THE METABOLISM OF IBUPROFEN BY SPHINGOMONAS IBU-2
A Dissertation
Presented to the Faculty of the Graduate School
of Cornell University In Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
by
Robert W. Murdoch
January, 2012
© 2012 Robert W. Murdoch
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2 THE METABOLISM OF IBUPROFEN BY SPHINGOMONAS IBU-2
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4 Robert W. Murdoch, Ph. D. Cornell University 2012
5
6 Pharmaceutical have been detected in bodies of water all over the world and their
7 environmental fates are the subject of increasing scientific attention. Sphingomonas Ibu-
8 2 was isolated from a wastewater treatment plant based on its ability to use ibuprofen as a
9 sole carbon and energy source. A yellow color indicative of meta-cleavage accumulated
10 in the culture supernatant when Ibu-2 was grown on ibuprofen. When 3-flurocatechol
11 was used to poison the meta-cleavage system, isobutylcatechol was identified in the
12 culture supernatant via GC/MS analysis. Ibuprofen-induced washed cell suspension also
13 metabolized phenylacetic acid to catechol. A chromosomal library of Ibu-2 was
14 generated in Escherichia coli and yielded a single fosmid clone ( E. coli epi300
15 pFOS3G7) capable of metabolizing ibuprofen to isobutylcatechol. Transposon
16 mutagenesis of pFOS3G7 and screening for loss of function revealed insertions in five
17 open reading frames ipfABDEF whose predicted amino acid sequences bore similarity to
18 the large and small units of aromatic dioxygenases, an SCPx thiolase, domain of
19 unknown function 35 (DUF35), and an aromatic coenzyme A ligase, respectively.
20 Complementation of the E. coli epi300 pFOS3G7 ipfD deletion mutant restored catechol-
21 production. An E. coli subclone expressing ipfABDEF from pFOS3G7 produced trace
22 catecholic metabolites from phenylacetic acid derivatives, suggesting that these genes
23 were sufficient for the observed activity. The co-expression of ipfABDEF with ipfH and
1 ipfI, a ferredoxin reductase and a ferredoxin respectively which were both identified on
2 pFOS3G7, greatly enhanced the production of catecholic metabolites. Further analysis
3 revealed that cell-free extracts of E. coli harboring a high copy number ipfF expression
4 vector catalyzed the ATP and magnesium-dependent ligation of coenzyme A to both
5 phenyacetate and ibuprofen. Uptake of phenylacetic acid was also found to be driven by
6 the ibuprofen coenzyme A ligase IpfF; when ipfF was knocked out, uptake was decreased
7 to negative control levels as determined by radiolabelled phenyletate uptake assays.
8 These results are consistent with ibuprofen uptake occurring via a vectorial acylation
9 mechanism. Taken toghether, the results described in this dissertation demonstrate that in
10 contrast to the widely distributed coenzyme A ligase, homogentisate, or
11 homoprotocatechuate pathways for metabolism of phenylacetic acids, Ibu-2 removes the
12 acidic side chain of ibuprofen and related compounds prior to ring-cleavage. The
13 involvement of a novel SCPx thiolase (IpfD) and a DUF35 protein (IpfE) in the
14 generation of the catechol suggest that this pathway is distinct from others described to
15 date and represents a new aerobic paradigm for the metabolism of phenylacetic acids
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1 BIOGRAPHICAL SKETCH
2 Robert was raised near Houston where he spent his childhood exploring the bayous and
3 piney woods of East Texas. He had the honor of attending the Texas Academy of Math
4 and Science north of Dallas, where he simultaneously completed two years of high school
5 and two years of college courses at the University of North Texas. Robert then attended
6 the University of Texas at Austin where he earned his Bachelor’s degree in Zoology. For
7 the following two years, Robert worked as a fisheries biologist for the National Marine
8 Fisheries Service. The majority of this time was spent on board commercial deep sea 9 fishing vessels. He earned his sea legs on the Bering Sea, the North Pacific off the coast
10 of Washington, and the Pacific Ocean south of Hawaii, racking up a total of ten months
11 worth of days at sea. Robert then entered the Cornell University Environmental
12 Toxicology PhD program. In addition to diverse coursework in risk studies, toxicology,
13 and microbiology, Robert learned the molecular biology and biodegradation ropes under
14 the mentorship of Dr. Anthony Hay. After five years, Robert took a job with Dr. Andria
15 Costello at Syracuse University for two years studying environmental microbiology, after 16 which he returned to Cornell University to complete his PhD dissertation. 17
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1 ACKNOWLEDGMENTS
2 Many people have provided me with the guidance, moral support, and technical
3 assistance required to get me to this point. I especially appreciate my advisor Dr.
