P450d,t, however at this time we have not characterized the product of this reaction. We hypothesize that the product is 7-hydroxy-dehydroabietic acid. r 64 2.6 References 1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped B L A S T and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389-402. 2. Ausubel, F. M. 1992. Short protocols in molecular biology, 2nd ed. Green Publishing Associates ; John Wiley & Sons, New York, N . Y . 3. Bicho, P. A., V. Martin, and J. N. Saddler. 1995. Growth, induction, and substrate specificity of dehydroabietic acid-degrading bacteria isolated from a kraft mill effluent enrichment. Appl Environ Microbiol 61:3245-50. 4. Clarke, A. R. 1996. Analysis of Ligand Binding by Enzymes, p. 199-221. In Enzymology Labfax. Academic Press Inc. and BIOS Scientific Publishers, San Diego. 5. Gerhardt, P. 1994. Methods for general and molecular bacteriology. American Society for Microbiology, Washington, D.C. 6. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557-80. 7. Hawkes, D. B., G. W. Adams, A. L. Burlingame, P. R. Ortiz de Montellano, and J. J. De Voss. 2002. Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization. J Biol Chem 277:27725-32. 8. Hoang, H. P. S. a. T. T. 1995. An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158:15-22. 9. Jefcoate, C. R. 1978. Measurement of substrate and inhibitor binding to microsomal cytochrome P-450 by optical-difference spectroscopy. Methods Enzymol 52:258-79. 10. Martin, V. J., and W. W. Mohn. 2000. Genetic investigation of the catabolic pathway for degradation of abietane diterpenoids by Pseudomonas abietaniphila BKME-9 . J Bacterid 182:3784-93. 11. Martin, V. J., and W. W. Mohn. 1999. A novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium Pseudomonas abietaniphila B K M E - 9 . J Bacteriol 181:2675-82. 12. Mohn, W. W. 1995. Bacteria obtained from a sequencing batch reactor that are capable of growth on dehydroabietic acid. Appl Environ Microbiol 61:2145-50. 13. Morgan, C. A., and R. C. Wyndham. 2002. Characterization of tdt genes for the degradation of tricyclic diterpenes by Pseudomonas diterpeniphila A19-6a. Can J Microbiol 48:49-59. 14. Peterson, J. A., and Y . Ishimura. 1971. Reaction mechanism of bacterial cytochrome P-450-catalyzed camphor methylene hydroxylation. Chem Biol Interact 3:300-2. 65 15. Peterson, J. A . , J. Y. Lu, J. Geisselsoder, S. Graham-Lorence, C. Carmona, F. Witney, and M. C. Lorence. 1992. Cytochrome P-450terp. Isolation and purification of the protein and cloning and sequencing of its operon. J Biol Chem 267:14193-203. 16. Schweizer, H. P., and T. T. Hoang. 1995. An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158:15-22. 17. Simons, R., U. Priefer, and A . Punier. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Techonology 1:784-790. 18. Smith, P. K., R. I. Krohn, G. T. Hermanson, A . K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N . M. Goeke, B. J. Olson, and D. C. Klenk. 1985. Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85. 19. Stassi, D., S. Donadio, M. J. Staver, and L. Katz. 1993. Identification of a Saccharopolyspora erythraea gene required for the final hydroxylation step in erythromycin biosynthesis. J Bacteriol 175:182-9. 20. Unger, B., I. Gunsalus, and S. Sligar. 1986. Nucleotide sequence of the Pseudomonas putida cytochrome P-450cam gene and its expression in Escherichia coli. J. Biol. Chem. 261:1158-1163. 21. Wust, M., and R. B. Croteau. 2002. Hydroxylation of specifically deuterated limonene enantiomers by cytochrome P450 limonene-6-hydroxylase reveals the mechanism of multiple product formation. Biochemistry 41:1820-7. 22. Yanisch-Perron, C , J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103-19 23. Zon, L. I., D. M. Dorfman, and S. H. Orkin. 1989. The polymerase chain reaction colony miniprep. Biotechniques 7:696-8. 66 3. The LB400 dit Cluster 1 3.1 Introduction Bacteria of the genus Burkholderia have been isolated throughout the environment and have significant interactions with both plants and animals. The ability to colonize both plant and animal tissue gives this group of bacteria interesting ecological niches. In humans, bacteria comprising the Burkholderia cepacia complex (Bcc) have been associated with respiratory tract 5 infections in patients with cystic fibrosis (8). In plants, the relationship can be beneficial, preventing disease and contamination or promoting growth, nodule formation or nitrogen fixation (7), or deleterious, causing disease (28). However, the likely role for the majority of Burkholderia species is a non-pathogenic interaction with plant rhizospheres (8). Burkholderia xenovorans LB400 was isolated from a PCB-contaminated landfill in New 10 York State (15). The genome of B. xenovorans LB400 was recently sequenced and annotated (5). This large bacterial genome containing -9000 coding sequences is 9.7 Mbp and is comprised of three replicons - chromosome 1 (4.87 Mbp), chromosome 2 (3.36 Mbp) and a megaplasmid (1.47 Mbp). Functional genomics of LB400 have focused on biphenyl, benzoate, and CI metabolic pathways (10-12). During the course of research summarized in Chapter 2, a cluster of 15 genes in LB400 encoding proteins with high sequence identity to those encoded by the Pseudomonas abietaniphila B K M E - 9 dit cluster, was discovered. Other bacteria from the genus Burkholderia have been found to mineralize abietane diterpenoids (23), however, this is the first bacterium for which complete genomic information is available allowing for the first genomic investigation of abietane diterpenoid catabolism. 20 This chapter describes growth of LB400 on 4 abietane diterpenoids and changes in the LB400 dit cluster mRNA levels between cultures grown on DhA or succinate, using microarray transcriptomic analysis. The necessity of the LB400 dit cluster was established through mutation 1 A version of this chapter will be submitted for publication. 67 25 oiditAl encoding a ring-hydroxylating dioxygenase. Using analysis of metabolites in cell suspensions, we show the similarity between the degradation of abietane diterpenoids by LB400 and B K M E - 9 . 3.2 Methods and Materials Table 3.1 Strains and plasmids used in this study Genotype or Description Reference or Source 30 35 Strains Burkholderia xenovorans LB400 DitA1 KO Escherichia coli DH5a S17-1 Plasmids pEXIOOT pX1918G pDS2 pDS3 Wild type; grows on abietane diterpenoids (15) ditA1 ::accC1; Gm r This study endA1 hsdR17 (rk" mk') supE44 thiA recA1 gyrA (Nalr) Gibco BRL relA1 A(laclZYA-argF) U169 deoR (08OdlacA(lacZ)M15) recA pro thi hsdR with integrated RP4-2-TcMu::Kna::Tn7; (31) Tra+ Trr Sm r sacB conjugable plasmid for gene replacement; Ap r (30) xylE-accC1 fusion cassette-containing plasmid; Ap r Gm r (30) 1.8 kbp PCR amplicon containing LB400 ditA1 cloned into This study the unique Xmal site of pEXIOOT 1482-bp EcoR\/-Sma\ of pDS1 cloned into the Smal site This study ofpEXIOOT Bacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 3A.E. coli was cultured on Luria-Bertani (LB) medium and incubated at 37°C, and B. xenovorans strains were cultured at 30°C on L B without NaCl or K I mineral medium (11) supplemented with biphenyl vapors, 1 g per litre succinate, 90 mg per litre AbA, 90 mg per litre DhA, 90 mg per litre PaA, or 95 mg per litre 7-oxo-DhA. LB400 was streaked out from frozen stock on 1.5 % purified agar K I plates, which were inverted over a petri dish lid containing biphenyl crystals and incubated for 3-4 days. D i tAIKO was streaked out as above or on 1.5 % agar L B without NaCl plates containing 10 mg/ml of gentamycin and incubated for 2-3 days. Liquid cultures were incubated on a rotary shaker at 200-250 rpm. 68 40 Growth was monitored by analysis of optical density (OD) or protein concentration. Protein concentrations were determined using the micro-bicinchoninic acid (BCA) protein assay kit (Sigma) as described in Chapter 2 (pg. 48). 1 litre K I was prepared by combining 100 ml of 10X K I stock with 20 ml of Hunter mix and adding autoclaved distilled and 880 ml deionized H 2 O (ddH 20) for liquid medium or ddH 2 0 plus 1.5% purified agar (Becton Dickinson, Cockeysville, 45 MD) for plates. 10X K I stock was prepared by adding 31.8 g K 2 H P 0 4 , 5 g (TSIH4)2S04, and 3.54 g N a H 2 P 0 4 » H 2 0 to 700 ml of ddH 2 0 while stirring, followed by addition of ddH 2 0 to a final volume of 1 litre. The solution was then equally divided and transferred to 2-1 litre bottles and autoclaved for 30 minutes. After cooling 250 /xl (10 mg/ml in water) of the following vitamins, thiamine, nicotinic acid, inositol, and riboflavine were added to each 500-ml autoclaved portion. 50 In addition 25 jul of 2 mg/ml biotin in methanol was added to each 500-ml portion. Bottles were then wrapped with aluminium foil and stored at room temperature. 1 litre Hutner mix was prepared by adding 10 g of nitrilotriacetic acid to 600 ml of ddH 2 0. K O H pellets were then added slowly while stirring until all nitrilotriacetic acid had dissolved. Next 14.45 g of MgS0 4 »7H 2 0 , 3.33 g Ca(N0 3 ) 2 , 9.25 mg (NH 4 ) 6 Mo 7 0 2 4 «24H 2 0 , 99 mg FeS0 4 «7H 2 0 , and 50 55 ml of Metals 44 (see below), were combined with ddH 2 0 to 1 litre. The pH of the solution was adjusted to 6.6 to 6.8 and then the solution was equally divided and transferred to 3-500 ml bottles with screw caps and autoclaved for 30 minutes. Solutions were stored at room temperature. Metals 44 was prepared by combining l g EDTA, 4.4 g of Z n S 0 4 # 7 H 2 0 , 2 g FeS0 4 »7H 2 0 , 620 mg of MnS0 4 «H 2 0 , 119 mg of CuS0 4 «5H 2 0 , 99.4 mg of Co(N0 3 ) 2 «6H 2 0 60 and 70.8 mg of Na 2 B 4 0 7 «10H 2 O and adding ddH 2 0 to 400 ml, with the addition of a few drops of concentrated H 2 S 0 4 to dissolve, and autoclaved for 30 minutes and stored at room temperature. LB400 from frozen stock was initially grown on biphenyl vapors as a means of selection. For cultures used in transcriptomic analysis, LB400 colonies grown on biphenyl vapors were used to 69 65 inoculate 50 ml of K I containing 1 g per litre of succinate. After 24 hours the culture reached late log-phase (ODeoo ~ 0.9) and cells were transferred to fresh K I containing 1 g per litre of succinate at an initial OD600 of 0.001. After approximately 18 hours, this second succinate culture reached mid-log phase (OD600 0.68). For analysis of succinate-grown LB400 and R N A extraction, cells from the second succinate culture were used to inoculate 2 -200-ml cultures of 70 K I containing l g per litre succinate. From this point on, each 200-ml culture was treated separately and each represented a biological replicate. After 18 hours of incubation, cultures had reached mid-log phase (OD600 of 0.5) and cells were harvested as described below. For analysis of DhA-grown LB400 and R N A extraction, cells from a second succinate culture (as described above) were transferred to a K I medium containing 90 mg per litre of DhA 75 with an initial OD600 of 0.01. After approximately 140 hours, the culture had reached late-log phase (OD600 of 0.15) and cells were transferred to a fresh K I medium containing DhA at an initial OD of 0.001. After approximately 48 hours of incubation the culture had reached a mid- to late-log phase (ODgoo of 0.07 to 0.1). Cells from this second DhA culture were then used to inoculate 2 -300-ml K I cultures containing 90 mg per litre of DhA with an initial OD600 of 80 0.001. From this point on, each 300-ml culture was treated separately and each represented a biological replicate. After approximately 45 hours, each culture reached mid-log phase (OD600 0.065 and 0.057) and cells were harvested. Harvesting cells. Plate counts revealed that each succinate-grown culture contained approximately 108 colony-forming units (CFU)/ml while DhA-grown cultures contained 85 approximately 10 CFU/ml of culture. Several aliquots of approximately 10 cells were collected for R N A extraction from each culture (10 ml of succinate-grown culture and 100 ml of DhA-grown culture). Cells were harvested as follows: immediately after incubation was halted, cultures were cooled on ice and an amount equal to 10% of the volume of 5% phenol in ethanol was added and mixed by inversion. Cells were harvested by eentrifugation at 8800 rpm at 4°C 70 90 for 10 min. One ml of supernatant from each culture was removed and transferred to a 1.7 ml Eppendorf tube. The remainder of the supernatant was decanted, and the pellets of succinate- or DhA-grown cells were suspended in 10 or 100 pi, respectively, of the supernatant (removed in the last step) and 10 or 100 pi of R N A later (Qiagen). The cell suspensions were then transferred to 1.7 ml Eppendorf tubes, frozen in liquid nitrogen and stored at -80°C. 95 R N A Extraction. Each sample for R N A extraction was subdivided into 5 aliquots to optimize R N A extraction using the RNAeasy R N A extraction kit (Qiagen) as per manufacture's instructions. Nucleic acid was eluted from the column using 35 pi of RNase-free water two times. Nucleic acid content was quantified by U V spectroscopy. Following R N A extraction, samples were combined and D N A was removed by treatment with DNase I (Roche) as per 100 manufacture's instructions for 30 min at room temperature. The sample was then phenol/chloroform extracted and centrifuged for 5 min at 6000 rpm. The water phase was recovered and 1/10 volume of 3M NaOAc (pH5.2 Sigma) was added, mixed and supplemented with 1 volume of cold (4°C) isopropanol (Sigma). After thorough mixing by inversion, the tube was centrifuged for 20 min at 4°C and the supernatant was removed. The pellet was then washed 105 with 80% ethanol followed by a 20-min eentrifugation at 4°C. The supernatant was then removed and allowed to air dry for ~5 min. The pellet was then suspended in 35 pi of nuclease-free water and heated at 50°C for 5-10 min to dissolve RNA. U V spectroscopy was used to determine R N A quantity and purity. R N A Aminoallyl labelling. To generate cDNA with incorporated aminoallyl dUTP, 2 pg of 110 R N A plus 2 pi of Random Hexamers (Invitrogen) (3 mg/ml) were combined and adjusted to a final volume of 15 pi with RNase-free water. The solution was mixed well and incubated at 70°C for 10 minutes and then placed immediately on ice. Next, 6 pi of 5X first strand buffer, 3 pi of 0.1 M DTT, 1.2 pi of 25X aminoallyl-dNTP mix (5 pi of 100 m M dATP, 5 pi of 100 m M dCTP, 5 ul 100 m M of dGTP, 3 ul 100 m M of dTTP, plus 2 ul 100 m M of aminoallyl-dUTP), 2 ul of 115 Superscript II RT (Invitrogen) (200 U/ul) and 2.8 ul of Mi l l iQ water were combined and incubated at 42°C for 3 hours. To hydrolyze RNA, 10 ul 1 M NaOH, and 10 ul of 0.5 ul EDTA, were combined and mixed and incubated at 65°C for 15 minutes. To neutralize pH, 25 ul 1 M Tris (pH 7.4) or 10 ul of 1 M HC1 was added. To remove unincorporated aa-dUTP and free amines, a modified Qiagen Qiaquick PCR purification kit protocol was used. A phosphate wash 120 buffer and elution buffer (EB) were substituted for the Qiagen supplied buffer, because Qiagen buffers contain free amines, which compete with the Cy-dye coupling reaction. One hundred ml of phosphate buffer (5 m M KPO4, pH 8.0, 80% ethanol) was prepared by mixing 0.5 ml 1 M KPO4, 15.25 ml of Mil l iQ water, and 84.25 ml 95% ethanol. Phosphate EB was prepared by diluting 1 M K P 0 4 (pH 8.5) to 4.0 m M with Mil l iQ water. The cDNA was eluted twice with 30 125 ul of phosphate EB. The sample was then dried in a speed vacuum system. Coupling of Cy ester to aminoallyl-labeled c D N A . The cDNA was suspended in 4.5 ul of 0.1 M sodium carbonate buffer (Na2C03), pH 9.0. 4.5 ul of the appropriate NHS-ester Cy dye (prepared as per manufacturer's instructions in DMSO) and incubated 1 hour in the dark at room temperature. Uncoupled dye was removed using the Qiagen Qiaquick PCR purification kit 130 protocol and eluted twice with 30 ul of EB. The quantity of labelled cDNA and the fluorophore incorporation efficiency were determined by using UV-visible spectrophotometry. Cy3/Cy5 labelled probes were dried in a speed vac and stored at -80°C. Hybridization. Hybridization and scanning were conducted at Michigan State University in the lab of Dr. J. Tiedje using microarray technology developed by Xeotron Technologies (14) and 135 chips described in (10) and (5). Two biological replicates were used for hybridizations. cDNA generated from LB400 grown on DhA was labelled with Cy3 in one hybridization and Cy5 in the other and visa versa for LB400 grown on succinate^ The LB400 array comprises 2 chips 72 containing a total of-16000 probes. These represent -8450 CDS's of the -9000 in the current annotation. Briefly, hybridizations were conducted in a Xeotron M-2 micro fluidic hybridization 140 station as per manufacturer's instructions. A l l buffers were passed through a 0.22 micron filter (Corning Costar Corporation, Cambridge, Massachusetts) prior to hybridization to minimize -particulate matter interference with the chips. Two hundred pmol of labelled cDNA per dye per chip were used for hybridization. Labelled cDNA was suspended in 100 pi of hybridization mix containing 33 pi of 18X SSPE (pH 6.6), 25 pi of 100% formamide, 4 pi of 10% Triton-XlOO, 145 plus labelled cDNA and nuclease-free water to 100 pi. Samples were heated at 95°C for 3 minutes, placed immediately on ice for 1 min and then filtered through 0.22 micron filters as above. Samples were hybridized at 32°C at a flow rate of 300 pl/minute for 18 hours. Scanning and data analysis. Microarray scanning and data analysis were conducted as previously reported (10). Briefly, microarrays were scanned using an Axon 4000B (Axon 150 Instruments) scanner and data files were extracted using Genepix 5.0 (Axon Instruments). Data from two genomic sub-chips were merged and median signal intensity for both 635-nm (Cy5) and 532-nm (Cy3) were imported into Genespring 7.0 (Silicon Genetics) and normalized using Lowess intensity-dependent normalization. GeneSpring's one sample Student Mest algorithm was used to test whether the mean log2 ratio (DhA/Succinate) values of biological replicates 155 were significantly different from zero. In general, a gene transcript was considered up-regulated when the log2 ratio was greater than one and thep value was less than 0.05, and, was considered down-regulated when the log2 ratio was less than 0.5 and the p value was less than 0.05. Values M. [M— log2(Cy5/Cy3)] versus A [A = l o & C v / W x Cl/3)] were determined for each biological replicate according to the method of Dudoit et al. (13) and plotted to assess normalized data 160 quality. 73 Cell suspensions. LB400 or Di tAIKO colonies were used to inoculate 100 ml of K I containing 1 g per litre succinate. When the OD600 reached between 0.4 and 0.8, cells were transferred to 100 ml of fresh K I medium containing 1 g per litre succinate with an initial OD600 of 0.001 and incubated as above. When the OD600 reached between 0.3 and 0.4, cells were transferred to 1 165 litre of K I containing 1 g per litre of succinate for an initial OD600 of 0.001 in a 2-litre Erlenmeyer flask. When the OD 6 0o of these cultures' reached 0.4-0.6, cells were harvested by centrifugation for 10 minutes at 8800 X g, washed with K I salts containing no vitamins or Hunter mix, and suspended at an OD of ~ 3.0 in K I medium. The culture was then divided into 4 equal samples of approximately 50 ml each and 90 mg per litre AbA, 90 mg per litre DhA, 90 170 mg per litre PaA, or 95 mg per litre 7-oxo-DhA was added to each culture. Aliquots of 1.5 ml were collected at various time points and after addition of 2-3 drops of I N HC1 samples were frozen at -20°C for later analysis by gas chromatography-mass spectrometry (GCMS). Cell suspension assays were conducted in duplicated with representative curves presented in the results (Fig 3.2). 175 After thawing samples, an internal standard of 12,14-dichlorodehydroabietic acid was added to each 1.5-ml aliquot to a final concentration of 50 uM and samples were extracted twice with equal volumes of ethyl acetate and dried over anhydrous Na2S04. Samples were derivatized using diazomethane. GC electron impact (EI) mass spectrometry of methyl ester derivatives was conducted as previously described in Chapter 1, using an Agilent Technologies 6890N Network 180 GC system equipped with an Agilent 5973 Mass Selective Detector. National Institute of Standards and Technology MS Search (2.0) was used to analyze mass spectral data. Disruption of ditAl. LB400 genomic D N A was used as the template in a PCR with primers PI (5 ' -ATA G C C C G G G A A C A G TTG CGC C T A CCT G A A G-3') and P4 (5'-TTA G C C C G G G T A T A G A T C A G G TCC TCC GCA-3') , both containing 5' Xmal restriction site extensions 185 (underlined), to amplify a 1797-bp fragment containing ditAl. The resulting amplicon was ( digested with Xmal and then ligated to the unique dephosphorylated Xmal site of pEXIOOT, containing the sacB counter selectable marker, and used to transform E. coli DH5oc. Electroporation and selection of white colonies were used as described in Chapter 2. Plasmid D N A was then extracted with QIAprep Spin Miniprep columns as above, digested with Sphl, , 190 and run on a 0.7 % agarose gel to identify clones containing inserts with the correct orientation. The successful ligation was designated pDS2. Next, the xylE-accCl transcriptional fusion antibiotic cassette of pX1918G was amplified using primer CI (5 '-TAG G C G CGC C G A G A G C A C CGC GAT C A A GGA-3') containing a 5' Ascl restriction site extension (underlined) and C2 (5'-CAT G A A T T C C G A ATT C C G ATC CGT C G A GA-3') containing a 5'EcoRl 195 restriction site extension (underlined). The resulting 2.1-kbp amplicon and pDS2 were digested with EcoRl and Ascl. Digested pDS2 was run on a 1.0% agarose gel and a 6939-bp fragment was extracted from the gel as described in Chapter 2. The 2.1-kbp amplicon and 6.9-kbp pDS2 fragment were ligated to generate pDS3 and this was used to transform, by electroporation, mobilization strain Escherichia coli SI7-1. Successful transformants were selected as described 200 in Chapter 2. Homologous recombination of the mutated allele into strain LB400 was accomplished by diparental conjugation using a filter membrane essentially as described in (9). In general SI7-1 containing pDS3 and LB400 were grown overnight on L B with 10 mg/ml gentamycin and L B (-NaCl), respectively. Fresh L B with 10 mg/ml gentamycin and L B (-NaCl) were inoculated (10%) with the respective overnight cultures and the new cultures were grown 205 until the OD6oo reached 1-1.5. One ml of the donor cells S17-1 containing pDS3 was washed 2 times with L B and suspended in 1 ml of L B . Next, 100 ul of donor cells and 100 ul of recipient cells were suspended in 0.8% sterile saline and vortexed for 10 seconds at medium speed. The mating mixture was then transferred to a 5-ml syringe and filtered with a 0.22 uM cellulose filter (Millipore G V type) in a reusable filter case. The filter was then removed with forceps and 210 placed with the cells on the upper face on an L B (-NaCl) plate and incubated for ~ 24 hours at 75 30°C. The filter was then removed with forceps and washed by vortexing in 5 ml of sterile saline, which was then plated on K I purified 1.5% agar plates supplemented with 1 g per litre pyruvate plus 10 mg/ml gentamycin, followed by a two step selection method as previously described (20) and Chapter 2. Homologous recombination was confirmed by PCR amplification 215 of a 3350-kb fragment using primers PI and P4 at an annealing temperature of 56.3°C and identified on an agarose gel. The lack of a 1.7-kbp band, and gentamycin resistance further confirmed the desired construct. The 3350-kbp fragment was also sequenced using primers PI and P4, and the resulting sequence was compared to the expected insertion sequence. 76 3.3 Results 220 Table 3.2 Growth characteristics of LB400 on four abietane diterpenoids 7-oxo-DhA n=4 DhA n=4 AbA n=3 PaA n=3 Lag phase (hours) 121 (4) 134 (6) 258 (21) 248 (5) Doubling time (hours) 23 (4) 17(1) 37 (2) 19 (1) Maximum Protein Concentration (ug /ml culture) 18.3(1.2) 25.5 (0.9) 10.9(1.3) 26.0 (1.1) Growth Yield g of protein/ g of substrate 0.19 (0.01) 0.28 (0.01) 0.12 (0.01) 0.29 (0.01) Initial inoculum for each culture was ~2 x 106 cells/ml grown to mid-log phase on succinate. Numbers in brackets indicate standard error. A l l growth was carried out in K I mineral salt media with an initial abietane diterpenoids concentration of 300 pM. 225 3.3.1 LB400 growth on abietane diterpenoids The genome of B xenovorans LB400 was found to contain a large cluster of genes with high similarity to those encoding abietane diterpenoid catabolism by P. abietaniphila B K M E - 9 230 (32). To determine i f LB400 can catabolize diterpenoids, growth assays were conducted. LB400 grew on the abietane diterpenoids, AbA, DhA, PaA, or 7-oxo-DhA as sole sources of carbon and energy (Fig. 3.1, Table 3.2, see Fig. 3.5 for chemical structures). LB400 failed to grow on the pimerane diterpenoids, isopimaric acid and pimaric acid. Initial lag phases preceding growth on the abietanes were long and variable. The lag phase was shortest on the more soluble, aromatic 235 compounds, 7-oxo-DhA and DhA and longer on the less-soluble, non-aromatic compounds, AbA and PaA. While the lag phase on PaA was almost double of that on DhA, the doubling times and yields were similar on the two compounds. 7-Oxo-DhA and A b A supported much lower maximum final protein concentrations and growth yields than DhA or PaA. Growth on A b A and DhA was confirmed by monitoring substrate removal and protein increase. 77 CD O c '(D -i—> o CL CO 100 150 200 250 Time (hours) 300 240 Figure 3.1 Growth curves of LB400 on four abietane diterpenoids. Initial inoculum for each culture was ~2 x 106 cells/ml grown to mid-log phase on succinate. Data are . representative growth curves (n= 3-4). A l l growth was carried out in K I mineral salt media with an initial abietane diterpenoid concentration of 300 uM. Symbols, LB400 growth on 0 - AbA, • - DhA, A - PaA, and O.- 7-oxoDhA. Inset: Substrate removal during growth. Symbols, • - A b A , 245 • -DhA. Rates of LB400 growth coincided with maximum rates of substrate removal, and entry into stationary phase coincided with complete removal of the substrates (Fig. 3.1). Inocula for initial LB400 growth assays on abietane diterpenoids were grown on 250 succinate as described in the Materials and Methods. When cells grown on DhA were transferred to fresh medium with DhA, the lag phase was reduced from over 130 hours to less than 24 hours, and the doubling time was reduced from 17 to approximately 8 hours. Further transfers on DhA resulted in minimal change in growth kinetics. A similar effect was observed with serial transfers on AbA, PaA and 7-oxo-DhA. To confirm that the LB400 dit cluster contains genes required for 255 abietane degradation we generated a knockout of the gene encoding the alpha-subunit of the 78 ring-hydroxylating dioxygenase, ditAl, producing strain D i t A l K O . During 400 hours of incubation D i t A l K O did not grow on AbA, DhA, PaA or 7-oxo-DhA as sole organic substrates. 3.3.2 Metabolic analysis of LB400 and DitAlKO cell suspensions Cell suspensions of LB400 removed AbA, DhA, PaA or 7-oxo-DhA completely; 260 whereas, D i t A l K O only slightly reduced concentrations of the substrates (Fig. 3.2). As previously reported (22), boiling the cells prior to incubation, nearly abolished removal of LB400 DitAl KO 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Time (hours) Figure 3.2 Cell suspensions of LB400 and DitAlKO with AbA, DhA, and PaA. A . AbA cell suspension, B. DhA cell suspension, C. PaA cell suspension. Left hand charts correspond to LB400 and right hand charts are D i t A l K O . Open symbols indicate values corresponding to the 265 left y-axis and open or stick symbols indicate values corresponding to the right y-axis. Symbols, • -AbA, • and n-DhA, • - PaA, O - 7-oxoDhA, +- dimethyl heptanedioic acid, and X -Unknown I. 79 abietane diterpenoids; however, some initial removal (< 20%) was detected. In these cases no metabolites were formed, therefore removal was likely the result of sorption of diterpenoids to 270 culture tubes or to the cell debris. 7-Oxo-DhA was detected in both LB400 and D i t A l K O cell suspensions. LB400 transiently accumulated small amounts of 7-oxo-DhA during incubations with AbA, DhA, or PaA (Fig 3.2). A l l 7-oxo-DhA was completely removed within 144 hours by LB400 in all cases (not shown). D i t A l K O increased 7-oxo-DhA concentrations during incubations with AbA, PaA, 275 and particularly with DhA, but did not remove it, even after 144 hours of incubation. A second peak, observed only in LB400 cell suspensions, was identified as 2,4-dimethyl heptanedioic acid (DMHDA) by a NIST library search. LB400 accumulated small amounts of D M H D A in all cases and did not remove the accumulated D M H D A by 144 hours. Other metabolites were detected in both LB400 and Di tAIKO cell suspensions on AbA, DhA, or PaA. 280 A peak with a retention time relative to the internal standard DiCl DhA of 0.96 (Unknown I) accumulated in both LB400 and Di tAIKO cell suspensions with A b A and PaA but not on DhA. Unknown I could not be identified using a NIST library search of the MS spectrum. The 5 largest peaks of the MS spectrum are 255, 330, 256, 163, and 271 with the molecular ion at 330. Comparison of the mass spectrum of 7oxo-DhA methyl ester (MW 328) with that of the 285 Unknown I (m/z 330) showed similar fragmentation patterns differing by an m/z of 2 (not shown), suggesting structural similarity. Major MS peaks from Unknown I correspond to loss of the methyl group of the methyl ester (-CH 3) (315/313), the carboxyl group (-COO) (271/269) and the ketone ( - 0 ) (255/253). In PaA cell suspensions (Fig 3.2 C), DhA present as a contaminant in the initial substrate 290 mixture decreased slightly during incubation with LB400 for 6 hours, but after this time, the DhA concentration increased and reached a maximum level at 72 hours. By 96 hours all accumulated DhA was removed by LB400 (not shown). In PaA cell suspensions, Di tAIKO 80 increased the DhA concentration without an initial detected decrease for 72 hours. The DhA concentration then remained steady for at least 144 hours. The DhA concentration did not 295 increase during incubation of boiled cell controls for 144 hours, indicating that this transformation from PaA to DhA is enzymatic. In 7-oxo-DhA cell suspensions, the substrate was removed within 24 hours by LB400 and not removed by the D i t A l K O ; however, the 7-oxo-DhA concentration did initially decrease (data not shown). 3.3.3 T r a n s c r i p t o m i c analysis 300 Changes in gene regulation between LB400 growing exponentially on DhA or succinate were monitored by analysis of differences in mRNA levels using microarray transcriptomic analysis. Normalized data quality was evaluated using M-versus-A plot of the combined genomic chip set (Fig. 3.3), which indicated the absence of signal intensity-dependent bias. 5-. 4 -3 -2 -1 -0--1 -- 2 -- 3 --4--5-6 ° o ° 0 ° 0 8 1 0 12 14 16 305 F i g u r e 3.3 P lots o f M versus A o f the c o m b i n e d genomic c h i p set a f te r Lowess i n tens i t y d e p e n d e n t n o r m a l i z a t i o n . M= log 2(DhA signal intensity/Succinate signal intensity, A = log2(A/DhA signal intensity x Succinate signal intensity) 81 Comparison of raw and normalized signal intensity revealed that although the growth rate of 310 LB400 on succinate was substantially higher than that on DhA, R N A expression levels for several housekeeping genes showed only minor variations and therefore did not have an effect on overall transcriptomic analysis. 3.3.3.1 Dit cluster Of the 72 genes from BxeC0578 to BxeC0649, 43 are up regulated, with a > 2 fold 315 increase (p < 0.05) in expression, during growth.on DhA versus on succinate (Fig. 3.4). This cluster, found on the LB400 megaplasmid, has been named the LB400 dit cluster for diterpenoid degradation encoding genes. (Fig. 3.4, Table 3.3). Based on similar expression levels, the same gene orientation and proximity, it is possible to predict several putative mRNA transcripts up-regulated during growth on DhA including, BxeC0583-BxeC0585, BxeC0586-BxeC0587, 320 BxeC0590, BxeC0591, BxeC0592, BxeC0594-BxeC0597, BxeC0599, BxeC0600-BxeC0606, BxeC0609-BxeC0610, BxeC0616-BxeC0620, BxeC0621-BxeC0623, BxeC0624-BxeC0627, BxeC0638-BxeC0640, BxeC0641-BxeC0648. The most highly induced genes during growth on DhA versus succinate were BxeC0605 (encoding a putative acyl Co A dehydrogenase), BxeC0606 (encoding a conserved hypothetical protein), BxeC0642 (encoding a putative 325 dehydrogenase), and BxeC0643 (encoding a putative enoyl CoA hydratase/isomerase). 82 o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O (U(DO(D00a)<i>(i)<D<u(U(U(i)(i)<D(D(i)o<U(i}a}(ua)Q)(UQ}<u<i)<U(U(U<ua)oa} x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x [ i i f l i c Q C Q a i i i i a j i i i f f l a i a i c Q i a m a i f f l f l i t D f f l c D C D f f l i D f f l c Q C Q c i i c D f f l f f i f f i f f l i i i i i i i i i f f l / • V Figure 3.4 A. Plot shows M values (LOG2(DhA/Succinate) for signal intensities for genes of the LB400 dit cluster. Error bars indicate standard error. B. Physical map of the LB400 dit cluster of genes. The 3 rows of arrows represent one contiguous cluster of genes and show the 330 orientation of each gene. Grey arrows represent genes with > 2-fold up-regulation during growth on DhA (p < 0.05) compared to growth on succinate. 