4 Anthony Hay who took me in with no microbiological or molecular biological experience 5 and took the time to patiently guide me towards this destination. My early labmates and
6 mentors Lauren Junker and Jeanne Kagle provided a very helpful and cheerful
7 environment in the first years of my time here at Cornell. My assistant Anastasia
8 Kazimirova provided invaluable technical support. My later labmates Andreas Toba,
9 Abigail Wise-Porter, Amy Risen, Giomar Rivera, Chris DeRito, Kevin George, Karl
10 Rueggeberg, Mitch Thompson, Mary Thompson, Jennie Sanders, Hanh Nguyen, and
11 Fadime Kara all helped to foster a positive work environment. Field administrative 12 assistant Cindy Urovcik was always ready to help me to navigate beaurocratic hurdles
13 and provide moral support. My first two years of funding were provided by the SUNY
14 Minority Fellowship and I also received a year of generous funding from the Cornell
15 Provost’s Diversity Dissertation Fellowship program. I would particularly like to thank
16 my parents Bruce and Carol Murdoch.
17
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1 TABLE OF CONTENTS
2 3 CHAPTER 1 INTRODUCTION ...... 1 4 PHARMACEUTICALS IN THE ENVIRONMENT ...... 1 5 IBUPROFEN ...... 6 6 The fate of ibuprofen in the environment ...... 9 7 Ibuprofen Ecotoxicology ...... 13 8 BACTERIAL METABOLISM OF AROMATIC CHEMICALS ...... 17 9 Classical double-dioxygenation metabolism of aromatics...... 18 10 Alternative aerobic ring cleavage substrates ...... 22 11 Anaerobic metabolism of aromatics ...... 23 12 METABOLISM OF PHENYLACETATE ...... 25 13 Fungal metabolism of phenylacetate...... 26 14 Early research into the bacterial metabolism of phenylacetate ...... 26 15 Phenylacetyl coenzyme A ligase pathway ...... 28 16 Bacterial metabolism of 2-phenylpropionate ...... 37 17 Metabolism of ibuprofen ...... 37 18 TRANSPORT OF AROMATICS AND RELATED CHEMICALS BY BACTERIA ...... 38 19 CONCLUSIONS ...... 42 20 CHAPTER 2 FORMATION OF CATECHOLS VIA ACID SIDE CHAIN 21 REMOVAL FROM IBUPROFEN AND RELATED AROMATIC ACIDS ...... 64 22 ABSTRACT ...... 64 23 INTRODUCTION ...... 65 24 METHODS ...... 66 25 Materials and Strains ...... 66 26 Isolation of Ibu-2 via enrichment of sewage sludge ...... 67 27 Stereospecificity ...... 67 28 Substrate specificity analysis with washed cells ...... 68 29 Growth substrate analysis ...... 68 30 Analysis of culture supernatants by GC/MS ...... 69 31 Analysis of catecholic ibuprofen intermediates ...... 70 32 Characterization of catechols produced from ibuprofen analogs ...... 70 33 Cell-free extract assays ...... 71 34 Assaying sidechain oxidation ...... 71 35 RESULTS AND DISCUSSION ...... 72 36 Stereospecificity ...... 72 37 GC/MS analysis of culture supernatant extracts ...... 73 38 Substrate specificity analysis ...... 76 39 Growth substrate analysis ...... 76 40 Characterization of catechols produced from ibuprofen analogs ...... 79 41 Characterization of meta-cleavage products of ibuprofen analogs...... 79 42 Cell-free extract activities upon ibuprofen...... 79 43 CONCLUSIONS ...... 80
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1 ACKNOWLEDGEMENTS ...... 81 2 CHAPTER 3 GENETIC AND CHEMICAL CHARACTERIZATION OF 3 IBUPROFEN AND PHENYLACETIC ACID METABOLISM BY 4 SPHINGOMONAS IBU-2 ...... 86 5 ABSTRACT ...... 86 6 INTRODUCTION ...... 87 7 MATERIAL AND METHODS ...... 91 8 Materials ...... 91 9 Creation and screening of Ibu-2 fosmid library...... 92 10 Creation and metabolic screening of fosmid clone transposon libraries ...... 92 11 Functional analysis of pFOS3G7 clones via HPLC...... 95 12 Fosmid subcloning ...... 96 13 Metabolic analyses of subclones ...... 97 14 Complementation of pFOS3G7 ipfABDEF mutants...... 99 15 In silico analyses ...... 100 16 Expression of ipfD and ipfE in E. coli K12 paa operon mutants ...... 101 17 Metabolism of Phenylacetyl Coenzyme A in cell-free extracts ...... 101 18 RESULTS ...... 103 19 Ibu-2 fosmid library ...... 103 20 Metabolic analyses of clones and constructs ...... 106 21 Expression of ipfDE in E. coli K12 paa mutants...... 113 22 DISCUSSION ...... 115 23 ipfA ...... 115 24 ipfB ...... 118 25 ipfD ...... 119 26 ipfE ...... 126 27 ipfF ...... 129 28 ipfH ...... 129 29 ipfI ...... 130 30 Ipf pathway overview ...... 130 31 ACKNOWLEDGEMENTS ...... 136 32 CHAPTER 4 UPTAKE OF PHENYLACETIC ACID BY SPHINGOMONAS IBU- 33 2 FOSMID LIBRARY CLONE 3G7 ...... 146 34 ABSTRACT ...... 146 35 INTRODUCTION ...... 147 36 METHODS ...... 150 37 Strains and Clones: Construction and Identity ...... 151 38 Coenzyme A ligase activity ...... 152 39 Uptake in Ibu-2 and E. coli constructs ...... 154 40 Effect of metabolic poisons on phenylacetic acid uptake by E. coli epi300 pFOS3G7, 41 and E. coli epi300 pFOS3G7Tn:ipfF ...... 154 42 Effect of pH on uptake by E. coli epi300 pFOS3G7...... 155 43 Uptake Assays ...... 155 44 RESULTS ...... 157 45 Assaying coenzyme A ligase activity ...... 157