83 Table 3.3 Dit cluster annotation and gene expression on DhA G e n e I D G e n e N a m e P r o d u c t d e s c r i p t i o n C O G F o l d u p - p - V a l u e F u n c t i o n a l r e g u l a t i o n o n G r o u p D h A BxeC0578 Putative Transcriptional Regulator of the K 1.77 0.002 TetR/AcrR Family BxEC0579 Putative Pyridine nucleotide-disulphide R 6.40 0.066 oxidoreductase BxeC0580 ORF2 Putative Transporter of the Major Facilitator GEPR 1.52 0.004 Superfamily BxeC0581 ditF Conserved hypothetical R 4.35 1E-04 BxeC0582 Conserved hypothetical 1 2.03 0.002 BxeC0583 ditG Putative dehydrogenase QR 5.10 2E-05 BxeC0584 Putative Hemerythrin P 6.78 2E-04 BxeC0585 ditH Putative Fumarylacetoacetate Hydrolase Q 6.76 1E-04 BxeC0586 ditAl Ring-hydroxylating dioxygenase alpha PR 2.93 0.131 subunit BxeC0587 ditA2 Ring-hydroxylating dioxygenase beta subunit PR 2.93 3E-04 BxeC0588 Putative Glyoxalase Family Protein E 1.68 0.040 BxeC0589 Putative Short Chain Dehydrogenase QR 1.13 0.23 BxeC0590 Conserved hypothetical No COG 2.28 7e-04 BxeC0591 ditl Putative Short Chain Dehydrogenase QR 5.31 3e-04 BxeC0592 ditJ Putative CoA Ligase IQ 7.43 3e-04 BxeC0593 ditK PutativeTranscriptional Regulator, TetR K 1.60 0.002 family BxeC0594 Conserved hypothetical R 2.08 1E-04 BxeC0595 ditM Putative Fumarylacetoacetate Hydrolase Q 8.20 0.020 BxeC0596 ditN Putative 3-hydroxyacyl CoA Dehydrogenase 1 7.43 0.064 BxeC0597 ditO Putative Thiolase 1 6.53 0.029 BxeC0598 ditP Conserved hypothetical No COG 0.99 0.955 BxeC0599 ditQ Cytochrome P450 Q 2.43 3E-05 BxeC0600 Putative CaiB/BaiF family protein C 3.95 0.008 BxeC0601 Putative Ferredoxin C 2.48 0.163 BxeC0602 Putative Acyl CoA Dehydrogenase 1 3.35 0.006 BxeC0603 Putative Enoyl CoA Hydratase 1 3.31 2E-04 BxeC0604 Putative Acyl CoA Dehydrogenase 1 6.70 0.030 BxeC0605 Putative Acyl CoA Dehydrogenase 1 17.4 0.001 BxeC0606 Conserved hypothetical No COG 23.4 9E-06 BxeC0607 ditR Transcriptional Regulator (IcLR family) K 1.72 0.004 BxeC0608 ditD Putative Fumarylacetoacetate Hydrolase Q 1.23 0.182 BxeC0609 Putative MFS transporter GEPR 3.61 6E-04 BxeC0610 Putative short chain dehydrogenase QR 10.8 4E-04 BxeC0611 Hypothetical No COG 1.13 0.255 BxeC0612 Putative Methyl-Accepting Chemotaxis N •1.43 0.022 Protein BxeC0613 Putative Transcriptional Regulator of LysR K 1.286 0.002 Family BxeC0614 Putative Oxidoreductase C 2.11 0.169 BxeC0615 Putative Transporter of the Major Facilitator E 1.70 0.005 Superfamily BxeC0616 Putative Dehydrogenase QR 2.98 2E-05 BxeC0617 Putative Acyl CoA Dehydrogenase 1 3.16 2E-04 BxeC0618 Putative Phosphotransferase R 3.17 3E-05 BxeC0619 Putative Short chain dehydrogenase QR 2.85 2E-05 BxeC0620 Putative Dehydrogenase QR 3.76 3E-04 BxeC0621 Conserved hypothetical No COG 6.73 4E-05 BxeC0622 Putative Rieske Iron Sulphur Protein PR 2.73 0.003 Gene ID Gene Name Product description COG Fold up- p-Value Functional regulation on Group DhA BxeC0623 Conserved hypothetical PR 3.50 3E-04 BxeC0624 Conserved hypothetical I 1.98 0.012 BxeC0625 Conserved hypothetical Q 1.78 1E-03 BxeC0626 Putative Methyl Transferase H 2.28 0.002 BxeC0627 Conserved hypothetical E 2.57 0.011 BxeC0628 Putative Transcriptional Regulator of K 1.10 0.251 TetR/AcrR Family BxeC0629 Conserved hypothetical No COG 1.04 0.458 BxeC0630 Conserved hypothetical No COG 0.99 0.857 BxeC0631 ditU Cytochrome P450 Q 1.11 0.241 BxeC0632 Putative AraC type transcriptional regulator K 1.24 0.123 BxeC0633 Conserved hypothetical No COG 1.34 0.004 BxeC0634 Conserved hypothetical No COG 1.46 0.201 BxeC0635 Putative Dehydrogenase QR 1.23 0.135 BxeC0636 Putative Transcriptional Regulator of the K 1.07 0.442 TetR/AcrR Family BxeC0637 Conserved hypothetical Q 1.16 0.729 BxeC0588 Hypothetical No COG ND ND BxeC0638 ditA3 Ferredoxin C 7.38 1E-04 BxeC0639 ditB Putative Dehydrogenase QR 5.10 2E-04 BxeC0640 ditC Aromatic Ring Cleavage Dioxygenase No COG 5.60 7E-04 BxeC0641 Conserved hypothetical R(l) 3.15 3E-04 BxeC0642 Putative Dehydrogenase QR 21.2 2E-09 BxeC0643 Putative Enoyl CoA Hydratase/lsomerase I 18.4 0.035 BxeC0644 Conserved hypothetical E 9.15 0.032 BxeC0645 Putative Transporter of the RND Superfamily R 7.34 2E-04 BxeC0646 Conserved hypothetical No COG 7.88 1E-04 . BxeC0647 Conserved hypothetical No COG 5.54 0.003 BxeC0648 Conserved hypothetical No COG 6.38 2E-04 BxeC0649 Putative Transcriptional Regulator of the K 1.12 0.358 TetR/AcrR Family 335 3.3.3.2 Global analysis - COG distribution 3.3.3.2.1 Up-regulated genes There were 97 up-regulated genes with greater than 2-fold increase in signal intensity (p . < 0.05) on DhA compared to on succinate (Table 3.4, Supplementary Table 3.1 pg. 98). A l l genes encoding proteins of the secondary metabolite biosynthesis, transport, and catabolism 340 COG functional group (COG Q) are encoded in the dit cluster (Fig. 3.4, Table 3.3). The COG functional group containing the greatest number of up-regulated genes (16) was involved in lipid metabolism (COG I), ten of which are encoded by the dit cluster. 85 Table 3.4 Summary of transcriptional analysis based on COG protein classification Up-regulated Down-regulated COG _. . ,. genes genes D e s c r | P t l o n (97 total) (39 total) Information Storage and Transport K Transcription 1 3 L DNA replication, recombination and repair 1 1 Cellular Processes M Cell envelope biogenesis, outer membrane 4 1 N Cell motility and secretion 0 4 T Signal transduction mechanisms 0 4 Metabolism C Energy Production and Conversion 8 5 E Amino acid transport and metabolism 8 3 F Nucleotide transport and metabolism 0 1 G Carbohydrate transport and metabolism 3 1 H Coenzyme metabolism 2 1 I Lipid metabolism 16 0 P Inorganic ion transport and metabolism 11 2 Q Secondary metabolite biosynthesis, transport, and . , Q catabolism Poorly Characterized R General function prediction only 8 5 S Function unknown 1 0 None No match 23 345 Values are the number of up- or down-regulated [> 2X change in expression (t-test, p-value < 0.05)] genes based on transcriptional analysis of DhA-grown cells compared to succinate-grown cells. Genes up-regulated on DhA involved in energy production and conversion (COG C) 350 include genes BxeB2301 and BxeB2302, which are embedded in a cluster of genes involved in citrate/aconitate metabolism. On DhA, gene BxeB2301, encoding an aconitate hydratase, was up-regulated 3.1-fold (p = 0.019) and BxeB2302, encoding a 2-methyl citrate synthase, was up-regulated greater than 10-fold (p = 0.042). Gene BxeB2300 in this citrate/aconitate metabolism cluster was also up-regulated but is included in the COG S group of unknown functions. Two 355 clusters of genes up-regulated on DhA encode several proteins putatively involved in inorganic ion transport and metabolism (COG P). Genes BxeA2467, BxeA2469 and BxeA2470 (COG P) are putatively involved in sulphate uptake. Genes BxeA3659 (COG H), BxeA3660 (COG P), BxeA3663 (no COG), BxeA3664 (COG P), and BxeA3671 (COG R) encode proteins putatively involved in sulphate metabolism. The relationship between sulfate and abietane diterpenoid 86 360 metabolism is unclear. Another COG P protein, BxeA3458, is a putative catalase, which likely is up-regulated to detoxify byproducts of uncoupled oxygenase activity. The other up-regulated genes in the COG P group are involved in phosphate, iron, or oxygen metabolism. Four of the eight up-regulated genes encoding proteins involved in amino acid transport and metabolism (COG E) are located in a single cluster of genes, BxeB2702, BxeB2704, 365 BxeB2705, and BxeB2706. Also included in this cluster is a COG C group member, BxeB2708, encoding a putative class II pyridine nucleotide-disulphide oxidoreductase. Genes BxeB2702-BxeB2705 encode a putative A B C transport system. Genes BxeB2705-BxeB2706 have similarity to those encoding A and B subunits of an opine oxidase. Genes BxeB2702-BxeB2709 are conserved in Bradyrhizobium japonicum, with deduced amino acid identity of 48% to 56% 370 and identical gene order. In combination, the transport genes and the A and B subunit of the opine oxidase could function in the import and oxidation of opines. Opine oxidase causes the oxidative cleavage of opines to pyruvate or 2-ketoglurate and L-arginine (38). Opines are plant-produced amino acids resulting from infection of Agrobacterium tumefaciens in plant rhizospheres. The only up-regulated gene with a product involved in transcription (COG K) is 375 BxeB1739, a putative AraC family transcriptional regulator. Two genes downstream of this gene, BxeB1741 and BxeB1742, and 1 gene directly upstream, BxeB1738, were also up-regulated. Both BxeB1741 and BxeB1742 encode putative proteins involved in dimethyl sulfoxide (DMSO) reductase activity, while BxeB1738 encodes a hypothetical protein. 3.3.3.2.2 Down-regulated genes 380 There were 39 down-regulated genes, with > 2 fold decrease in signal intensity (p < 0.05) on DhA compared to on succinate (Supplementary Table 3.2 pg. 101). None, of the down-regulated genes are contained in the dit cluster. Four signal transduction (COG T) proteins are encoded by genes that are down-regulated, compared to no up-regulated genes in this group. Two of these genes, BxeA4195 and BxeA4196, are adjacent and immediately downstream of a 87 385 down-regulated gene encoding a putative C4-dicarboxylate transport protein BxeA4197 (COG C) possibly involved in succinate transport. Other COG C genes include, BxeB1810 encoding a putative oxygen-dependent terminal cytochrome bd oxidase subunit 1, BxeA0283 encoding a putative malate dehydrogenase which catalyzes the oxidative decarboxylation of malate to pyruvate, and BxeA3313, encoding a putative cytochrome c. Overall, the down-regulated genes 390 might indicate slower metabolism compared to metabolism during relatively rapid growth on succinate. Additionally, four cell motility and secretion proteins (COG N) encoded by genes BxeA0132, BxeA0132, BxeA0143, and BxeA0157, are down-regulated. 3.4 Discussion 395 This report presents the first genomic analysis of bacterial abietane diterpenoid catabolism. Based on greater than 60% deduced amino acid sequence identity with 17 of the 23 proteins encoded by the P. abietaniphila B K M E - 9 dit cluster (32), and transcriptional analysis of up-regulated genes, we have identified an 80.5-kb cluster of genes from BxeC0578 to BxeC0649 400 in LB400 involved in abietane diterpenoid catabolism (Fig. 3.4). We have designated this region of the genome the LB400 dit cluster. We also identified several smaller clusters of genes outside the dit cluster, which may be important for abietane diterpenoid degradation. This greatly increases the number of genes known to be associated with diterpenoid degradation and will facilitate further understanding of this complex catabolic process. A knockout of a key gene 405 coding for the alpha subunit of the ring-hydroxylating dioxygenase, ditAl, confirmed the necessity of dit cluster genes in the degradation of abietane diterpenoids. Metabolites identified in cell suspensions indicate a degradation pathway similar to the proposed convergent pathway of B K M E - 9 (19, 20). 88 3.4.1 Competitiveness of LB400 410 Several bacteria with abietane degradation capabilities have been isolated (reviewed in (23) and (25)). Typically isolates were enriched for degradation of abietane diterpenoids from environmental samples such as pulp and paper mill effluent, forest soil, or batch sequencing reactors. The isolates, members of the community growing relatively fast on diterpenoids under the enrichment conditions, were then partially characterized genetically and biochemically. In 415 this study, we took a different approach by first identifying homologues of genes required for abietane diterpenoid degradation from the genome sequence of LB400, and then assessing growth on those compounds. LB400 has a longer doubling time and lag phase than other characterized diterpenoid degrading bacteria (23, 32), but it affords a much more comprehensive analysis of the degradative process than in other bacteria. It is possible that a bacterium such as 420 LB400 would have been missed using enrichment cultures because of its long lag phase and long doubling time compared with other isolates. Growth yields of LB400 on DhA, however, were similar to those of Sphingomonas sp. DhA-33, Zoogloea sp. DhA-35 and P. abietaniphila B K M E - 9 (23, 32). It is interesting to note that when LB400 grown on succinate is transferred to medium 425 with different diterpenoids, the lag phase correlates with solubility of the compounds. At neutral pH, the solubilities of AbA and DhA are 3-5 mg/ per litre and 4-6 mg per litre respectively (18). The solubilities of 7-oxo-DhA and PaA are not available. However, based on turbidity of uninoculated culture medium containing 300 p M of each individual compound, 7-oxo-DhA is the most, and PaA is the least soluble. Thus, bioavailability may limit induction of diterpenoid 430 degradation. In agreement with this interpretation, after transfer of a culture grown on a particular resin acid to fresh medium containing the same resin acid, substrate transformation begins after a shorter lag phase than in the initial culture probably because genes required for degradation are still up-regulated. Therefore, after induction, under conditions where abietane 89 diterpenoids are continuously available to the cells, such as wastewater treatment systems or 435 perhaps some plant-associated environments, LB400 would grow at similar rates as other abietane diterpenoid degrading bacteria and potentially be competitive with them. 3.4.2 The dit clusters As reported previously, the 72 genes of the LB400 dit cluster contain homologues of all 22 genes of the previously characterized B K M E - 9 dit cluster (32) and 7 genes of the previously 440 characterized Pseudomonas diterpeniphila A19-6a tdt cluster (24), with the sole exception of ditE.from BKME-9 , encoding a putative permease of the major facilitator superfamily. Of the 52 genes that are included in the LB400 dit cluster and not found in the B K M E - 9 dit cluster, 30 were up-regulated during growth on DhA, indicating that these genes are functional members of the LB400 dit cluster. The newly described genes of the LB400 dit cluster encode proteins 445 putatively involved in beta-oxidation, transport, general catabolism, and several conserved hypothetical proteins. Through gene knockout analysis, several genes of the B K M E - 9 dit cluster (19) and A19-6a tdt cluster (24) were shown to be required for abietane diterpenoid metabolism, including ditAl. A B K M E - 9 strain with a Tn5 insertion in ditAl lost the ability to grow on DhA (20), 450 which is in agreement with results obtained with the LB400 mutant strain D i t A l K O . Other homologues of genes shown to be required for abietane diterpenoids metabolism in B K M E - 9 and A19-6a were up-regulated during growth on LB400 including, BxeC0581 (ditF, encoding a conserved hypothetical protein), BxeC0591 (ditl, encoding a putative short chain dehydrogenase), BxeC0585 (ditH, encoding a putative hydrolase), BxeC0638 (ditA3 encoding a 455 ferredoxin), and BxeC0640 (ditQ encoding a meta cleavage dioxygenase) from B K M E - 9 and BxeC0592 (tdtL, encoding a putative CoA ligase) from A19-6a. Previously we showed that the gene in B K M E - 9 encoding the P450 monooxygenase, ditQ, was not required for growth on certain abietane diterpenoids (32). Growth of a B K M E - 9 • - ' • 90 ditQ mutant on AbA and 7-oxo-DhA was similar to that of the wild type, whereas growth on 460 DhA and PaA was severely impaired. The ditQ orthologue in LB400 was up-regulated on DhA, which supports its involvement in DhA metabolism. We previously suggested that a paralogue of ditQ in B K M E - 9 might complement the ditQ gene knockout, allowing for limited growth of the knockout strain on DhA. In the LB400 dit cluster, we found a ditQ paralogue, ditU, which encodes a second P450. However, ditU was not up-regulated on DhA, and therefore likely would 465 not complement ditQ. This suggests that gene regulation may differ between B K M E - 9 and LB400. This is supported by the different gene arrangement in the two organisms and the fact that homologues in the B K M E - 9 and LB400 dit clusters with the lowest amino acid identity are genes involved in transcriptional regulation. This also suggests that the substrate ranges of the P450s in the two bacteria may differ. Knockouts of both ditQ and ditU in LB400 are discussed in 470 the next chapter. 3.4.3 Catabolic transposon The genomes of Burkholderia spp., including LB400, are rich in insertion sequences (17). Catabolic transposons have a wide variety of sizes and have been reported to be as large as 90 kb in length (34). The LB400 megaplasmid contains several transposases including a group 475 flanking the dit cluster BxeC0485-BxeC0490 and BxeC0519 and BxeC0670-BxeC0672, while no genes encoding transposases have been identified within the dit cluster. In addition, the GC content of the LB400 dit cluster is 64.9% versus 61.7% for the megaplasmid and 62.6% for the total genome. Chain et al. (5) reported the presence of the dit cluster of genes in two additional B. xenovorans strains, LMG21720 and LMG16224. Interestingly one of these strains did not 480 contain a megaplasmid, implying that the dit cluster was encoded on chromosome 1 or 2 of that strain. This further implies that the dit cluster is mobile and raises the possibility that the LB400 dit cluster is part of a catabolic transposon. Interestingly, a cluster of genes with high sequence identity and similar gerte arrangement to that of the LB400 and B K M E - 9 dit clusters was 91 identified on the recently sequenced genome of Pseudomonas aeruginosa 2192 (Pseudomonas 485 aeruginosa 2192 Sequencing Project, Broad Institute of Harvard and MIT (http://www.broad.mit.edu)), suggesting broad distribution of the dit cluster among Proteobacterial genomes. To date, however, there is no evidence that P. aeruginosa 2192 can grow on abietane diterpenoids. 3.4.4 Uptake of diterpenoids 490 The mechanism of uptake of abietane diterpenoids is unknown, however several putative transport proteins were up-regulated during growth on DhA. Interestingly, one gene of the B K M E - 9 characterized dit cluster which does not have a homologue in the LB400 dit cluster is ditE, encoding a putative permease. This suggests the strains may differ in the mechanism of uptake of diterpenoids. Two LB400 A B C transport systems, not encoded in the dit cluster, were 495 up-regulated during growth on DhA, one associated with sulfate uptake and the other associated with opine uptake. It is possible that one or both of these transport systems is involved in abietane diterpenoid uptake; however, it is more likely that one of the putative transport proteins identified in the dit cluster is responsible for this activity. The genes of the dit cluster code for four putative permeases, 3 putative members of the major facilitator superfamily (MFS), and one 500 putative member of the Resistance Nodulation (RND) Cell Division Superfamily. One of the MFS permeases, BxeC0609, and the putative RND permease BxeC0645, were up-regulated during growth on DhA (See Fig. 3.4). Based on COG comparison, and a Transport Classification Data Base (TCDB) B L A S T search analysis, gene BxeC0645 is a member of the RND Superfamily. This gene encodes a 505 protein of 835 amino acids with 12 predicted transmembrane segments (TMS) and 2 large hydrophilic extracytoplasmic domains between TMS 1 and 2 and between TMS 7 and 8. Members of this family are found across diverse phyla and participate in a wide range of transport activities (36). A Prosite search of the protein encoded by BxeC0645 identified a 5 92 TMS sterol sensing domain from TMS 2 to TMS 6 (residues 278-400). This domain consists of 510 ~180 amino acids that form five predicted membrane-spanning helices with short intervening loops. Recent work in vitro has shown direct binding of cholesterol to an 8 TMS containing the sterol sensing domain of a hamster protein involved in lipid homeostasis (29). Recently another member of the RND family also containing a sterol-sensing domain, NPC1L1, was found to be responsible for intestinal cholesterol absorption in mice (1). NPC1L1 is predominately expressed 515 in the epithelial layer bordering the luminal space. Given the structural similarity between steroids and DhA, these findings raise the possibility that BxeC0645 encodes a diterpenoid transport protein responsible for uptake of abietane diterpenoids. Genes BxeC06464BxeC0648, downstream of BxeC0645, were also up-regulated during growth on DhA and encode conserved hypothetical proteins containing signal peptides, which may also participate in an abietane 520 diterpenoid transport system. 