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1 Uptake of phenylacetic acid in all strains, clones, and knockouts ...... 158 2 Effect of metabolic poisons on E. coli epi300 pFOS3G7, and E. coli epi300 3 pFOS3G7Tn:ipfF ...... 159 4 Effect of pH on uptake ...... 160 5 DISCUSSION ...... 161 6 ACKNOWLEDGEMENTS ...... 170 7 CHAPTER 5 CONCLUSIONS ...... 177 8 PHARMACEUTICALS IN THE ENVIRONMENT : THE SCOPE OF THE CHALLENGE TO THE 9 SCIENTIFIC COMMUNITY ...... 178 10 METABOLISM OF IBUPROFEN BY SPHINGOMONAS IBU -2 ...... 181 11 POINTS OF UNCERTAINTY IN THE IBU -2 IBUPROFEN METABOLISM MODEL ...... 183 12 ENVIRONMENTAL RELEVANCE OF THE IBU -2 IBUPROFEN PATHWAY ...... 185 13 FUTURE WORK ...... 188 14 Future Work: Environmental ...... 188 15 Future Work: Microbiological ...... 190 16 APPENDIX 1 A NOVEL MECHANISM FOR THE BIODEGRADATION OF 17 IBUPROFEN ...... 203 18 PREFACE ...... 203 19 ABSTRACT ...... 203 20 INTRODUCTION ...... 204 21 MATERIALS AND METHODS ...... 207 22 RESULTS AND DISCUSSION ...... 210 23 APPENDIX 2 E. COLI EPI00 PFOS3G7 METHODOLOGICAL 24 CONSIDERATIONS ...... 221 25 THE EFFECT OF P H ON FOSMID ACTIVITY ...... 221 26 FOSMID AERATION ...... 223 27 ADDRESSING POSSIBLE INSTABILITY OF THE FOSMID ...... 226 28 REFERENCES ...... 230 29 APPENDIX 3 IDENTITY AND INITIAL CHARACTERIZATIONS OF OTHER 30 GENES OF INTEREST IN SPHINGOMONAS IBU-2 FOSMID LIBRARY 31 CLONES ...... 231 32 ABSTRACT ...... 231 33 ADDITION OF THE ELECTRON TRANSPORT GENES TO SUBLCONED IPF ABDEF (PJ25) 233 34 ADDITIONAL GENES OF INTEREST ...... 242 35 DIOL -DEHYDROGENASE IPF L ...... 242 36 AAA+ ATP ASE IPF R ...... 245 37 ACYL -COA RACEMASE IPF T AND ENOYL -COA HYDRATASE IPF Y ...... 251 38 CONCLUSIONS ...... 254 39 PERIPLASMIC BINDING PROTEIN IPF G ...... 255 40 APPENDIX 4 ADDITIONAL EXPERIMENTS AND LINES OF INQUIRY ...... 264 41 PREFACE ...... 264
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1 SCREENING , METABOLIC , AND GENETIC WORK DONE PRIOR TO THE FOSMID LIBRARY 2 ...... 264 3 Screening assays ...... 264 4 Mineralization assays ...... 265 5 Investigation of megaplasmids in Sphingomonas Ibu-2 ...... 268 6 Sphingomonas Ibu-2 antibiotic resistances and attempts to create knockouts ...... 271 7 Generating an isobutylcatechol standard ...... 272 8 Early attempts to gain a foothold in the Ibu-2 ibuprofen catabolic genes ...... 272 9 Activity of ibuprofen-induced aerobically grown Ibu-2 under anoxic conditions .. 277 10 CONSTRUCTS AND CELL -FREE ASSAYS ...... 278 11 Meta-cleavage product degradation assay ...... 278 12 Catechol 2,3 dioxygenase ...... 281 13 Phenylacetyl-CoA disappearance when incubated with Ibu-2 cell-free extract ..... 283 14 Cloning ipfFG ...... 284 15 Cloning the ferredoxins ...... 284 16 Cloning pJ25 into Novosphingomonas aromaticivorans F199...... 286 17 ipfF toxicity assay...... 286 18 ENVIRONMENTAL WORK ...... 290 19 Spiking sewage sludge with ibuprofen and 3-fluorocatechol...... 290 20 Spiking bacterial fractionated sludge with ibuprofen ...... 291 21 Isolation of additional ibuprofen-growing organisms ...... 291 22 Incubation of environmental bacterial fractions with ibuprofen and m-tolylacetic 23 acid ...... 292 24 APPENDIX 5 IBU-2 DNA SEQUENCES AND ADDITIONAL PUTATIVE 25 PROTEIN CHARACTERIZATION...... 296 26 ASSEMBLED SPHINGOMONAS IBU -2 DNA SEQUENCES ...... 296 27 PHYLOGENETIC ANALYSES OF PUTATIVE GENE PRODUCTS OF IPF A AND IPF B ...... 303
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1 LIST OF FIGURES