3.4.5 Diterpenoid metabolism Results with cell suspensions of LB400 and D i t A l K O show that the abietane diterpenoid catabolic pathway of LB400 is similar to the proposed pathway for B K M E - 9 (32), and differs from those proposed for Arthrobacter sp. and Flavobacterium resinovorum (as reviewed in (16)) 525 (Fig. 3.5). The former involves a 7-oxo-DhA derivative, while the latter proceeds through a 3-oxo-DhA derivative and decarboxylation. Previously, we proposed that in BKME-9 , DhA, AbA, and PaA are hydroxylated at C7 by the DitQ P450 monooxygenase, leading to 7-hydroxy-DhA. This metabolite was identified in the supernatant of Alcaligenes eutrophus growing on DhA (2). Oxidation of the C7 alcohol to a C7 ketone via the activity of an as yet unidentified 530 dehydrogenase would lead to the formation of 7-oxo-DhA. 7-Oxo-DhA has been reported to be a substrate for the B K M E - 9 DitA ring-hydroxylating dioxygenase (20). In both B K M E - 9 and LB400, 7-oxo-DhA was removed by the wild type, but ditAl mutants of both organisms 93 Figure 3.5 Proposed convergent pathway for abietane diterpenoid degradation by LB400.1, 535 abietic acid; II, palustric acid; III, dehydroabietic acid; IV, 7-oxo-palustric acid; V , 7-hydroxy-dehydroabietic acid; VI, 7-oxo-dehydroabietic acid; VII, 7-oxo-ll,12-dihydroxy-8,13-abieadien acid; VIII,7-oxo-11,12-dihydroxydehydroabietic acid. failed to remove 7-oxo-DhA in cell suspensions with DhA, AbA and PaA or 7-oxo-DhA, 540 indicating that DitA systems in both organisms likely play a similar role in DhA metabolism. Formation of 7-oxo-DhA during A b A and PaA metabolism may proceed through a 7-oxo-PaA intermediate (Fig. 3.5). The unknown substance I that was detected in both LB400 and 94 Di tAIKO cell suspensions with A b A or PaA was tentatively identified as 7-oxo-PaA (Fig 3.5, IV). 7-oxo-PaA could potentially be an oxidized product of both AbA and PaA degradation. The 545 same metabolite was also detected in cell suspensions of a B K M E - 9 ditAl mutant incubated with PaA (19). These results are consistent with Unknown I being 7-oxo-PaA and confirm the common pathway used by both B K M E - 9 and LB400. Previously, we reported the transformation by B K M E - 9 of PaA to DhA, involving aromatisation of the C ring (32). Increases of DhA concentration in both LB400 and Di tAIKO 550 cell suspensions indicate that LB400 also catalyzes this transformation. Accumulation and removal of 7-oxo-DhA by LB400 incubated with PaA further suggests that catabolism of the accumulated DhA follows the same initial pathway as in cells with DhA as the substrate (Fig. 3.5). However, the flux of PaA through this pathway is uncertain, and an additional pathway may exist. If Unknown I is 7-oxo-PaA, this would indicate that PaA can follow two routes of 555 catalysis, either oxygenation of C7 prior to aromatisation of the C-ring or aromatisation of the C-ring prior to oxygenation of C7. 3.4.6 A putative oxygenase-driven electron transport system In several cases, genes encoding the ferredoxin and ferredoxin reductase components of oxygenase electron transport systems are located immediately downstream of the catalytic 560 subunit genes. This type of gene organization is seen in the LB400 biphenyl ring-hydroxylating dioxygenase, encoded by BxeCl 197 and BxeCl 196 (respectively encoding BphA and BphE, the alpha and beta subunits of the biphenyl ring-hydroxylating dioxygenase), BxeCl 194 (encoding BphF, the ferredoxin), and BxeCl 193 (encoding BphG, a ferredoxin reductase)(l 1). This is not the case, however, for dit cluster oxygenase genes. The alpha and beta subunit genes of the DitA 565 ring-hydroxylating dioxygenase are adjacent (BxeC0586, BxeC0587); however, no ferredoxin or ferredoxin reductase genes are located near these subunit genes. Martin and Mohn (20) were able to express catalytically active DitA in E. coli using the ditAl, ditA2 and dit A3 of B K M E - 9 . The 95 ditA3 gene codes for a 3Fe-4S ferredoxin, which is approximately 9.2 kb upstream of the alpha and beta subunits of B K M E - 9 . No heterologous expression of a ferredoxin reductase was -i 570 required; therefore, catalysis likely occurred through a surrogate ferredoxin reductase from E. coli (20). The LB400 dit cluster contains two genes that encode ferredoxins: BxeC0601, and BxeC0638. Sequence analysis revealed that BxeC0638 encodes a 3Fe-4S ferredoxin that shares 56.7% amino acid identity with B K M E - 9 ditA3. By contrast BxeC0601 codes for a plant-type 575 2Fe-2S ferredoxin. Typically, ring-hydroxylating dioxygenases utilize 2Fe-2S type ferredoxins, however, a knockout of dit A3, a 3Fe-4S ferredoxin, showed that it was required for growth on abietane diterpenoids by B K M E - 9 (19). This combined with dit A3 participation in the expression of DitA activity in E. coli support its proposed role in the DitA dioxygenase electron transport system in BKME-9 . Overall sequence identity and putative structural similarities support the 580 same role in LB400 for the 3Fe-4S-type ferredoxin encoded by BxeC0638. A ferredoxin reductase homologue was identified in B K M E - 9 through Tn5 transposon mutagenesis (21). The mutant lost the ability to grow on DhA. Sequencing of the flanking regions of the transposon revealed part of an ORE with similarity to a ferredoxin reductase gene. The location of this transposon insertion, relative to the B K M E - 9 dit cluster, was not determined. 585 The deduced 119 base amino acid sequence obtained from the sequence of the flanking regions of the Tn5 insert shares 65% amino acid identity with residues 261-380 of the deduced amino acid product of BxeC0579 of the LB400 dit cluster. During growth on DhA, BxeC0579 was up-regulated 6.4-fold (p = 0.066). The protein encoded by BxeC0579 shares 51% amino acid sequence identity with ThcD from Rhodococcus erythropolis (26) and 50% with EthA from 590 Rhodococcus ruber (6), which serve as ferredoxin reductases in P450 systems involved in the degradation of S-ethyl dipropylcarbamothioate and ethyl tert-butyl ether, respectively. We speculate that BxeC0579 encodes a ferredoxin reductase that functions in DitA electron 96 transport. The transcriptomic analysis did not show up-regulation of any other ferredoxin reductase gene during growth on DhA, indicating that the ferredoxin reductase encoded by 595 BxeC0579 may additionally function in electron transport for the P450 monooxygenase encoded by ditQ, which was also up-regulated on DhA. An electron transport system shared between monooxygenase and dioxygenase systems involved in a catabolic pathway for naphthalene has been reported for Ralstonia sp. U2 (39). 3.4.7 Role of lipid metabolism genes 600 Mineralization of abietane diterpenoids likely involves CoA-dependent metabolism. As mentioned previously, Morgan et al. (24) showed that tdtL, a homologue"of BxeC0592, encoding a putative Co A ligase, was required for growth of P. diterpeniphila A19-6a on AbA or DhA. During growth of LB400 on DhA, BxeC0592 was up-regulated, along with two clusters of genes that encode enzymes putatively catalyzing beta-oxidation-like reactions, BxeC0594 to 605 BxeC0597 and BxeC0600 to BxeC0606. The BxeC0594 to BxeC0597 cluster shares high sequence identity and gene arrangement in common with regions in both the B K M E - 9 dit and A19-6a tdt clusters, whereas the BxeC0600 to BxeC0606 cluster is newly identified in the LB400 dit cluster. Cleavage of the abietane diterpenoid rings followed by hydrolysis would result in branched short chain alkenes with acid, alcohol and ketone substituents, compounds 610 expected to be degraded in the same manner as fatty acids. Possible products of a beta-oxidation-like reaction will require further transformation to T C A intermediates for complete catabolism. Based on transcriptional analysis, propionyl-CoA may be an intermediate in abietane diterpenoid degradation. The DhA up-regulated genes, BxeB2301, BxeB2302, and BxeB1203 encode enzymes that catalyze all steps required in the propionyl-CoA catabolic pathway. The induction 615 of these genes during growth on DhA suggests that beta-oxidation processes during degradation of DhA produce propionyl-CoA that is transformed to pyruvate and enters the T C A cycle. 97 In conclusion, this chapter presents the first genomic study of a bacterium during growth on an abietane diterpenoid. Through the transcriptomic analysis, we were able to characterize the LB400 dit cluster during growth on DhA. A mutation of the DitA ring-hydroxylating 620 dioxygenase showed that this gene, and presumably also the others of the dit cluster are required for catabolism of abietane diterpenoids. A convergent pathway similar to that seen in B K M E - 9 and utilizing 7-oxo-DhA as a central intermediate was presented. In the following chapter, the focus will return to the initial steps of abietane diterpenoid metabolism, which putatively leads to formation of 7-oxo-DhA. 98 625 S u p p l e m e n t a r y T a b l e 3.1 U p - r e g u l a t e d genes o u t s i d e o f the dit c l u s t e r Gene ID Gene Annotation COG Functional Groups Fold Up-regulated on DhA P-Value CoA and Transport Cluster I BxeC0694 BxeC0698 BxeC0700 BxeC0701 Transposase BxeC0821 Hypothetical Protein Putative Porin Protein Putative Enoyl CoA hydratase/isomerase family protein Putative Acyl CoA dehydrogenase Putative Transposase No CoG M I NO CoG 2.314 2.6 2.249 2.431 3.337 0.00941 0.000313 0.049 0.00107 0.00814 Mannose Metabolism Cluster BxeC1071 Putative NUDIX LR hydrolase (gmm) BxeC1073 Hypothetical protein No CoG BxeC1085 Putative GDP M Mannoase dehydratase (gmd) BxeC1090 Putative Mannose 6- M phosphate isomerase (manA BxeC1103 Hypothetical Protien No CoG BxeC1104 Conserved Hypothetical No CoG 2.966 2.236 2.011 2.393 3.176 2.751 0.0051 0.000902 0.00447 0.0481 0.0168 0.000533 Porin BxeC1231 CoA Metabolism II Cluster BxeB0961 BxeB0963 2-methvl citrate lyase BxeB1203 Phosphoglucomutase BxeB1518 DMSO Reductase Cluster Putative Outer membrane protein Putative CoA carboxyltransferase (propionyl - C o A carboxylase) Putative Biotin Carboxylase Putative Isocitrate lyase and phosphorylmutase family protein Putative Phosphoglucomutase/p hosphomannomutase (ManB) BxeB1738 Hypothetical Protien BxeB1739 Putative AraC transcriptional regulator M 2.128 6.65 2.981 4.041 2.254 2.116 2.937 0.000326 1.39E-06 0.0144 0.0277 0.000574 0.000546 0.0197 99 BxeB1741 BxeB1742 DMSO reducatase anchor subunit C Putative Fe-S containing protein-(similar to DMSO subunit B) R C 2.336 2.001 0.0116 0.0018 BxeB2212 Putative Pyridoxamine 5'-phosphate oxidase family protein R 3.057 0.00482 Aconitate/Citrate Cluster 1 BxeB2300 Conserved Hypothetical S 2.914 0.0015 BxeB2301 Aconitate hydratase C (4, 5) 3.102 0.0187 BxeB2302 2-methyl citrate synthase C (36) 10.76 0.0421 Porin BxeB2364 Putative Outer Membrane Protein No COG 2.885 0.00436 ABC Transport Cluster BxeB2702 BxeB2704 BxeB2705 BxeB2706 BxeB2708 Bacterial extracellular solute-binding protein ABC transporter permease component ABC transporter permease component FAD dependent oxidoreductase Putative Pyridine nucleotide-disulphide oxidoreductase, class-ll E E/P E/P E C/R 5.123 5.667 3.221 2.924 2.936 6.12E-05 0.000126 0.000974 0.000862 0.00116 Methvl Citrate Synthase BxeB2900 2-methyl citrate synthease C 2.317 0.0069 ABC Transport Protein BxeA0019 ABC-type, periplasmic component E 2.635 0.00397 CoA-lipase BxeA0042 Putative AMP dependent synthease and ligase IQ 2.249 0.000314 Polyphosphate Kinase BxeA1237 Putative polyphosphate kinase P 2,069 7.11E-05 CoA Tranferase BxeA1367 3-oxoacid CoA Transferase beta subunit I (28, 34) 2.097 0.000444 Isocitrate Lvsase BxeA1651 Putative Isocitrate Lysase C (38) 2.759 0.000234 Sulfate Transport Cluster BxeA2467 Putative ABC-type P 2.315 0.0332 sulfate/molybdate transport systems, ATPase component BxeA2469 Putative ABC-type P 2.824 0.00153 sulfate/molybdate transport systems, permease components BxeA2470 Putative ABC-type P 4.795 0.00114 sulfate transport system, periplasmic component- sulfate binding protein Catalase BxeA3458 Putative catalase P 2.128 0.0018 ulfate Metabolism Cluster BxeA3659 Putative Uroporphyrin- H 3.428 0.00342 III C-methyltransferase BxeA3660 Putative GTPases - P 3.141 0.00176 Sulfate adenylate transferase subunit 1 (SelB) BxeA3663 Conserved hypothetical No COG 3.435 0.0424 BxeA3664 Putative Sulfite P . 2.666 0.018 reductase hemoprotein beta-component BxeA3671 Putative R 2.619 0.00668 oxidoreductase, Gfo/ldh/MocA family. Hypothetical BxeB1651 Conserved Hypothetical No COG 2.742 0.00215 Protein BxeB2794 Conserved hypothetical No COG 3.506 1.78E-05 BxeB2795 Conserved hypothetical No COG 3.113 0.000485 BxeA3322 • Conserved Hypothetical No COG 2.046 0.00122 protein BxeA3394 Conserved Hypothetical No COG 7.833 4.47E-05 protein BxeA3475 Conserved hypothetical No COG 2.624 0.000262 BxeA4260 Conserved Hypothetical E/R 2.042 0.00532 BxeA0586 Conserved hypothetical No COG 2.605 0.00241 BxeA0268 Hypothetical Protein No COG 2.779 0.0414 Supplementary Table 3.2 Down-regulated genes Gene ID Gene Annotation COG Functional Group Fold up-regulation on DhA P-value BxeC033 Putative ATPase R 0.433 0.00669 BxeB0477 Putative Protein Phosphatase T 0.448 0.0096 BxeB0695 Putative GntR Bacterial KE 0.518 0.00105 Regulator BxeB0773 Putative FAD/FMN containing C 0.403 0.00319 oxidoreductase BxeB0871 Putative MFS Transporter P 0.548 0.00474 BxeB0922 Putative Sulfite/Nitrite P 0.503 0.0131 Reductase BxeB1250 Putatvie methionie synthase E 0.122 0.000638 cobalamin independent BxeB1493 Putative hydrolase (HAD R 0.443 0.0149 superfamily) BxeB1810 Putative Cytochrome bd type C 0.168 0.0129 BxeB1813 Putative Response Regulatory TK 0.473 0.00212 Protein BxeB2567 Putative GntR Bacterial K 0.258 0.00172 Regulator BxeB2600 Hypothetical protein No COG 0.321 0.00274 BxeB2662 Hypothetical protein F 0.484 0.0144 BxeB2786 Putative OmpC M 0.0967 0.00733 BxeB2858 Putative Protein Phosphatase No COG 0.398 0.0332 BxeB0923 Hypothetical Protein No COG 0.323 0.0283 BxeB0921 Hypothetical Protein No COG 0.296 0.00162 BxeA0132 Putative Flagellar GTP binding N 0.549 0.000949 protein BxeA0141 Putative flagellar Basal Body N 0.351 0.00439 protein BxeA0143 Putative Flagellar Hook protein N ' 0.36 0.0136 BxeA0157 Putative Flagellar Motor Switch N 0.436 0.0426 protein BxeA0178 Putative S- H 0.37 6.48E-05 adenosylhomocysteine hydrolase BxeA0277 N-acetyl-gamma-glutamyl- 0.37 0.0128 phosphate reductase BxeA0283 Putative Malic Enzyme C 0.495 0.000356 BxeA1538 Hypothetical Protein No COG 0.446 4.70E-05 BxeA2843 Hypothetical Protein No COG 0.395 0.000216 BxeA2986 Putatvie acetyltransferase, KR 0.269 0.00529 GNAT family BxeA3155 Putative Phospholipase R 0.175 0.00158 BxeA3313 Putative Cytochrome C C 0.335 0.00627 BxeA3972 Putative DNA gyrase R 0.14 0.000104 modulator BxeA3984 Putative ferritin DPS-family L 0.42 0.000451 DNA binding protein BxeA4109 Putative membrane protein R 0.446 0.00126 BxeA4195 Putative Transport T 0.499 0.00903 transcriptional regualtory protein BxeA4196 Putative Signal Transduction T 0.525 0.0239 Histadine Kinase BxeA4197 C4 -Dicarboxylate Symporter C 0.33 0.00453 BxeA4365 Putative MFS Transporter GEPR 0.534 0.00223 BxeA4445 Putative Amino Acid E 0.43 0.0276 Transporter BxeA0219 Hypothetical Protein No COG 0.449 0.0445 BxeA2598 Hypothetical Protien No COG 0.39 0.0116 102 Altmann, S. W., H. R. Davis, Jr, L.-J. Zhu, X. Yao, L. M. Hoos, G. Tetzloff, S. P. N. Iyer, M. Maguire, and A. Golovko. 2004. Niemann-Pick CI Like 1 protein is critical for intestinal cholesterol absorption. Science 303:1201-1204. Biellmann, J. F., G. Branlant, M. Gero-Robert, and M. Poiret. 1973. Degradation bacterienne de l'acide dehydroabietique par un Pseudomonas et une Alcaligenes. Tetrahedron 29:1237-1241. Bramer, C. O., L. F. Silva, J. G. C. Gomez, H. Priefert, and A. Steinbuchel. 2002. Identification of the 2-methylcitrate pathway involved in the catabolism of propionate in the polyhydroxyalkanoate-producing strain Burkholderia sacchari IPT101T and analysis of a mutant accumulating a copolyester with higher 3-hydroxyvalerate content. Appl. Environ. Microbiol. 68:271-279. Bramer, C. O.^  and A. Steinbuchel. 2001. The methylcitric acid pathway in Ralstonia eutropha: new genes identified involved in propionate metabolism. Microbiology 147:2203-2214. Chain, P. S. G., V. J. Denef, K. Konstantinidis, L. M. Vergez, L. Agullo, V. L. Reyes, L. Hauser, M. Cordova, L. Gomez, M. Gonzalez, M. Land, V. Lao, F. Larimer, J. J. LiPuma, E. Mahenthiralingam, S. A. Malfatti, C. J. Marx, J. J. Parnell, A. Ramette, P. Richardson, M. Seeger, D. Smith, T. Spilker, S. W., T. V. Tsoi, L. E. Ulrich, I. B. Zhulinlgor, and J. M. Tiedje. 2006. Burkholderia xenovorans LB400 harbors a multi-replicon, 9.7 M bp genome shaped for versatility. Submitted to PNAS. Chauvaux, S., F. Chevalier, C. Le Dantec, F. Fayolle, I. Miras, F. Kunst, and P. Beguin. 2001. Cloning of a genetically unstable cytochrome P-450 gene cluster involved in degradation of the pollutant ethyl tert-butyl ether by Rhodococcus ruber. J. Bacteriol. 183:6551-6557. Chen, W.-M., L. Moulin, C. Bontemps, P. Vandamme, G. Bena, and C. Boivin-Masson. 2003. Legume symbiotic nitrogen fixation by P-Proteobacteria is widespread in Nature. J. Bacteriol. 185:7266-7272. Coenye, T., and P. Vandamme. 2003. Diversity and significance of Burkholderia species occupying diverse ecological niches. Environmental Microbiology 5:719-729. de Lorenzo, V., and K. N. Timmis. 1994. Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and TnlO-derived minitransposons. Methods Enzymol 235:386-405. Denef, V. J., J. A. Klappenbach, M. A. Patrauchan, C. Florizone, J. L. M. Rodrigues, T. V. Tsoi, W. Verstraete, L. D. Eltis, and J. M. Tiedje. 2006. Genetic and genomic insights into the role of benzoate-catabolic pathway redundancy in Burkholderia xenovorans LB400. Appl. Environ. Microbiol. 72:585-595. Denef, V. J., J. Park, T. V. Tsoi, J.-M. Rouillard, H. Zhang, J. A. Wibbenmeyer, W. Verstraete, E. Gulari, S. A. Hashsham, and J. M. Tiedje. 2004. Biphenyl and benzoate metabolism in a genomic context: outlining genome-wide metabolic networks in Burkholderia xenovorans LB400. Appl. Environ. Microbiol. 70:4961-4970. Denef, V. J., M. A. Patrauchan, C. Florizone, J. Park, T. V. Tsoi, W. Verstraete, J. M. Tiedje, and L. D. Eltis. 2005. Growth substrate- and phase-specific expression of biphenyl, benzoate, and CI metabolic pathways in Burkholderia xenovorans LB400. J. Bacteriol. 187:7996-8005. 103 13. Dudoit, S., Y. H. Yang, J. C. Callow, and T. P. Speed. 2002. Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experiments. Statistica Sinica 12:111 -13 9. 675 14. Gao, X., E. LeProust, H. Zhang, O. Srivannavit, E. Gulari, P. Yu, C. Nishiguchi, Q. Xiang, and X. Zhou. 2001. A flexible light-directed D N A chip synthesis gated by deprotection using solution photogenerated acids. Nucl. Acids Res. 29:4744-4750. 15. Goris, J., P. De Vos, J. Caballero-Mellado, J. Park, E. Falsen, J. F. Quensen, 3rd, J. M. Tiedje, and P. Vandamme. 2004. Classification of the biphenyl- and polychlorinated 680 biphenyl-degrading strain LB400T and relatives as Burkholderia xenovorans sp. nov. Int J Syst Evol Microbiol 54:1677-81. 16. Kieslich, K. 1976. Microbial transformations of non-steroid cyclic compounds. J. Wiley, Chichester, Eng.. 17. Lessie, T. G., W. Hendrickson, B. D. Manning, and R. Devereux. 1996. Genomic 685 complexity and plasticity of Burkholderia cepacia. FEMS Microbiol Lett 144:117-28. 18. Liss, S. N., P. A. Bicho, and J. N. Saddler. 1997. Microbiology and biodegradation of resin acids in pulp mill effluents: a minireview. Can J Microbiol 43:599-611. 19. Martin, V. J., and W. W. Mohn. 2000. Genetic investigation of the catabolic pathway for degradation of abietane diterpenoids by Pseudomonas abietaniphila BKME-9 . J 690 Bacteriol 182:3784-93. 20. Martin, V. J., and W. W. Mohn. 1999. A novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium Pseudomonas abietaniphila B K M E - 9 . J Bacteriol 181:2675-82. 21. Martin, V. J. J. 1999. Molecular Genetic Investigation of the Abietane Diterpenoid 695 Degradation Pathway oi Pseudomonas abietaniphilaBKME-9. Doctor of Philosophy. University of British Columbia, Vancouver. 22. Mohn, W. W. 1995. Bacteria obtained from a sequencing batch reactor that are capable of growth on dehydroabietic acid. Appl Environ Microbiol 61:2145-50. 23. Mohn, W. W., A. E. Wilson, P. Bicho, and E. R. Moore. 1999. Physiological and 700 phylogenetic diversity of bacteria growing on resin acids. Syst Appl Microbiol 22:68-78. 24. Morgan, C. A., and R. C. Wyndham. 2002. Characterization of tdt genes for the degradation of tricyclic diterpenes by Pseudomonas diterpeniphila A19-6a. Can J Microbiol 48:49-59. 25. Morgan, C. A., and R. C. Wyndham. 1996. Isolation and characterization of resin acid 705 degrading bacteria found in effluent from a bleached kraft pulp mill. Can J Microbiol 42:423-30. 26. Nagy, I., G. Schoofs, F. Compernolle, P. Proost, J. Vanderleyden, and R. de Mot. 1995. Degradation of the thiocarbamate herbicide EPTC (S-ethyl dipropylcarbamothioate) and biosafening by Rhodococcus sp. strain NI86/21 involve an 710 inducible cytochrome P-450 system and aldehyde dehydrogenase. J. Bacteriol. 177:676-687. 27. Parales, R. E., and C. S. Harwood. 1992. Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonasputida. J Bacteriol 174:4657-66. 104 715 28. Partida-Martinez, L. P., and C. Hertweck. 2005. Pathogenic fungus harbours endosymbiotic bacteria for toxin production. 437:884-888. 29. Radhakrishnan, A., L.-P. Sun, H. J. Kwon, M. S. Brown, and J. L. Goldstein. 2004. Direct binding of cholesterol to the purified membrane region of SCAP: Mechanism for a sterol-sensing domain. Molecular Cell 15:259-268. 720 30. Schweizer, H. P., and T. T. Hoang. 1995. A n improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158:15-22. 31. Simons, R., U . Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Techonology 1:784-790. 725 32. Smith, D. J., V. J. Martin, and W. W. Mohn. 2004. A cytochrome P450 involved in the metabolism of abietane diterpenoids by Pseudomonas abietaniphila BKME-9 . J Bacteriol 186:3631-9. 33. Steinmann, D., R. Koplin, A. Puhler, and K. Niehaus. 1997. Xanthomonas campestris pv. campestris IpsI and IpsJ genes encoding putative proteins with sequence similarity to 730 the a and P-subunits of 3-oxoacid CoA-transferases are involved in LPS biosynthesis. Archives of Microbiology 168:441-447. 34. Tan, H.-M. 1999. Bacterial catabolic transposons. Applied Microbiology and Biotechnology 51:1-12. 35. Textor, S., V. F. Wendisch, A. A. D. Graaf, U . Muller, M. I. Linder, D. Linder, and 735 W. Buckel. 1997. Propionate oxidation in Escherichia coli: evidence for operation of a methylcitrate cycle in bacteria. Archives of Microbiology 168:428-436. 36. Tseng, T. T., K. S. Gratwick, J. Kollman, D. Park, D. H. Nies, A. Goffeau, and M. H. Saier, Jr. 1999. The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins. J Mol Microbiol 740 Biotechnol 1:107-25. 37. Wang, Z.-X., C. O. Bramer, and A. Steinbuchel. 2003. The glyoxylate bypass of Ralstonia eutropha. FEMS Microbiology Letters 228:63-71. 38. Zanker, H., G. Lurz, U . Langridge, P. Langridge, D. Kreusch, and J. Schroder. 1994. Octopine and nopaline oxidases from Ti plasmids of Agrobacterium tumefaciens: 745 molecular analysis, relationship, and functional characterization. J. Bacteriol. 176:4511-4517. 39. Zhou, N.-Y., J. Al-Dulayymi, M. S. Baird, and P. A. Williams. 2002. Salicylate 5-hydroxylase from Ralstonia sp. Strain U2: a monooxygenase with close relationships to and shared electron transport proteins with naphthalene dioxygenase. J. Bacteriol. 750 184:1547-1555. 105 4. DitQ and DitU1 4.1 Introduction The involvement of DitQ in the initial steps of the catabolism of abietane diterpenoids by B K M E - 9 was examined in Chapter 2. While a knockout of ditQ in B K M E - 9 led to impaired 5 growth on DhA and PaA, it affected growth on A b A or 7-oxo-DhA minimally. Substrate binding of DhA to DitQ supported its role in DhA catabolism; however, binding assays with other substrates were inconclusive. These findings raised the possibility of a second monooxygenase that was able to partially complement the ditQ knockout in the metabolism of DhA and PaA and possibly responsible for the transformation of AbA. This hypothesis was supported by the dit 10 cluster of LB400, which contains two genes encoding cytochromes P450 sharing greater than 60% amino acid identity with Di tQ-BKME-9. As noted in Chapter 3, transcriptomic analysis of LB400 cells confirmed that ditQ (encoding CYP226A1) was up-regulated during growth on DhA while the second putative P450 encoding gene, ditU (encoding CYP226A2), was not. This chapter describes the use of 2D gel-based proteomics to characterize the expression 15 of proteins during growth of Burkholderia xenovorans LB400 on each of DhA and AbA. This analysis was aimed at gaining a clearer understanding of the initial steps of abietane diterpenoid catabolism and the involvement of two cytochromes P450, DitQ and DitU. The results revealed a key difference in the catabolism of DhA and AbA by LB400: the differential expression of DitU. DitQ is expressed during growth on both DhA and AbA, whereas DitU is only expressed 20 during growth on AbA. Phenotypic studies of the knockouts containing an insertional mutation of ditQ or ditU showed that ditQ is required for growth on DhA and substrate transformation, both DhA and PaA but not 7-oxo-DhA. An in vitro P450 assay confirmed that DhA is a substrate whereas ditU is required for AbA catabolism. Substrate binding assays revealed that DitQ binds for DitQ and transforms DhA to 7-hydroxy-DhA. 1 A version of this chapter will be submitted for publication. • • ' 106 25 4.2 Materials and Methods 30 Bacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 4.1. E. coli was cultured on Luria-Bertani (LB) medium and incubated at 37°C and B. xenovorans strains were cultured on L B without NaCl, or K I mineral medium (see Chapter 3 for details) supplemented with 1 g per litre succinate, 90 mg per litre AbA, 90 mg per litre DhA, 90 mg per litre PaA, or 95 mg per litre 7-oxo-DhA. A l l B. xenovorans strain incubations were conducted at 30°C. A l l liquid cultures were incubated on a rotary shaker at 200-250 rpm. Table 4.1 Bacterial strains and plasmids used in this study Genotype or Description Reference or Source Strains Burkholderia xenovorans LB400 DitQKO DitUKO Escherichia coli DH5a S17-1 Plasmids pEX18AP pX1918G pEX5906 pEX5906KO pEX5938 pEX5938KO Wild type; grows on abietane diterpenoids (9) ditQ:: xylE-accd; Gm r This study ditU:: xylE-accC1'; Gm' This study endA1 hsdRM (rk" mk") supE44 thi-1 recA1 gyrA (Naf) Gibco BRL rel A1 A(laclZYA-argF) U169 deoR (<^80dlacA(lacZ)M15) recA pro thi hsdR with integrated RP4-2-TcMu::Kna::Tn7; (26) Tra+ Trr Sm r sacS conjugable plasmid for gene replacement; Ap r (23) xy/E-Gmr fusion cassette-containing plasmid; Ap r Gm' (23) 1.3 kbp PCR amplicon containing LB400 ditQ cloned into This study the Xbal HindWl of the multiple cloning site of pEX18AP PCR amplified xylE-Grri fusion cassette of pX1918G This study cloned into Kas\ Nod site of pEX5906 1.6 kbp PCR amplicon containing LB400 ditU cloned into This study the EcoRl BamH\ of the multiple cloning site of pEX18AP Xmal digested fragment of pX1918G containing the xylE- This study accC1 fusion cassette cloned into the unique Xma\ site of peX5938 disrupting ditU 35 DNA Manipulation DitQKO. Primers HISLEFT5906 and HISRIGHT5906 (Table 4.2) were used to amplify a 1312 bp fragment including ditQ from LB400 genomic DNA. The Nhel and HindUl digested amplicon was ligated into Xbal and HindUl digested p E X l 8AP (11) and used to transform 107 DH5oc to produce pEX5906. Next, the xylE—accCl transcriptional fusion antibiotic cassette of 40 pX1918G was amplified using primers XyLE5906L and XyLE5906R and ligated into the Kasl and Noel digested pEX5906, which disrupted ditQ. The ligation product was used to transform SI7-1 to produce pEX5906KO. Table 4.2 Oligonucleotide primers used in PCR Oligo nucleotide Sequence3 Restriction Site HISLEFT5906 5'-CGC GCG CTA GCA TGG AGA CCG GAA TGA CCA-3' Nhe\ HISRIGHT5906 5'-GCG CGA AGC TTG TCG ACG GAC TAC CGC TCA-3' Hind\\\ XyLE5906L 5'-GAT CCC ATG GAC CGT GAT CGG CGA ACT GGA-3' Nco\ XyLE5906R 5'- ATA TGG CGC CCTCGG CCA CCG TCA TCT TCC-3' Kas\ KOLBcep5938 5'-CGC GGA TCC TGG TGA GTT GCA GGC CGT A-3' BamH\ KORBcep5938 5'-CCG GAA TTC ACG CCG TTA AGC TGC ACG A-3' EcoR\ HISLEFT5938 5'-CGC GCA AGC 1 1 1 GGT GAG TTG CAG GCC GTA-3' Hind\\\ HISRIGHT5938 5'-CGC GCG CTA GCA TGA GCA CCA CCC TCG AAA-3' Nhe\ a Restriction sites are underlined. 45 DitUKO. Primer KOLBcep5938 and KORBcep5938 were used in an LB400 colony PCR (annealing temp. 58.9°C) to amplify a 1609 bp fragment including ditU. A BamHl and EcoRl digest of the amplified fragment was ligated into the EcoRl and BamHl digested pEX18AP to produce pEX5938. Next, an Xmal digested xylE-Gvci transcriptional fusion antibiotic cassette of 50 pX1918G (23) was ligated into the dephosphorylated unique Xmal site of pEX5938, which disrupted the ditU gene, and was used to transform SI 7-1 to generate pEX5938KO. Successful transformants of pEX5906KO and pEX5938KO were selected as described in Chapter 2. Homologous recombination of the mutated allele into strain LB400 was accomplished by diparental conjugation as in (4) and described in Chapter 3. Successful strains were 55 designated DitQKO and DitUKO. 108 t Homologous recombination of pEX5906KO was confirmed by PCR of a 2.7-kbp PCR product from strains growing on L B (-NaCl) agar plates containing 10 pg of gentamycin/ml using PCR primers XylE5906R and HISLEFT5906 under annealing condition of 57.8°C for 30 seconds and 30 cycles. Homologous recombination of pEX5938KO was verified by 60 amplification of a ~ 3kp fragment from gentamycin resistant strains using primers HISLEFT5938 and HISRIGHT5938 at 60.0°C for 30 seconds. Amplicons generated from both DitQKO and DitUKO were sequenced and compared to the expected product. Cell suspension and growth assays. Cell Suspensions and growth assays were conducted as described in Chapter 3 using LB400, DitQKO or DitUKO. 65 Proteome Cultures Succinate. LB400 was streaked out from frozen stock on 1.5 % purified agar K I plates, which were inverted over a petri dish lid containing biphenyl crystals and incubated for 3-4 days. The initial growth on biphenyl was used as a selection. Colonies were then used to inoculate 50 ml of K I containing 1 g per litre of succinate. After 24 hours the culture reached late-log phase with an 70 OD600 o f - 0.9, and cells were transferred to fresh K I containing 1 g per litre of succinate for an initial OD6oo of 0.001. After 18 hours, the culture reached mid-log phase (OD6oo 0.68). Cells from this culture were used to inoculate 1 litre Erlenmeyer flasks of K I containing l g per litre succinate. After 18 hours of incubation, cultures reached mid-log phase growth with an OD6oo of - 0.5 and were collected as described below. 75 Abietic acid. LB400 cells used for proteomic analysis of AbA catabolism were first grown on biphenyl as described above for succinate grown cultures. A colony from biphenyl grown cells was used to inoculate 100-200 ml K I medium containing l g per litre of succinate. After the culture reached late-log phase (OD600 ~0.9), cells were transferred to fresh succinate-containing K I for an initial OD of 0.001 and allowed to reach mid-log phase with an OD6oo of-0.7 (-18 80 hours). Cells from this second succinate culture were used to inoculate a 200 ml culture of K I 109 containing 90 mg per litre of A b A for an initial OD600 of 0.01. After an incubation of-300 hours, A b A grown cells were used to inoculate fresh K I medium containing 90 mg per litre of A b A at an initial O D 6 0 0 of 0.001 or 0.01 and incubated until OD6oo reached 0.04-0.0475. Cells from the second AbA culture were used to inoculate l-3Titres of K I containing 90 mg per litre 85 of A b A for an initial OD600 of 0.001 and incubated for 30-35 hours until mid-log phase (OD600 ~ 0.05) and harvested as described below. Dehydroabietic Acid. LB400 cells used for proteomic analysis of growth on DhA were first grown on biphenyl and subsequent succinate cultures as described above for both succinate and AbA grown cultures. As described above for AbA, succinate-grown cells were used to inoculate 90 100-300 ml of K I medium for an initial OD600 of 0.01 containing 90 mg per litre of DhA. After incubation of approximately 150 hours these DhA grown cells were used to inoculate 50-100 ml of fresh K I containing 90 mg per litre of DhA for an initial OD600 of 0.001-0.005 and incubated until cultures had reached late-log phase (~ 48 hours). Cells from the second DhA culture were used to inoculate 1 litre of K I containing 90 mg per litre of DhA for an initial OD600 of 0.001. 