2 3 Figure 1.1. Common non-steroidal anti-inflammatory pharmaceuticals with a 4 phenylacetic acid moiety (bolded)...... 7 5 Figure 1.2. General action of cyclooxygenase enzymes (Fent, Weston et al. 2006)...... 8 6 Figure 1.3. Human primary metabolites of ibuprofen (Rudy, Knight et al. 1991; Lee, 7 Peart et al. 2005)...... 10 8 Figure 1.4. Basic features of the double-dioxygenation paradigm for the aerobic 9 metabolism of aromatic chemicals by bacteria. “Upper pathway” broadly refers to the 10 reactions leading up to initial ring dioxygenation while “lower pathway” refers to all 11 reactions following the initial dioxygenation...... 20 12 Figure 1.5. General scheme of the 1,2 dioxygenation cmt pathway (Eaton 1996; Eaton 13 1997)...... 21 14 Figure 1.6. General scheme of the benzoyl-CoA paradigm for the anaerobic metabolism 15 of aromatic chemicals by bacteria (Carmona, Zamarro et al. 2009)...... 24 16 Figure 1.7. The phenylglyoxylate pathway (Dangel, Brackmann et al. 1991), which 17 channels phenylacetate to the central benzoyl-CoA pathway under anaerobic conditions 18 in Pseudomonas (Dangel, Brackmann et al. 1991), Thauera aromatica via (Hirsch, 19 Schagger et al. 1998) and Azoarcus evansii (Hirsch, Schagger et al. 1998) ...... 29 20 Figure 1.8. The paa pathway for the aerobic metabolism of phenylacetate (Teufel, 21 Mascaraque et al. 2010)...... 35 22 Figure 2.1. The enantiomeric fraction (EF) of R-ibuprofen (-○-) and overall ibuprofen 23 concentration (-♦-) in a growing Ibu-2 culture in which ibuprofen is the sole carbon and 24 energy source. The concentrations of both ibuprofen enantiomers were determined by 25 chiral CE analysis...... 73 26 Figure 2.2. Proposed pathway for the metabolism of ibuprofen by Ibu-2. Metabolites b-d 27 were all detected via GC/MS. b: isobutylcatechol; c: 5-formyl-2-hydroxy-7-methylocta- 28 2,4-dienoic acid; d: 2-hydroxy-5-isobutylhexa-2,4-dienedioic acid ...... 75 29 Figure 2.3. Average final culture density (n=3) as measured by OD 600 when Ibu-2 was 30 inoculated in liquid MSM culture containing ibuprofen or its analogs (ipf ibuprofen, paa 31 phenylacetic acid, 2ppa 2-phenylpropionic acid, 3taa 3-tolylacetic acid, 4taa 4-tolylacetic 32 acid, 2(4t)pa 2-(4-tolyl)propionic acid)...... 78 33 Figure 3.1. Catechols and corresponding meta-cleavage products absorbance maxima 34 and the substrate chemicals as detected in ibuprofen-induced resting Sphingomonas Ibu-2 35 (Chapter 2)...... 90 36 Figure 3.2 expression vector forward primer strategy. RBS; ribosomal binding site .... 100 37 Figure 3.3. Mass-spectrum of acetylated isobutylcatechol detected in extracts of E. coli 38 epi300 pFOS3G7 supernatant...... 104 39 Figure 3.4. Open reading frames found on pFOS3G7. Large black arrows represent 40 ORFs with high similarity to conserved transposase genes. Small black arrows represent 41 the location and orientation of loss-of-function transposon insertions. ORFs described in 42 this study are named...... 106 43 Figure 3.5. PCR of indicated ipf genes using either intact pFOS3G7 or makerless 44 mutants. All products were of the predicted size for targeted gene deletion...... 107
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1 Figure 3.6. Percentage of 50ppm ibuprofen remaining after two days of incubation in E. 2 coli epi300 pFOS3G7, loss-of-function mutants, and the two successful complementation 3 constructs as determined by HPLC analysis of the cultures shown in Figure 3.17. n = 3, 4 standard deviations were too small to be visualized effectively...... 107 5 Figure 3.7. Catecholic polymer accumulation in E. coli epi300 pFOS3G7, loss-of- 6 function mutants, and the two successful complement constructs grown in LB broth with 7 50 ppm ibuprofen and 1.5mM ferric chloride...... 108 8 Figure 3.8. Centrifuged knockout and complement strains showing the tendency of 9 catecholic polymers created in the presence of 1.5mM ferric chloride to pellet along with 10 the cell mass...... 108 11 Figure 3.9. % of 50ppm ibuprofen metabolizied by E. coli epi300 pFOS3G7, E. coli 12 epi300 pFOS3G7Tn: ipfH, and epi300 negative control after four days as determined by 13 HPLC analysis...... 109 14 Figure 3.10. 150ul of E. coli epi300 harboring the indicated plasmids following 18 hours 15 of incubation with 1mM ibuprofen (IPF) or phenylacetate (PAA) with 1.5 mM ferric 16 chloride for catecholic metabolite visualization...... 111 17 Figure 3.11. Phenylacetate and catechol concentration in E coli epi300 cultures 18 harboring pJ25 and/or pGEM: ipfHI (pHI) following 18 hours of incubation 1 mM 19 phenylacetate...... 112 20 Figure 3.12. Ibuprofen and isobutylcatechol concentration in E coli epi300 cultures 21 harboring pJ25 and/or pGEM: ipfHI (pHI) following 18 hours of incubation 1 mM 22 ibuprofen...... 112 23 Figure 3.13. Disappearance of phenylacetyl CoA (PAA-CoA) in cell-free extract 24 experiments. PAA-CoA was quantified via HPLC. n=3. All data points are significantly 25 different from one another as calculated by student’s t-test (p<.05)...... 113 26 Figure 3.14. A; Concentration of phenylacetate remaining after three days in E. coli K12 27 cultures (7mM starting concentration). The strains are WT, wild type; G, ∆ paaG , J, 28 ∆paaJ ; Z, ∆ paaZ. “DE” indicates presence of pGEM: ipfDE rbs. All strains without 29 pGEM: ipfDE rbs harbored the empty pGEMt-easy vector. All strains were grown in 30 0.2% glycerol MSM with 1mM IPTG induction at 37C. Asterics indicate significantly 31 higher 2-hydroxyphenylacetate (p<0.05) ...... 114 32 Figure 3.15. Reactions catalyzed by sterol carrier protein X (SCPx) in animals. A; the 33 thiolytic depropionation of 24-oxo-3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA 34 (Takeuchi, Chen et al. 2004): B; the thiolytic depropionation of 3-ketopristanoyl-CoA 35 (Westin, Hunt et al. 2007 )...... 121 36 Figure 3.16. Phylogenetic tree of characterized thiolase-type proteins found in the Swiss- 37 prot database and listed in Table 3.5...... 122 38 Figure 3.17. General schema of bacterial metabolic pathways that require the action of 39 SCPx thiolases: A; DitF, B; PhlC, C; camphor ORF2, D; BbsB...... 124 40 Figure 3.18. Alignment of conserved catalytic residues of IpfD, bacterial and eukaryotic 41 SCPx thiolases, and FadA fatty acid thiolases. Conserved catalytic residues are 42 highlighted in light grey, variations from the conserved catalytic residues are highlighted 43 in dark grey. Created using MegAlign (DNAStar, Inc., Madison, WI)...... 126 44 Figure 3.19. Three dimensional model of the predictied amino acid sequence of ipfE 45 modelled on the structure of Sulfolobus DUF35 (Krishna, Aravind et al. 2010) with
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1 conserved domains labeled. Image created using Pov-Ray (Persistence of Vision 2 Raytracer Pty. Ltd., Victoria, Australia)...... 128 3 Figure 3.20. Proposed pathway by which Sphingomonas Ibu-2 metabolizes ibuprofen to 4 isobutylcatechol. I: Ibuprofen, II: Ibuprofen-CoA, III: 1,2-cis-diol-2-hydroibuprofen- 5 CoA, IV: 4-isobutylcatechol. The creation of II is suggested by the fact that IpfF was 6 shown to CoA-ligate ibuprofen in an enzyme assay and by the analagous CoA ligation of 7 phenylacetic acid as confirmed by HPLC analysis (discussed in Chapter 4). Additionally, 8 the creation of phenylacetyl-coenzyme A, catalyzed by the same enzyme, was confirmed 9 via HPLC. III has not been detected and is hypothesized based upon the putative 10 function of IpfAB. The identity of IV was confirmed via GC/MS...... 132 11 Figure 4.1. Phenylacetic acid coenzyme A ligase activity in the cell-free extracts of 12 different strains. “F” represents the pGEMt-easy ipfF clone while “AB” is the respective 13 ipfAB clone. (n=3)...... 158 14 Figure 4.2. Uptake of phenylacetic acid by strains E. coli epi300 pFOS3G7, E. coli 15 epi300 E. coli and epi300 pFOS3G7Tn :ipfF over a range of phenylacetic acid 16 concentrations. Uptake was standardized to total cell protein. E. coli epi300 17 pFOS3G7Tn :ipfF was significantly less (n=3, p < 0.05) than E. coli epi300 pFOS3G7 at 18 all concentrations tested...... 159 19 Figure 4.3. Uptake of 25 µM phenylacetic acid by E. coli epi300 pFOS3G7 and E. coli 20 epi300 pFOS3G7Tn: ipfF exposed to 0.5mM of the metabolic poisons 2,4 dinitrophenol 21 or potassium cyanide. Both pFOS3G7 poisoning treatments exhibited significantly less 22 uptake than the non-poisoned control (n=3, p < 0.05) while no other differences were 23 significant...... 160 24 Figure 4.4. Phenylacetic acid uptake by E. coli epi300 pFOS3G7at pH 5.5 divided by 25 uptake at pH 7. All data points other than for 0.5mM phenylacetate demonstrate that 26 uptake was higher at pH 5.5 vs pH 7 as is consistent with the vectorial acylation 27 mechanism (n=3, p < 0.05)...... 