95 The culture was incubated until it reached mid-log phase (OD600 ~ 0.06) and harvested as described below. Cell harvest and washing. Cells were harvested by centrifugation at 10,000 rpm for 20 min at 25°C, decanted, frozen using liquid nitrogen, and stored at -80°C. Pellets were thawed on ice and then kept cool on ice throughout the remaining steps. Pellets were suspended in a small amount 100 of chilled sterile saline (0.8%) and then saline was added to the original culture volume prior to centrifugation, and mixed vigorously by shaking. The suspension was then centrifuged at 10,000 rpm at 4°C for 7 min and decanted immediately. The pellet was then suspended in 3 ml of chilled TE (10 m M Tris/HCl, 1 m M EDTA, pH 8.0) and aliquoted into pre-chilled 1.7 ml Eppendorf tubes. Suspensions were then centrifuged for 5 min at 13,200 rpm at 4°C, decanted with pipetting 105 to remove all remaining fluid, and stored at -80°C. 110 Protein extraction and quantification. Pellets were thawed on ice and suspended in 300 ul of lysis buffer (4% cholamidopropyldimethylammoniopropanesulfoate [CHAPS], 30 m M Tris pH 6.5) by vortexing. Next, protease inhibitor cocktail [one tablet of Mini Complete per 10 ml solution; Roche] was added to the cell pellet (1:100, vol/vol) and incubated on ice for 20 min. 110 Lysate was then added to 0.3 g of sterile beads (0.1-mm zirconia/silica beads; BioSpec Products Inc., Bartlesvilles, OK). The lysate was bead beaten using a Fast Prep Bio 101 Thermo Savant bead beater 5 times for 15 seconds at speed setting 6.0 with a 3 minute incubation on ice between runs to facilitate lysate cooling. The lysate was then centrifuged for 10-20 min at 13,200 rpm at 4°C to remove unbroken cells and debris. The supernatant was then removed and placed 115 in a 1.7 ml Eppendorf tube. This centrifugation step was repeated until supernatant was clear and free of beads and then aliquoted into pre-chilled Eppendorf tubes and stored at -80°C. Proteins were quantified using 2D Quant kit (Amersham Biosciences) as per manufacturer's instructions. Proteomic analysis. Proteomic analysis was carried out as described previously (5, 21). Briefly, after protein extraction, 2D gels were run for each of three biological replicates for LB400 cells 120 grown on succinate, dehydroabietic acid, or abietic acid. The first-dimension separation was carried out using non-linear IPG strips (24 cm, pH 3 to 7). The IPG strips were rehydrated "in-gel" using 90 mg protein extract suspended in 400 ul rehydration solution (10 M urea, 2 M thiourea, 30 m M dithiothreitol, 3% CHAPS, Pharmalyte pH 3 to 10). To minimize carbamylation, the temperature was maintained between 20 and 25°C during protein 125 solubilization. Isoelectric focusing in the IPG strips was carried out for atotal of 73.5 kVh at 20°C under mineral oil using E T T A N IPGphor (Amersham Biosciences). The IPG strips were then equilibrated and run into 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels (24 by 20 cm) using the E T T A N DALTtwelve system (Amersham Biosciences). Broad-range molecular mass markers (Invitrogen) were run on each side of the gel. 130 Protein was detected using the fluorescent stain Sypro Ruby, and the gels were imaged using a 111 variable mode imager Typhoon 9400 (excitation 488 nm, emission 610 nm; Amersham Biosciences). A n a l y s i s o f 2D g e l s a n d p r o t e i n i d e n t i f i c a t i o n . The 2D gel images were differentially analyzed using Progenesis Workstation software (Nonlinear Dynamics, Durham, NC). A signal intensity 135 was assigned to each spot; and the signal intensity of each spot was normalized against the total signal intensity of all spots on the gel. The normalized signal intensity of each spot was then averaged over gels obtained from three different cultures. Averaged gels included only proteins spots that were present in at least two of three replicate gels. Only spots with a minimum normalized signal intensity of 0.002 or greater were analyzed further. Molecular mass values 140 were assigned using the broad-range molecular mass markers (Invitrogen) run on each side of the gel. Isoelectric point values were assigned using an application provided with the Progenesis Workstation software. Theoretical molecular mass and isoelectric point values were predicted based on protein sequence using Expasy compute pI/Mw tool, available at http://ca.expasy.org/tools/pi_tool.html. For proteins appearing on the gel as a horizontal series of 145 spots, likely due to carbamylation, the pi and mass of only the major spot in the series were recorded, and the difference in abundance was calculated based on the summed signal intensities of all the spots in the series. Protein spots whose intensities were at least twofold higher or lower versus the control (succinate-grown cells) were recorded as more or less abundant, respectively. Spots of interest were excised from gels and digested in-gel using trypsin (12). Mass 150 spectrometry analyses were performed using a Voyager DESTR matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) (Applied Biosystems). Proteins were identified as described previously (21) using the MASCOT search engine (www.matrixscience.com) using a database generated by in silico digestion of the total LB400 proteome predicted from the complete genome sequence (http://genome.ornl.gov/microbial/bfun/). A protein was considered 155 identified i f the hit fulfilled four criteria: 1) the hit was statistically significant (a M A S C O T search score above 52 for the LB400 database); 2) the number of matched peptides was five or higher; 3) the protein sequence coverage was above 20%; and, 4) the predicted mass and pi values were consistent with the experimentally determined ones. When two or more significant hits were returned, it was usually possible to narrow the identification to a single hit based on 160 listed criteria (e.g., the pi and mass of some hits did not match that of the spot). Otherwise, the identification was excluded from the data set. Substrate Binding. Cultures of E. coli DH5cc containing pEX5938, pEX5906, or pEX18Ap were prepared and analyzed as described in Chapter 2 with the following exception. Instead of a French Press, two passes through an Emulsiflex C-5 cell disrupter operated at 15000 psi 165 homogenizing pressure were used for cell lysis. DitQ was assayed at concentrations from 0.46 to 0.77 uM and DitU was assayed at concentrations for 0.61 to 0.70 uM. In vitro P450 Assay. DitQ-LB400, or DitU-LB400 was collected and quantified in E. coli crude lysate as described for substrate binding assays in Chapter 2. Both ferredoxin DitA3 from P. abietaniphila - B K M E - 9 and ferredoxin reductase BphG from Comamonas testosteroni were 170 purified to homogeneity and kindly provided by L. Eltis. Vials containing both DitA3 and BphG were sparged with argon for approximately 1 minute prior to the activity assay. BphG (1.8 uM), DitA3 (3.6 uM), E. coli crude lysate containing DitQ or DitU (1.0 uM), N A D H (350 uM) and 100 u M abietane diterpenoid were combined and incubated for 5 min at room temperature or 30 min at 30°C. Controls included acidified reaction mixture or assays using IPTG induced E. coli 175 crude lysate containing pEX18Ap. 113 4.3 Results 4.3.1 Succinate, DhA and AbA proteomes Approximately 800 protein spots were detected in each of the proteomes of LB400 grown on succinate, AbA, and DhA as sole organic substrates (Fig. 4.1, See Appendix). Of the 180 551 protein spots common to the three proteomes, 52 were greater than 2-fold more abundant on both AbA and DhA than on succinate. Of the 679 protein spots common to the DhA and AbA proteomes, 128 were not detected in the succinate proteome. Of the protein spots significantly more abundant on the abietane diterpenoid substrates, 23 were identified (Table 4.3). Of these, genes of the LB400 megaplasmid dit cluster encode 16 proteins. They include two P450 185 monooxygenases, one of which is DitQ, encoded by BxeC0599, and detected in both AbA and Figure 4.1 Global analysis of 3 LB400 proteomes. Circles represent proteomes of LB400 grown on succinate, AbA, or DhA as sole organic substrates. Numbers within each circle represent protein spots («=3). Numbers outside circles represent the total number of protein 190 spots present on each proteome with greater than or equal to 0.002 normalized signal intensity. Overlap between circles indicates protein spots that were present on more than one proteome. Succinate 772 Spots Abietic Acid 794 Spots Dehydroabietic Acid 815 Spots 114 Table 4.3 Proteins involved in abietane diterpenoid catabolism identified by MASCOT-based analysis of MALDI-TOF spectra Gene no. a Gene name Protein name Theor. pl DTheor. M W b Exp. p l b Exp. MW b # P C Mascot score c SC C Normalized Siqnal Intensity Succ DhA AbA BxeC0585 ditH Putative Hydrolase 5.3 37.9 5.0 39.2 16 185 57 ND 0.47 0.57 BxeC0586 ditAl Alpha subunit of Ring Hydroxylating 5.7 52.5 5.6 54.6 9 72 26 0.70 1.00 1.26 Dioxygenase BxeC0587 ditA2 Beta subunit of Ring Hydroxylating 5.8 21.1 5.8 22.1 8 115 61 ND 0.89 0.72 Dioxygenase BxeC0592 ditJ Putative CoA Ligase 5.8 60.0 5.8 64.9 9 65 20 ND 0.57 0.19 BxeC0594 Conserved Hypothetical 6.0 40.7 6.1 39.3 9 61 32 0.34 1.06 1.24 BxeC0597 ditO Putative Thiolase 5.9 41.8 6.0 42.9 11 93 35 0.02 0.32 0.46 BxeC0599 ditQ Cytochrome P450 6.0 46.9 6.1 47.7 17 100 42 ND 0.15 0.34 BxeC0602 Putative Acyl CoA Dehydrogenase 5.9 41.8 6.0 41.8 12 128 38 ND 0.68 0.35 BxeC0606 Conserved Hypothetical 5.6 49.5 5.4 48.7 10 71 33 0.02 2.02 2.17 BxeC0619 Putative Short Chain Dehydrogenase 5.6 29.4 5.7 26.1 8 63 43 0.09 0.19 0.17 BxeC0630 Conserved Hypothetical 5.8 39.1 5.8 39.6 7 56 23 ND ND 0.42 BxeC0631 ditU Cytochrome 450 5.9 47.6 6.1 48.1 23 153 64 ND ND 0.50 BxeC0639 ditB Putative Dehydrogenase 6.1 26.7 5.0 29.5 11 123 66 ND 0.40 0.86 BxeC0640 ditC Ring Cleavage Dioxygenase 10.4 49.6 6.4 36.7 13 88 35 ND 0.69 0.46 BxeC0644 Conserved Hypothetical 6.1 37.2 6.0 37.4 13 113 51 ND 1.41 LR BxeC0647 Conserved Hypothetical 6.0 51.2 5.5 47.7 17 137 49 ND 0.42 0.24 BxeC1186 bphD Hydrolase 6.1 32.1 4.8 38.5 6 61 25 0.01 0.12 0.26 BxeB0962 Putative CoA Hydratase 5.2 28.5 4.5 26.8 14 120 70 ND 0.16 0.19 BxeB0963 Putative Biotin Carboxylase 5.4 72.9 5.0 76.4 19 127 36 ND 0.16 0.13 BxeB1203 Putative Isocitrate Lyase and 5.6 31.5 5.4 32.2 10 106 56 0.04 0.55 0.43 Phosphorylmutase family protein BxeB2301 Aconitate Hydratase 5.5 94.6 5.3 96.3 32 200 47 ND 0.56 0.50 BxeA1366 Putative 3-oxoacid CoA-transferase 5.7 25.5 5.7 27.1 7 67 67 0.05 0.43 0.27 alpha subunit BxeA2466 Putative LysR type Regulator 6.0 34.5 6.1 37.2 11 77 38 ND 0.42 LR Abbreviations, Theo., Theoretical; Exp., Experimental; MW, molecular weight; pl, Isoelectric point; Succ, succinate; DhA, dehydroabietic acid; AbA, abietic acid. a LB400 genome sequence available at http://genome.ornl.gov/microbial/bfun/ b See Methods and Materials for description of how Theor. And Exp. pl and MW were calculated c Number of peptides matched (#P) and sequence coverage (SC) in MASCOT analysis. MASCOT-generated probability-based Mowse score. In the LB400 protein database scores 200 greater than 52 are significant (P < 0.05) Average normalized signal intensities from three biological replicates under each of the tested growth conditions; Succ, succinate. ND, not detected. LR, low resolution in area where spot was expected. DhA proteomes (Fig.4.2). The second one is DitU, encoded by BxeC0631, unique to the AbA proteome. DitQ and DitU have similar pis and molecular weights and, as expected, are located 205 next to each other on the AbA proteome map (Fig 4.2). Citrate synthase I Succinate Citrate synthase I DitQ DhA AbA Figure 4.2 Differential expression of DitU. Three dimensional representation of 2D PAGE of the Succinate, DhA, and AbA proteomes of LB400 grown on the respective substrates showing the same relative area of each gel. 210 The DhA proteome contained a protein of low abundance with similar mobility as DitU, but this protein was identified as citrate synthase I, with a tryptic digest containing no peptide fragments corresponding to DitU (Fig. 4.2). An additional seven proteins not encoded within the dit gene cluster were unique to the AbA and DhA proteomes or were up-regulated on those 116 substrates relative to the succinate proteome (Table 4.3). These include proteins encoded by 215 BxeCl 186, BxeB0962, BxeB0963, BxeB1203, BxeB2301, BxeA1366, andBxeA2466. 4.3.2 Growth of mutant strains To investigate the function of the identified P450s in abietane diterpenoid metabolism, two mutant strains were prepared, DitQKO and DitUKO, whose corresponding genes were disrupted by insertion. The ditQ disruption completely abolished the ability of LB400 to grow on 220 DhA, but did not affect its growth on 7-oxo-DhA (Fig. 4.3). DitQKO grew on A b A with a similar doubling time and similar maximum protein concentration as LB400, but the lag phase was extended by approximately 100 hours. DitQKO grew on PaA to a similar maximum protein concentration as the LB400, but the lag phase was longer, and the doubling time greater. DitUKO was not able to grow on AbA; however, it showed similar lag phases, doubling times 225 and maximum protein concentrations as LB400 on 7-oxo-DhA and DhA. Similar to DitQKO, growth of Di tUKO on PaA reached a maximum protein concentration similar to the LB400, but the lag phase was longer and the doubling time greater. 4.3.3 Cell suspension assays Cell suspensions assays were conducted to examine substrate removal and potential metabolite 230 accumulation and/or removal. AbA. LB400 and DitQKO were able to completely remove AbA within 72 hours (Fig. 4.4A). By contrast, Di tUKO was only able to reduce the initial concentration of A b A by 34%, which indicates that ditU is involved in the catabolism of AbA. Trace amounts of 7-oxo-DhA were detected in all the cell suspensions on A b A at time zero, as this was a contaminant of the 235 A b A reagent. LB400 and DitQKO increased amounts of 7-oxo-DhA, whereas, DitUKO did not increase 7-oxo-DhA above the initial concentration. This suggests that 7-oxo-DhA is a metabolite formed during AbA catabolism, and its formation requires DitU. A second metabolite 117 400 -| 350 W T D i t Q K O D i t U K O DhA AbA PaA 7-oxo DhA DhA AbA PaA 7-oxo DhA DhA AbA PaA 240 Figure 4.3 Growth characteristics of LB400 and two mutant strains, D i t U K O and D i t Q K O on four abietane diterpenoids. Initial inoculum for each culture was ~2 x 106 cells/ml grown to mid-log phase on succinate. N G denotes no growth. Error bars indicate standard error. 118 with similar accumulation and removal patterns seen with 7-oxo-DhA could not be confidently identified using a NIST library search. This same metabolite was previously observed in cell suspensions of Di tAIKO on AbA or PaA (Chapter 3) and was named Unknown I, and 245 tentatively identified as 7-oxo-PaA (see Fig. 4.7). A third metabolite, identified as 2,4-dimethyl heptanedioic acid (DMHDA), was observed in LB400, DitQKO, and DitUKO cell suspensions. This same metabolite was also identified in an LB400 cell suspension assay reported in Chapter 3. D h A . LB400 and DitUKO completely removed DhA within 72 hours, whereas DitQKO 250 decreased concentrations of DhA by 40%, but was not able to remove it after 72 hours (Fig. 4.4 B). No 7-oxo-DhA or D M H D A accumulation was observed in DitQKO cell suspensions incubated on DhA, whereas LB400 and DitUKO with DhA showed the accumulation of both metabolites. P a A . LB400 and DitQKO respectively removed 91% and 80% of the initial PaA by 72 255 hours; whereas, Di tUKO removed only 22% by the same time (Fig. 4.4 C). With a longer incubation of 144 hours, DitUKO decreased the PaA concentration by 73%. A l l strains showed the accumulation of DhA, 7-oxo-DhA, D M H D A and Unknown I during PaA removal, which was not observed in heat-killed cell controls, supporting an enzymatic transformation of substrates and intermediates. 260 7-oxo-DhA. Cell suspensions of all strains completely removed 7-oxo-DhA within 24 hours (data not shown). This is an indication that neither DitQ nor DitU is essential for catabolism of 7-oxo-DhA. 119 265 Figure 4.4 Metabolite analysis of cell suspensions of LB400, DitQKO and DitUKO with AbA, DhA, and PaA. A , AbA cell suspension. B, DhA cell suspension. C, PaA cell suspension. Closed symbols indicate values corresponding to the left y-axis and open or stick symbols correspond to the right y-axis, Symbols: • - A b A , • and • - DhA, • - PaA, • and O - 7-oxo-DhA, +-dimethyl heptanedioic acid, x - Unknown I o 4.3.4 Binding assays 270 To confirm that ditQ and ditU encode functional cytochromes P450, carbon monoxide binding assays were conducted. Both DitQ and DitU, expressed in the crude lysate pf E. coli, bound carbon monoxide and produced a Soret maximum absorbance between 447 and 450 nm with little absorbance at 420 nm. This indicates that both DitQ and DitU are members of the P450 Superfamily and are expressed in E. coli in a native conformation. To investigate the 275 possible range of compounds that bind to DitQ and DitU, substrate-binding assays were conducted. Binding of DhA to DitQ produced a typical substrate binding spectrum with dissociation constants (Kd) of 0.98 + 0.01 uM (Fig. 4.5). This is a strong indication that DhA is a substrate for DitQ. Binding of PaA to DitQ produced a type I-like curve, which indicates that PaA is likely a substrate for DitQ, however an isosbestic point at 407 nm was not observed and 280 therefore the dissociation constant could not be determined (Fig. 4.5). Binding of A b A to DitQ perturbed the heme environment; however, the binding equation curve could not be fitted to the data points and therefore results were inconclusive. 7-oxo-DhA did not alter the spectrum of DitQ at concentrations up to 25 uM, indicating that it is not likely a substrate for DitQ. The same assay was used to investigate binding of abietane diterpenoids to DitU, however, results were 285 inconclusive. This may indicate that none of the assayed diterpenoids bind to DitU and therefore are not substrates for this enzyme, however other possibilities may exist, as discussed below. 4.3.5 P450 in vitro activity assay To investigate the catalytic activity of DitQ and DitU, in vitro P450 activity assays were conducted. The crude lysate of E. coli containing expressed DitQ was combined with purified 290 Comamonas testosteroni ferredoxin reductase BphG, purified P. abietaniphila B K M E - 9 ferredoxin Dit A3, N A D H , and either AbA, DhA, PaA or 7-oxo-DhA. BphG is the reductase component of the electron transport system of the biphenyl ring-hydroxylating dioxygenase. The gene encoding an LB400 homologue of DitA3 was up-regulated during growth on DhA versus 121 0.12 ~i 1 i ; 1 i 1 i 1 r~ 0 5 10 15 20 Dehydroabietic Acid (nM) 0.04 360 380 400 420 440 460 480 500 Wavelength (nm) 295 Figure 4.5 Binding spectra for DhA or PaA to DitQ. A . DhA binding to DitQ. Data points represent the difference in absorbance between 387 and 421 nm caused by increasing DhA concentrations. The curve represents a best fit of the binding equation to the data in which Kd = 0.98 ± 0.01 uM and A A m a x = 1.16 x 10"1 ± 6.55 x 10"3. Insets. UV-visible difference spectra of DitQ with increasing concentrations of DhA. B. PaA binding to DitQ. UV-visible difference 300 spectra of DitQ with increasing concentrations of PaA. Arrows indicate increases in the amplitude of maximum or minimum absorbance caused by increasing concentration of DhA or PaA. 122 305 310 on succinate in transcriptomic analysis (Chapter 3). GCMS analysis of the diazomethane-derivatized reactions incubated with DhA revealed the formation of a single peak at a retention time of 1.11 relative to that of DhA (Fig. 4.6). The mass spectrum of this peak indicated that the product is 7-hydroxy-DhA (Fig. 4.6, 4.7). Incubation of expressed DitQ with other diterpenoids did not yield a detectable product. The same assay was conducted using DitU expressed in E. coli under the same conditions and incubated with the same diterpenoids as used above, but no product was detected. B IOOH 0 4 162 55 67 87 105 128 145 J^ .IL.-^ L^  195 237 255 287 312 330 — i — , f— 287 312 100-195 255 162 \—\—i—I—i—i—i—r 237 330 "T—[—TT—I—I—|—I—I-\—I—| 1 1 I I—|— 50 80 T" 110 140 170 200 230 260 290 320 Figure 4.6 In vitro DitQ activity assay. A. Gas chromatograph of in vitro DitQ P450 reaction mixture incubated with DhA. B. Head to tail mass spectrum of peak with relative retention of 315 1.11. Fragmentation pattern above the zero line corresponds to the peak identified from DitQ with DhA in vitro activity assay. Fragmentation pattern below the zero line corresponds to 7-hydroxyTDhA mass spectrum from NIST library. 123 4.4 Discussion 320 This is the first proteomic investigation of bacteria grown on abietane diterpenoids (Fig. 4.1). This study provides the first conclusive evidence for the involvement of two cytochromes P450, DitQ (CYP226A1) and DitU (CYP226A2) of the LB400 dit cluster in the degradation of abietane diterpenoids. The results indicate that the P450s function in the catabolic pathways of AbA and/or DhA and are required in the formation of the central intermediate 7-oxo-DhA (Fig 325 4.7). We also provide the previously missing evidence for the C7 hydroxylating activity of DitQ. 4.4.1 Consistency of proteome and transcriptome The protein spots identified as being up-regulated in the DhA proteome agree with the up-regulated genes of the DhA transcriptome reported in Chapter 3. A l l identified protein spots, which are products of the LB400 dit cluster genes, were also up-regulated in the transcriptome. 330 Of the 7 identified proteins that are encoded by genes outside of the dit cluster, only one corresponding gene was not significantly up-regulated. Genes BxeB0962 and BxeA2466 (see Table 4.3) were up-regulated > 1.8 fold with p-values of < 0.01 and are clustered with up-regulated genes BxeB0961 and BxeB0963 involved in CoA-dependent metabolism, and BxeA2467, BxeA2469 and BxeA2470 involved in a possible sulfate transport cluster. Gene 335 BxeA1366 was not significantly up-regulated but clustered with the up-regulated gene B x e A l 367 both of which are also involved in CoA-dependent metabolism. Gene BxeC 1186, which encodes a serine hydrolase, BphD of the biphenyl catabolic operon, was up-regulated 1.4 fold with a p-value of 0.0007. These results indicate consistency between the proteome and transcriptome. 340 4.4.2 Cytochromes P450 DitQ and DitU Characterization of the LB400 dit cluster revealed two genes coding for cytochromes P450, ditQ and ditU (Chapter 3). The annotated LB400 genome revealed six putative cytochromes P450, four encoded on the megaplasmid and two on chromosome 1. Of these six 124 P450s, only DitU and/or DitQ were identified as having increased abundance during growth on 345 AbA or DhA relative to growth on succinate (Fig. 4.2). DitQ and DitU are closely related, sharing 60% amino acid sequence identity (see Table 4.4), while their identity with the other four P450s of LB400 ranges from 19.5 to 24.4%. While DitQ is expressed during growth on both DhA and AbA, DitQKO mutant pheno types showed that DitQ is required for catabolism of DhA and not of AbA, indicating non-essential expression of DitQ during growth of AbA (Figs. 4.2-350 4.4). On the other hand, DitU is expressed only during growth on AbA, with DitUKO mutant phenotypes confirming its essential role in the catabolism of AbA but not DhA. The expression of DitQ and not DitU during growth on DhA is consistent with transcriptomic results presented in Chapter 3. A reporter construct in B K M E - 9 demonstrated that ditQ was induced by AbA, DhA or 355 PaA. There is no conclusive evidence that a second P450 is involved in B K M E - 9 abietane diterpenoid catabolism. However, growth phenotypes of a B K M E - 9 ditQ knockout (Chapter 2) suggested that a second monooxygenase is able to complement DitQ during growth on DhA and PaA. This mutant strain showed that growth was only impaired and not abolished on both DhA and PaA. Note that a similar result was obtained for a knockout of the ditQ homologue identified 360 in P. diterpeniphila A19-6a (17). The mutant strain was still able to grow on both AbA and DhA, however removal of both substrates was impaired. This raised the possibility of a second monooxygenase in both B K M E T 9 and A19-6a, possibly homologous to DitU in LB400. If this were the case, these previous findings would imply that the DitU homologues in B K M E - 9 and A19-6a were up-regulated or constitutively expressed during growth on DhA, which was not the 365 case for LB400. This also raises the possibility that DitU-LB400 may also be able to transform DhA; however, because the enzyme is not expressed by LB400 during growth on DhA it cannot complement DitQ activity. 125 4.4.3 Binding properties of DitQ The binding of DhA to DitQ (Fig. 4.5) agrees with our previous report for DitQ-BKME-9 370 (Chapter 2). However, DitQ-BKME-9 has a lower Kd than DitQ-LB400, which indicates that the affinity of DitQ-BKME-9 for DhA is greater than that of DitQ-LB400. DitQ-LB400 showed a type I-like binding spectrum for PaA, whereas titration of PaA with DitQ-BKME-9 caused a perturbation of the heme environment but results were inconclusive. Recently, however, atypical type I red-shifted binding spectra, similar to the one observed for DitQ-BKME-9 bound to PaA, 375 have been reported for P450 EpoK bound to the polyketide epothilone D (19). EpoK has been shown to cause the epoxidation of epothilone D. This result raises the possibility that DitQ-B K M E - 9 , which shares 71 % amino acid identity with DitQ-LB400 (see Table 4.4), also binds PaA. Alternatively, it could be that the substrate range of DitQ-LB400 is wider than DitQ-B K M E - 9 . 380 Both proteomic and mutant phenotype analysis imply that DitU is involved in the catabolism of AbA,. however, substrate binding assays and in vitro activity assays were inconclusive. This may indicate that the natural substrate for this enzyme has not been identified or the in vitro assay system for this enzyme was ineffective. In a recent report, Simgen et al. (25) were able to catalyze the 15(3-hydroxylation of deoxycorticosterone with the purified P450 m e g 385 (CYP106A2), adrenodoxin and adrenodoxin reductase. They were not, however, able to show evidence of binding using UV-Vis methods (25). Similarly, results from an investigation of P450 m o r , isolated from Mycobacterium sp. Strain HE5, did not show evidence of binding of morpholine (up to 50 mM) to the purified HIS-tagged P 4 5 0 m O r . Morpholine is a putative native substrate for the enzyme. However, an in vitro P450 assay utilizing the purified enzyme in the 390 presence of its native ferredoxin and ferredoxin reductase showed turnover of this substrate [29 + 3.0 nmol morpholine"1 min"1 (nmol P450)"1] (24). Further investigation with purified catalytic 126 subunits and electron transfer components may be necessary to establish the substrate range and activity of DitU. 4.4.4 Demonstration of DitQ activity 395 This thesis provides the missing evidence that DitQ catalyzes the C7 hydroxylation of DhA (Fig. 4.6). This conclusion is supported by the mutant phenotypes. Both LB400 and DitUKO accumulated 7-oxo-DhA in cell suspensions with DhA, whereas, DitQKO was not able to transform DhA, indicating that DitQ is required for this activity. DhA showed the lowest Kd with DitQ, which was the only substrate yielding a detectable product. This product was reported 400 as a metabolite found in cultures of A. eutrophus incubated with DhA (2) and more recently as a biotransformation product oi Aspergillus niger (10), however, no enzyme responsible for this transformation has been identified. Even though PaA was shown to bind to DitQ-LB400, no transformation product from this substrate was observed in the in vitro activity assay. Inefficient electron transfer to the 405 catalytic subunit using non-native electron transport components may have contributed to the lack of turnover of PaA, as suggested for DitU. Further investigation is needed to determine the substrate range of DitQ with native electron transport components (see below). 4.4.5 Proposed initial steps of the diterpenoid pathway \ Both DitU and DitQ likely function in the conversion of abietane diterpenoids to 7-oxo-410 DhA (Fig. 4.7). During growth on DhA (V), ditQ is up-regulated and expressed. DitQ then binds DhA and catalyzes hydroxylation to 7-hydroxy-DhA (VII). The hydroxyl group is presumably further oxidized to 7-oxo-DhA (VIII) by an unidentified dehydrogenase. 7-oxo-DhA (VIII) is a substrate for DitA dioxygenase activity (14). Previous reports on B K M E - 9 suggested that A b A (I, Fig. 4.7) is transformed to DhA (V) 415 followed by catabolism as described above (13, 15, 16). However, there is no direct evidence of aromatization of the C ring or conversion of AbA to DhA prior to C7 hydroxylation neither in 127 B K M E - 9 nor in LB400. The current study provides new evidence that suggests a different route for AbA degradation (Fig. 4.7). The main route of AbA (I) degradation requires the activity of DitU, even though growth on AbA leads to the expression of both DitU and DitQ. Accumulation 420 of 7-oxo-DhA (VIII) by a ditAl knockout on A b A supports that 7-oxo-DhA is an intermediate in AbA catabolism (Chapter 3). DitUKO cell suspensions reported in this Chapter revealed that DitU is required for transformation of AbA to 7-oxo-DhA. This raises the possibility that DitU catalyzes a step in the transformation of AbA to 7-oxo-DhA. Others have speculated that transformation of A b A involves the formation of 7-hydroxy-AbA (3), and 7,8 epoxy-AbA (III) 425 (22). Chemical transformations of A b A have produced 7-acetoxy-DhA in a single step reaction with mercuric acetate (1). Also, fungal biotransformation of pimaradiene diterpenes, containing a B ring structure with a 7,8 double bond similar to AbA, revealed that the main products contained the epoxidation of the 7,8 double bond and 7-oxo derivatives (Chapter 1, Table 1.1) (6-8). Fraga et al. (6) speculated that the 7,8 epoxide rearranged to the 7-ketone by opening of 430 the oxirane ring. A 7-oxo product of AbA catabolism would require elimination or shift in the 7,8 double bond. Such a shift could result in 7-oxo-PaA (VI), which was tentatively identified in extracts of both PaA and AbA cell suspensions with the D i t A l K O mutant strain (Chapter 3) and in the present cell suspensions as Unknown I (Fig. 4.3). Perhaps 7-oxo-PaA (VI) is a common intermediate in AbA (I) and PaA (II) metabolism prior to aromatization of the C ring yielding 7-435 oxo-DhA (VIII) (see discussion below) (Fig. 4.7). A gene or enzyme leading to the aromatized ring C product has not been identified. Two pathways may exist for the degradation of PaA (II) by LB400 that can be catalyzed by either DitQ or DitU (Fig. 4.7). Cells incubated in the presence of PaA revealed that both DitQKO and DitUKO mutant strains were able to transform PaA to DhA (Fig. 4.4) as was 128 440 Figure 4.7 Proposed convergent pathway for abietane diterpenoid degradation by LB400.1. AbA; II, palustric acid; III, 7,8-epoxy-abietic acid; IV, 7-hydroxy-palustic acid ; V , dehydroabietic acid; VI, 7-oxo-palustric acid; VII, 7-hydroxy-dehydroabietic acid; VIII, 7-oxo-dehydroabietic acid; LX, 7-oxo-ll,12-dihydroxy-8,13-abieadien acid; X 7-oxo-ll,12-445 dihydroxydehydroabietic acid. Enzymes next to arrows indicate the reaction proposed to be catalyzed by that enzyme. 129 observed with D i t A l K O (Chapter 3). It follows that DhA (V) would then be degraded as described above via 7-hydroxy-DhA (VII). Alternatively, in Chapter 3 evidence was given for hydroxylation of PaA prior to aromatization of the C ring resulting in 7-oxo-PaA (VI) which 450 would be degraded as described above via 7-oxo-DhA (VIII). In LB400 the question is, is DitQ or DitU involved in one or both of these pathways or is there redundancy between the activities of the two P450s? Substrate binding assays showed that DitQ binds PaA and is therefore likely a substrate for the enzyme: DitQ could then catalyze the transformation of PaA (II) to 7-hydroxy-PaA (IV). However, i f only DitQ is expressed during growth on PaA this would not allow for 455 growth or substrate removal by DitQKO on PaA (Fig 4.3, 4.4 & 4.7). On the other hand, i f only DitU is expressed during growth on PaA this would not allow for growth or substrate removal by DitUKO on PaA. However, i f both DitQ and DitU are up-regulated, then during growth and in cell suspensions of DitQKO on PaA, DitU could transform PaA to 7-hydroxy-PaA (IV). It is also possible, as speculated in B K M E - 9 DitQ knockouts, that DitU is able to bind and transform DhA 460 (V) to 7-hydroxy-DhA (VII). During growth and cell suspensions of DitUKO, DitQ could catalyze the same transformations of PaA and DhA. More experimentation is required to determine the expression pattern of both P450s and transformations catalyzed by the two enzymes. 4.4.6. Electron transport ferredoxins 465 In the previous chapter, the possibility of a single ferredoxin reductase component functioning with both P450s and the ring hydroxylating dioxygenase was presented. The evidence there suggests that ditA3 encoding a 3Fe-4S ferredoxin functions with the dioxygenase. This class of ferredoxin can function in P450 electron transport (20), raising the possibility that DitA3 also functions with DitQ and/or DitU. The B K M E - 9 homologue of DitA3 was used in the 470 in vitro P450 assay for substrate transformation of DhA to 7-hydroxy-DhA, whereas assays which did not include DitA3 where not able to form a detectable product. This suggests that 130 DitA3 is used in both dioxygenase and monooxygenase electron transport systems. However, other evidence from a B K M E - 9 DitA3 mutant strain suggests that expression of DitA3 is not required for P450 activity (13). This mutant was not able to grow on DhA but did transform the 475 substrate to 7-oxo-DhA. This finding supports its role in DitA catalytic function, but also suggests that another ferredoxin must be capable of electron transport in any P450(s) involved in oxygenation of C7. BxeC0601, located two ORFs downstream of ditQ, is predicted to encode a typical plant-type 2Fe-2S ferredoxin component. Perhaps this second ferredoxin functions in electron transport for one or both of the dit P450s. If this were the case it would result in a 480 complex system of electron transfer involving a single reductase, two ferredoxins, one a 3Fe-4S and the other a 2Fe-2S, two cytochromes P450 and a ring hydroxylating dioxygenase. Further study is needed with purified electron transfer and catalytic components. 4.4.7.CYP226A family 1 DitQ-LB400 and DitU-LB400 are members of the cytochrome P450 CYP226A family. 485 Based on sequence identity, there are six identified members of the CYP226A family (Table 4.4) (18), four of which are from bacteria that grow on abietane diterpenoids as a sole source of carbon and energy. DitQ-LB400 and DitU-LB400 have been designated CYP226A1 and CYP226A2, respectively. The only other classified member of this family is TdtD (CYP226A3) from the abietane diterpenoid degrading Pseudomonas diterpeniphila A19-6a (17). The B K M E -490 9 homologue of DitQ has not yet been classified, but according to sequence identity of greater than 60% with other members of the family, DitQ from B K M E - 9 would also be included in this family. The remaining two P450s were identified in the recently sequenced genome of P. aeruginosa 2192 and are included in a putative 2192 dit cluster (Chapter 3). We suspect that this bacterium is also able to catabolize abietane diterpenoids, however this has not yet been 495 confirmed. It is also worth noting that homologues of both CYP226A1 and CYP226A2 were identified in the Sargasso Sea metagenome (27). It is possible that all members of the CYP226A 131 P450 family are involved in diterpenoid catabolism. This may allow for the prediction of other abietane diterpenoid degrading strains based on the presence of high sequence similarity with members of CYP226A. Table 4.4 CYP226A family 500 DitQ-LB400 DitQ-BKME-9 DitQ-A19-6a DitQ-2192 DitU-LB400 DitU-2192 DitQ-LB400 ID 70.8 72.2 70.8 60.9 56.7 DitQ-BKME-9 70.8 ID 84.1 84.4 62.1 60.7 DitQ-A19-6a 72.2 84.1 ID 93.8 63.0 61.6 DitQ-2192 70.8 84.4 93.8 ID 62.1 61.4 DitU-LB400 60.9 62.1 63.0 62.1 ID 64.7 DitU-2192 56.7 60.7 61.6 61.4 64.7 ID Deduced amino acid percent sequence identity matrix. ID, identical 4.5 Conclusion To our knowledge, this is the first clear demonstration of an enzyme, DitQ, which is responsible for C7 hydroxylation of an abietane diterpenoid in a degradation pathway. This type 505 of enzyme appears widespread, as similar transformation products are commonly identified in the lysates of organisms incubated with diterpenoids for the purpose of identifying possible metabolites in eukaryotic systems or for means of producing more effective drug candidates (Chapter 1). The evidence is less strong for an analogous role of DitU in the transformation of AbA, however, our findings are consistent for a role similar to DitQ. Further work will focus on 510 understanding the kinetics of the purified P450s as well as elucidating electron transport systems required for the degradation of abietane diterpenoids. 