161 28 Figure 4.5. Phylogenetic tree of the predicted aminoacid sequence of ipfF and 29 characterized coenzyme A ligases found in the Swiss-prot database and listed in Table 30 4.2...... 163 31 Figure 4.6 Location and probabilities of putative transmembrane helix motifs in PaaK1, 32 IpfF, and FadD as predicted by the TMHMM algorithm (Krogh, Larsson et al. 2001). 33 Bacteriorhodopsin, an extensively membrane associated protein (Haupts, Tittor et al. 34 1999), is presented for reference. Amino acid locations are shown on the x-axis while the 35 probability of membrane association is represented on the y-axis...... 167 36 Figure 4.7 Model illustrating the role of FadD in the vectorial transport of exogenous 37 fatty acids (Weimar, DiRusso et al. 2002). 1, free fatty acids become protonated in the 38 periplasmic space following FadL-dependent transport across the outer membrane (not 39 illustrated here) and then partition into the inner membrane. 2, the protonated fatty acid 40 flips from the periplasmic face of the inner membrane to the cytoplasmic face. 3, free 41 fatty acids within the membrane signal FadD to partition into the inner membrane, 42 presumably in an ATP-bound form. 4, FadD functions to abstract fatty acids from the 43 inner membrane concomitant with the formation of fatty acyl-CoA for further 44 metabolism...... 168
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1 Fig. A1.1. Growth of Ibu-1 on ibuprofen as measured by increase in Ibu-1 culture 2 density via spectrophotometry at 600nm ( u ). Concomittant loss of ibuprofen during
3 growth as measured via HPLC ( p )...... 210 4 Fig. A1.2. Spectra of putative metabolites identified via GC-MS analyses. Figures (a) 5 and (b) (Rt 22.7 min) from Ibu-1 culture supernatant and sludge respectively. Figures (c) 6 and (d) (Rt 27.4 min) from Ibu-1 culture supernatant and sludge respectively. These 7 spectra were only detected in extracts after 3-fluorocatechol poisoning followed by 8 aqueous acetylation. “Ac” is an acetyl group, “Me” is a methyl group...... 212 9 Figure A2.1. Percent ibuprofen remaining after growth of E. coli pFOS3G7 in LB media 10 with or without 25mM HEPES buffer and with or without additional acidification of the 11 media to pH6. Y-axis amplified to demonstrate the slight differences...... 223 12 Figure A2.2. Growth of E. coli pFOS3G7 in LB media with addition of 10mM 13 arabinose, 1.5mM ferric chloride, and 500ppm ibuprofen in two different types of vessel; 14 falcon tube on the left and 40mL conical on the right...... 224 15 Figure A2.3. Same cultures as shown in Figure A2.2 but with the falcon tube grown 16 culture transferred to conicals for the sake of visual comparison...... 225 17 Figure A2.4. One of each culture set shown in figures A2.2 and A2.3 centrifuged in 18 order to concentrate the dark catecholic polymer...... 225 19 Figure A2.5. Percent ibuprofen remaining in E. coli pFOS3G7 cultures grown in falcon 20 tubes (1) or 40mL conicial vials (2)...... 226 21 Figure A2.6. Comparison of remaining ibuprofen in E. coli pFOS3G7 cultures 22 descended from different colonies...... 227 23 Figure A3.1. ORFs found on the SeqMan assembly contig containing ferredoxin 24 reductase ipfH, ferrdoxin ipfI, and diol-dehydrodgenase ipfL. Black arrows represent 25 sequences with high similarity to conserved transposases. Regions A, B, and C have 26 similarities to 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase, and 4- 27 oxalocrotonate isomerase...... 232 28 Figure A3.2. Alignment between ferredoxin reductase IpfH (“query”) and several other 29 ferredoxin reductases found in the NCBI database showing the four conserved iron- 30 coordinating cysteine residues indicated by “#”...... 233 31 Figure A3.3. The FedoxSOE project. The sizes of the ipfI and ipfH amplicons are shown 32 above, while the successful strand overlap extension product is shown on the lower right. 33 ...... 235 34 Figure A3.4. E. coli epi300 pJ25, E. coli epi300 pJ25 pGEM: ipfHI , and E. coli JM109 35 pGEM: ipfHI , all with IPTG induction, grown in LB with 500 ppm ibuprofen and 1.5mM 36 ferric chloride. The slightly dark color that appeared in the pJ25 pGEM: HI culture 37 (poorly visible in this photograph) did not pellet in the same manner as the catecholic 38 polymers produced by E. coli epi300 pFOS3G7 (Figure 3.18). pGEM: ipfHI expression 39 in JM109 led to a dark coloration in the culture media and in deceased cell material 40 (indicated by arrow)...... 236 41 Figure A3.5. 2-hydroxyphenylacetete accumulation and phenylacetate disappearance by 42 two E. coli JM109 pGEM: ipfHI , E. coli epi300 pJ25, and E. coli epi300 pJ25 43 pGEM :ipfHI . Effect of lacZ induction on the metabolic behavior of J25F was examined
xii
1 by addition of 1mM IPTG. All data points are significantly different from one another as 2 determined by t-test (p<0.05) ...... 237 3 Figure A3.6. HPLC of E. coli epi300 pJ25 pGEM: ipfHI grown in LB with 5mM 4 phenylacetate for two days. Strains with single or no vector accumulated no detectable 5 catechol. This culture went on to turn dark brown as seen in Figure A3.7...... 238 6 Figure A3.7. E. coli pJ25 pGEM: ipfHI after two weeks incubation in LB media with 7 5mM phenylacetate compared to E. coli with only one or neither of the plasmids...... 239 8 Figure A3.8. 150ul of E. coli epi300 harboring the indicated vector exposed to either 2- 9 phenylpropionate (2PPA) or m-tolylacetate (mTAA). 1.5 mM ferric chloride has been 10 added as a catechol indicator. A standard curve of catechol with 1.5 mM ferric chloride 11 is presented for reference...... 240 12 Figure A3.9. m-tolylacetate and 3-methylcatechol concentration in E coli epi300 cultures 13 harboring pJ25 and/or pGEM: ipfHI (pHI) following 18 hours of incubation 1 mM 14 ibuprofen...... 241 15 Figure A3.10. 2-phenypropionate and catechol concentration in E coli epi300 cultures 16 harboring pJ25 and/or pGEM: ipfHI (pHI) following 18 hours of incubation 1 mM 17 ibuprofen...... 241 18 Figure A3.11. General reaction performed by the diol-dehydrogenase XylL...... 243 19 Figure A3.12. Dark catecholic accumulation by E. coli epi300 pFOS3G7 with ipfL 20 replaced by the pKD4 insertion cassette (confirmed by PCR in Figure A3.13). The media 21 consists of LB with 100 ppm ibuprofen and 1.5mM ferric chloride...... 244 22 Figure A3.13. PCR screening of several E. coli epi300 pFOS3G7 ipfL pKD4 insertion 23 cassette mutants using primers that flank the ipfL gene showing successful gene 24 replacement; ipfL is 432 bp while the insertion cassette is approximately 1.6kb...... 244 25 Figure A3.14. the ipfR transposon mutant HPLC assay showing increased phenylacetate 26 disappearance (top) and increased appearance of 2-hydroxyphenylacetate (bottom). Also 27 included in the assay was the ipfY pFOS4F6 transposon insertion mutant pFOS4F6B1. 28 ...... 247 29 Figure A3.15. SeqMan (DNAStar inc., Madison, WI) screencap of the fosmid library 30 sequence assembly project showing the proximity of the end of the pFOS3G7 DNA insert 31 to the vector; sequence “3G7 pCC1R yahong.seq” begins at the end of the vector...... 249 32 Figure A3.16. PCR of ipfT , ipfR , and ipfY from fosmid pFOS4F6 or pFOS3G7 template. 33 ...... 250 34 Figure A3.17. PCR of E. coli epi300 pFOS3G7 with ipfR replaced by the pKD4 35 kanamycin resistance cassette using primers that flank the region of ipfR targeted for 36 deletion. Note that the double bands correspond to both the pKD4 insertion cassette 37 (large band) and intact ipfR (small band)...... 251 38 Figure A3.18. Map of partial ipfY and ipfT ORFs showing the location of the pFOS4F6 39 vector, the location of the pFOS4F6Tn: ipfY transposon isertion, and the targets of the 40 ORF-specific and lambda red pKD4 primers...... 252 41 Figure A3.19. Disappearance of ibuprofen in E. coli epi300 pFOS3G7Tn: ipfG cultures 42 compared to intact pFOS3G7 and vectorless epi300. 3G7tn: ipfG showed significantly 43 reduced ibuprofen disappearance as determined by student’s t-test...... 256 44 Figure A3.20. Uptake of radiolabelled phenylacetate by E. coli epi300 harboring 45 pFOS3G7 or pFOS3G7 with transposon in ipfG or ipfF. Uptake by pFOS3G7Tn: ipfG 46 was higher than pFOS3G7 at 2.5 uM phenylacetate as indicated by the star (p<0.05). . 257
xiii