132 4.6. References 1. Barrero, A. F., E. J. AIvarez-Manzaneda, R. Alvarez-Manzaneda, R. Chahboun, R. 515 Meneses, and M. Aparicio B. 1999. Ring A functionalization of terpenoids by the unusual Baeyer-Villiger rearrangement of aliphatic aldehydes. Synlett:713-716. 2. Biellmann, J. F., G. Branlant, M. Gero-Robert, and M. Poiret. 1973. Degradation bacterienne de l'acide dehydroabietique par un Pseudomonas et une Alcaligenes. 520 Tetrahedron 29:1237-1241. 3. Cross, B. E., and P. L. Myers. 1968. The bacterial transformation of abietic acid. Biochem J 108:303-10. 525 4. de Lorenzo, V., and K. N. Timmis. 1994. Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and TnlO-derived minitransposons. Methods Enzymol 235:386-405. 5. Denef, V. J., M. A . Patrauchan, C. Florizone, J. Park, T. V. Tsoi, W. Verstraete, J. 530 M. Tiedje, and L . D. Eltis. 2005. Growth substrate- and phase-specific expression of biphenyl, benzoate, and CI metabolic pathways in Burkholderia xenovorans LB400. J. Bacteriol. 187:7996-8005. 6. Fraga, B. M., P. Gonzalez, M. G. Hernandez, M. C. Chamy, and J. A . Garbarino. 535 2003. Microbial transformation of 18-hydroxy-9,13-epi-ent-pimara-7,15-diene by Gibberella fujikuroi. J Nat Prod 66:392-7. 7. Fraga, B. M., P. Gonzalez, M. G. Hernandez, M. C. Chamy, and J. A . Garbarino. 1998. The microbiological transformation of a 9-epi-ent-pimaradiene diterpene by 540 Gibberella fujikuroi. Phytochemistry 47:211-215. 8. Fraga, B. M., M. G. Hernandez, P. Gonzalez, M. C. Chamy, and J. A . Garbarino. 2000. The biotransformation of 18-hydroxy-9-epi-ent-pimara-7,15-diene by Gibberella fujikuroi. Phytochemistry 53:395-399. 545 9. Goris, J., P. De Vos, J. Caballero-Mellado, J. Park, E. Falsen, J. F. Quenseh, 3rd, J. M. Tiedje, and P. Vandamme. 2004. Classification of the biphenyl- and polychlorinated biphenyl-degrading strain LB400T and relatives as Burkholderia xenovorans sp. nov. Int J Syst Evol Microbiol 54:1677-81. 550 . 10. Gouiric, S. C , G. E. Feresin, A . A . Tapia, P. C. Rossomando, G. Schmeda-Hirschmann, and D. A . Bustos. 2004. ip,7(3-Dihydroxydehydroabietic acid, a new biotransformation product of dehydroabietic acid by Aspergillus niger. World Journal of Microbiology and Biotechnology 20:281-284. 555 11. Hoang, T. T., R. R. Karkhoff-Schweizer, A . J. Kutchma, and H. P. Schweizer. 1998. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located D N A sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77-86. 560 133 12. Kinter, M., and N. E. Sherman. 2000. Protein sequencing and identification using tandem mass spectrometry. John Wiley, New York. 13. Martin, V. J., and W. W. Mohn. 2000. Genetic investigation of the catabolic pathway 565 for degradation of abietane diterpenoids by Pseudomonas abietaniphila BKME-9 . J Bacteriol 182:3784-93. 14. Martin, V. J., and W. W. Mohn. 1999. A novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium Pseudomonas abietaniphila 570 B K M E - 9 . J Bacteriol 181:2675-82. 15. Martin, V. J., Z. Yu, and W. W. Mohn. 1999. Recent advances in understanding resin acid biodegradation: microbial diversity and metabolism. Arch Microbiol 172:131-8. 575 16. Martin, V. J. J. 1999. Molecular genetic investigation of the abietane diterpenoid degradation pathway oi Pseudomonas abietaniphila BKME-9 . Doctor of Philosophy. University of British Columbia, Vancouver. 17. Morgan, C. A., and R. C. Wyndham. 2002. Characterization of tdt genes for the 580 degradation of tricyclic diterpenes by Pseudomonas diterpeniphila A19-6a. Can J Microbiol 48:49-59. 18. Nelson, D. R., L. Koymans, T. Kamataki, J. J. Stegeman, R. Feyereisen, D. J. Waxman, M. R. Waterman, O. Gotoh, M. J. Coon, R. W. Estabrook, I. C. Gunsalus, 585 and D. W. Nebert. 1996. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6:1-42. 19. Ogura, H., C. R. Nishida, U. R. Hoch, R. Perera, J. H. Dawson, and P. R. Ortiz de Montellano. 2004. EpoK, a cytochrome P450 involved in biosynthesis of the anticancer 590 agents epothilones A and B. Substrate-mediated rescue of a P450 enzyme. Biochemistry 43:14712-21. 20. O'Keefe, D. P., K. J. Gibson, M. H. Emptage, R. Lenstra, J. A. Romesser, P. J. Litle, and C. A. Omer. 1991. Ferredoxins from two sulfonylurea herbicide monooxygenase 595 systems in Streptomyces griseolus. Biochemistry 30:447-55. 21. Patrauchan, M. A., C. Florizone, M. Dosanjh, W. W. Mohn, J. Davies, and L. D. Eltis. 2005. Catabolism of benzoate and phthalate in Rhodococcus sp. strain RHA1: redundancies and convergence. J. Bacteriol. 187:4050-4063. 600 22. Prinz, S., U. Mullner, J. Heilmann, K. Winkelmann, O. Sticher, E. Haslinger, and A. Hufner. 2002. Oxidation products of abietic acid and its methyl ester. J Nat Prod 65:1530-4. 605 23. Schweizer, H. P., and T. T. Hoang. 1995. An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158:15-22. 24. Sielaff, B., and J. R. Andreesen. 2005. Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising 134 the morpholine mono-oxygenase from Mycobacterium sp. strain HE5. Febs J 272:1148-59. Simgen, B., J. Contzen, R. Schwarzer, R. Bernhardt, and C. Jung. 2000. Substrate binding to 15(3-Hydroxylase (CYP106A2) probed by FT infrared spectroscopic studies of the iron ligand CO stretch vibration. Biochemical and Biophysical Research Communications 269:737-742. Simons, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Techonology 1:784-790. Venter, J. C , K. Remington, J. F. Heidelberg, A. L. Halpern, D. Rusch, J. A. Eisen, D. Wu, I. Paulsen, K. E. Nelson, W. Nelson, D . E. Fouts, S. Levy, A. H. Knap, M. W. Lomas, K. Nealson, O . White, J. Peterson, J. Hoffman, R. Parsons, H. Baden-Tillson, C. Pfannkoch, Y.-H. Rogers, and H. O . Smith. 2004. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304:66-74. 135 5. Conclusion This dissertation greatly advances our understanding of the microbial metabolism of diterpenoids. Previous to the work presented here, a 16.7-kbp cluster of genes in BKME-9 , including those encoding ring hydroxylating and ring cleavage dioxygenases, (3) and a 9.18-kbp 5 cluster of genes, including a putative P450, (4) had been reported. We expanded knowledge of the B K M E - 9 dit cluster, showing that it contained a sequence of genes, including one encoding a cytochrome P450, with high deduced amino acid sequence identity and identical gene order to the tdt cluster. Using the characterized B K M E - 9 dit cluster, we identified an 80.5-kbp LB400 cluster of genes in LB400 containing many homologues with high sequence similarity to the 10 B K M E - 9 dit cluster. Finally, through transcriptomic and proteomic analysis we characterized the genes of the 80.5-kbp LB400 dit cluster up-regulated during growth on abietane diterpenoids. Recently, another putative dit cluster was identified in the sequenced genome of Pseudomonas aeruginosa 2192 (Pseudomonas aeruginosa 2192 Sequencing Project, Broad Institute of Harvard and MIT (http://www.broad.mit.edu)). The dit cluster is emerging as a common catabolic set of 15 genes in Proteobacterial genomes and likely will be found in more bacterial genomes as they become available. There is still much that remains unclear regarding the genes of the LB400 dit cluster. Of the 72 genes identified, we can confidently assign functions to only 6 catabolic gene products, which are involved in the transformation of abietane diterpenoids to an aromatic diol 20 intermediate, a substrate for ring cleavage (see Chapter 4 Fig. 4.7). The function of the other 66 genes is unclear, although they presumably encode all components required to import and transform abietane diterpenoids into common intermediates of the central catabolic pathways. Regarding uptake, the LB400 dit cluster encodes 4 putative transport proteins, which could 136 function in either uptake of substrate or export of toxic intermediates. Regarding downstream 25 catalysis of the cleaved C ring product, the dit cluster encodes 3 putative hydrolases, which could function in hydrolysis of carbon-carbon bonds splitting the C20 compound into smaller units, p-oxidation would likely then play a role with the dit cluster encoding several genes putatively involved in acyl-CoA metabolism including a CoA ligase, at least four acyl-CoA dehydrogenases, two putative enoyl-CoA hydratases, one 3-hydroxyacyl-CoA dehydrogenase, 30 one acetyl CoA acetyl transferase. Also encoded by the dit cluster are more than 20 genes encoding conserved hypothetical proteins with no known function. With such a large cluster of genes, many encoding homologous proteins, it raises questions regarding redundancy. Do homologous proteins catalyze the same reaction or are different homologues required for the catabolism of different substrates, possibly other varieties of diterpenoids? Further study is 35 . needed to answer these questions. Members of the CYP226A family play a key role in the initial steps of the catabolism of abietane diterpenoids. Previous reports implicated a P450 in the degradation pathway but gave no conclusive evidence for its role (4). We have shown that not one but two P450s are involved in the degradation of abietanes in LB400 and gave evidence that this is likely also the case in 40 B K M E - 9 . Using the information gained from these studies of the two P450s in abietane metabolism we were able to provide a clear model of the initial steps in the catabolism of DhA to 7-oxo-DhA. Although, we were able to show that DitU is involved in A b A and PaA catabolism, we were not able to confidently identify a substrate for DitU or show enzymatic activity. With respect to PaA catabolism, the involvement of DitQ was implicated through substrate binding 45 data using both DitQ-BKME-9 and DitQ-LB400, while our understanding of the involvement of 137 DitU is not clear. Further work on purified enzyme systems maybe necessary for a better understanding of the substrates and enzymes involved in this catabolic pathway. The major components of the anabolic pathway for abietane diterpenoid production in conifers is fairly well understood as reviewed in Chapter 1. We have recently begun to gain 50 insight into the catabolism of these natural products at a molecular level, leading to a better understanding of the complete carbon cycle of these abundant compounds. Previous to this study 3 pathways for DhA degradation had been identified, one via a 3-oxo intermediate (2), one via a 7-oxo-DhA intermediate (3), and a third via a combination of the 3-oxo and 7-oxo pathways (1). Does the newly gained knowledge presented here shed any light on the general scheme of 55 microbial degradation of the broad range of tricyclic diterpenoids via 7-oxo intermediates? In Chapter 1 we posed the question, does degradation rely on a few enzymes with broad specificities, or does there seem to be a proliferation of divergent degradative enzymes specific for the broad range of terpenoids? Similar to a divergent synthetic pathway requiring few relatively few enzymes for the synthesis of a broad range of diterpenoids, (Chapter 1, Fig. 1.5) 60 could the same scheme be utilized in their catabolism, that is relatively few enzymes are used to transform the broad range of diterpenoids into a common intermediate in a convergent degradation pathway. The limited range of substrates tested in this dissertation cannot clearly answer this question. However, with respect to abietane diterpenoid metabolism by B K M E - 9 and LB400, results are consistent with a few enzymes with broad substrate specificities required 65 for conversion to a central intermediate in a convergent pathway (Chapter 4, Fig. 4.7). What evidence exists for the catabolic pathways of other tricyclic diterpenoids? Included in this group is a class of compounds structurally related to abietane diterpenoids, the pimeranes. Pimeranes are synthesized from the same enzymes as abietanes, except that while the abietanes 138 have an isopropyl group at C13, the pimeranes have a methyl and a vinyl group (see Chapter 1 -70 Fig. 1.5). To date, we have very limited data regarding the pathway for pimerane catabolism. In general, gram negative bacteria that can grow on abietanes cannot necessarily utilize pimeranes, whereas strains that can grow on pimeranes usually utilize abietanes (5). Based on the observation that most pimerane-degrading strains can grow on abietane diterpenoids, it is possible that the pimeranes share a common intermediate with abietane catabolism. What are the 75 genetic traits distinguishing these capabilities? Do they have additional genes required for transformation of pimeranes to a common intermediate, or do pimerane-degrading strains possess genes encoding the similar enzymes as abietane-degrading strains with a wider substrate range. If it is in fact a convergent pathway, what is the common intermediate? As more genome sequence data becomes available, a strain capable of growth on both classes of diterpenoids will 80 likely be sequenced. Interestingly, the P. aeruginosa 2192 putative dit cluster possesses genes encoding a second set of alpha- and beta-subunits of a ring hydroxylating dioxygenase sharing greater than 60% amino acid identity with D i t A l and DitA2. Could this dioxygenase be required for conversion of pimeranes to a central intermediate of the diterpenoid pathway? Work presented in this dissertation has provided the background to experimentally investigate this and 85 other questions regarding abietane diterpenoid degradation. 5.1 Comments on future research Many possibilities exist for further work regarding the catabolism of abietane diterpenoids, including mechanisms of substrate transport, and genomic investigation of other putative abietane diterpenoid degrading strains, such as P. aeruginosa 2192. Recommendations 90 for future work, however, focus on the P450s involved in abietane diterpenoid metabolism. Preliminary, biochemical analysis of both DitQ and DitU in this dissertation was conducted in 139 crude lysate of E. coli. Purification of DitQ and DitU and their putative electron transport components should lead to clarification of the role of these enzymes in the catabolism of abietane diterpenoids. These enzymes represent a novel family of P450s involved in natural 95 product catabolism. Characterization of both DitQ and DitU and deciphering their complex electron transport system, potentially involving a single reductase functioning with 2 types of ferredoxins, a dioxygenase and 2 cytochromes P450 in a natural systems would be a novel finding and expand our understanding of electron transport in oxygenase systems. 100 5.4 References 1. Biellmann, J. F., G. Branlant, M. Gero-Robert, and M. Poiret. 1973. Degradation bacterienne de l'acide dehydroabietique par Flavobacterium resinovorum. Tetrahedron 29:1227-1236. 105 2. Kieslich, K. 1976. Microbial transformations of non-steroid cyclic compounds. J. Wiley, Chichester, Eng. 3. Martin, V. J., and W. W. Mohn. 2000. Genetic investigation of the catabolic pathway 110 for degradation of abietane diterpenoids by Pseudomonas abietaniphila BKME-9 . J Bacteriol 182:3784-93 4. Morgan, C. A . , and R. C. Wyndham. 2002. Characterization of tdt genes for the degradation of tricyclic diterpenes by Pseudomonas diterpeniphila A19-6a. Can J 115 Microbiol 48:49-59. 5. Wilson, A . E., E. R. Moore, and W. W. Mohn. 1996. Isolation and characterization of isopimaric acid-degrading bacteria from a sequencing batch reactor. Appl Environ Microbiol 62:3146-51. 120 140 A P P E N D I X BxeB2301 Aconitate Hydratase BxeA1362 Putative | Dehydrogenase BxeC0586 DitAl-Alpha subunit of Ring Hydroxylating Dioxygenase BxeC0585 DitH-Putative Fumarylacetoacetate Hydrolase | BxeC1186 BphD-Hydrolase BxeC0639 DitB-Putative Dehydrogenase BxeB0962 Putative CoA Hydratase BxeB1203 Putative Isocitrate Lyase and Phosphomutase family protein BxeA1366 Putative 3-oxoacid CoA Transferase alpha subunit BxeC0619 Putative Short Chain Dehydrogenase BxeC0587 DitA2-Beta subunit of Ring Hydroxylating Dioxygenase Figure A . l . Dehydroabietic Acid Proteome. BxeC0586 DitAl-Alpha subunit of Ring Hydroxylating Dioxygenase BxeC0606 Conserved Hypothetical BxeC0592 DitJ-Putative CoA Ligase BxeA1362 Putative Dehydrogenase . . . 41 — BxeA1651 Putative Isocitrate Lyase BxeA1651 Putative Isocitrate Lyase BxeC0631-DitU Cytochrome P450 | BxeA3660 Putative GTPase BxeC0585 DitH-Putative Fumarylacetoacetate Hydrolase BxeC1186 BphD-Hydrolase I BxeB0314 Putative Transketolase BxeC0630 Conserved Hypothetical BxeC0631-DitU Cytochrome P450 | !BB B)(gC0599-DitQ Cytochrome P450 BxeC0597 DitO-Putative Thiolase BxeC0602 Putative Dehydrogenase BxeC0594 Conserved Hypothetical BxeC0644 Conserved Hypothetical BxeC0639 DitB-Putative Dehydrogenase • . * • BxeB0962 Putative CoA Hydratase BxeC0640 DitC Ring Cleavage Dioxygenase j BxeA2466 Putative LysR type Regulator Hj BxeC0644 Conserved Hypothetical .j BxeC063Tj ( BxeB1203 Putative Isocitrate Lyase and Phosphomutase family protein 125 Figure A.2 Abietic Acid Proteome BxeC0587 DitA2-Beta subunit of Ring Hydroxylating Dioxygenase BxeA1366 Putative 3-oxoacid CoA Transferase alpha subunit BxeC0619 Putative Short Chain Dehydrogenase ro 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0093012/manifest

Comment

Related Items