1 Figure A4.1. The percent of total radiolabel present in sodium hydroxide trap following 2 growth of Ibu-2 in 500ppm ibuprofen MSM in the presence of 50,000 dpm 14 C- 3 ibuprofen. This experiment was performed in plastic culture tubes...... 266 4 Figure A4.2. The percent of total radiolabel present in sodium hydroxide trap following 5 growth of Ibu-2 in 500ppm ibuprofen MSM in the presence of 50,000 dpm 14 C- 6 ibuprofen. This experiment was performed in rubber-sealed glass tubes...... 267 7 Figure A4.3. PFGE of Ibu-2 total DNA (third lane) with two non-megaplasmid 8 containing E. coli controls in the first two lanes. Faint bands possibly representing 9 megaplasmids are outlined...... 269 10 Figure A4.4. Gel electrophoresis of PCR screening reactions of the nine putative 11 megaplasmid gel purifications. “1” employed the largest band as template, “2” the next 12 largest and so forth. “+” is positive control reactions (diluted Ibu-2 PCR products) and “- 13 “ is non-template controls. “c” is dmpC primed reactions while “b” is dmpB primed 14 reactions...... 270 15 Figure A4.5 Purified PCR products using the dmpB and dmpC primer sets on Ibu-2. . 273 16 Figure A4.6. RNA dot blot comparing induced and uninduced Ibu-2 probed with 17 ibu2cat23 radiolabeled PCR product...... 274 18 Figure A4.7. Dilution restriction enzyme method development. Fraction of one unit of 19 SmaI (shown above the lanes) incubated with Ibu-2 DNA for four hours...... 275 20 Figure A4.8. Representative pBBR1mcs-3 Ibu-2 chromosomal library probed with 21 ibu2cat23 radiolabelled PCR product. Positive hybridization is at the bottom of the 22 membrane...... 275 23 Figure A4.9. ibu2cat23 and dmpC primed PCR reactions using the six ibu2cat23 positive 24 pBBR1mcs-3 Ibu-2 DNA chromosomal library clones. Products match the expected 25 sizes...... 276 26 Figure A4.10. PCR products using ibu2cat23F and dmpCR primers on the six ibu2cat23 27 positive chromosomal library clones. The amplicon is approximately 1400bp...... 276 28 Figure A4.11. B; BamHI, E; EcoRI, S; SmaI. A: Overnight overdigestion of Ibu-2 29 chromosomal DNA using the enzymes indicated above the lanes. B: Result of probing 30 with radiolabelled Ibu2cat23 PCR product...... 277 31 Figure A4.12. UV-vis spectrum (350-400nm) of the supernatants of Ibu-2 resting cell 32 assays exposed to catechol, 4-methylcatechol, or 3-methylcatechol...... 279 33 Figure A4.13. Widely distributed bacterial strategy for the metabolism of metacleavage 34 products of catechol (top), 4-methylcatechol (middle), and 3-methylcatechol (bottom) 35 (Murray, Dugoleby et al. 1972)...... 281 36 Figure A4.14. Location of the putative catechol 2,3 dioxygenase relative to ipfA and 37 ipfB ...... 282 38 Figure A4.15. UV-vis spectrum of the supernatant of E. coli JM109 pGEM:Ibu2cat23 39 with 500ppm catechol. Maximum optical density = 377nm...... 282 40 Figure A4.16. Sequence of Ibu-2 ORF (shown in bold type) consistent with catechol 2,3 41 dioxygenase. Putative ribosomal binding site (underlined)...... 283 42 Figure A4.17. T7F / gene-specificR primed PCR product of pGEMt-easy ferredoxin 43 constructs...... 285 44 Figure A4.18. PCR screening of the fosmid transposon libraries for mutants with a 45 transposon inside of a ferredoxin-endocing ORF. The single positive result is shown. 286
xiv
1 Figure A4.19. PCR amplification of JM109 with the indicated plasmid using the 2 T7F/Ipf774F primers...... 288 3 Figure A4.20. Millions of colonies per microliter of overnight JM109 harboring the 4 indicated plasmid incoculated onto LB with the indicated 2-(4-tolyl)propionic acid 5 concentration. Significant inhibition of growth as determined by t-test is indicated by a 6 hashed bar...... 288 7 Figure A4.21. Millions of colonies per microliter of overnight JM109 harboring the 8 indicated plasmid incoculated onto LB with the indicated ibuprofen concentration. 9 Significant inhibition of growth as determined by t-test is indicated by a hashed bar. .. 289 10 Figure A4.22. UVvis spectra of the supernatants of sludge bacterial fractions exposed to 11 500ppm of the indicated aromatic acid for six days...... 293 12 Figure A4.23. UVvis spectra of the supernatants of Cayuga lake bacterial fractions 13 exposed to 500ppm of m-tolylacetic acid for six days. Raw supernatant is shown on the 14 left, while back extracted concentrated supernatant is shown on the right...... 294 15 Figure A5.1. Conserved domain architecture of the predicted protein encoded by ipfA 16 obtained via CDD analysis...... 303 17 Figure A5.2. Phylogenetic tree of the predicted amino acid sequence of ipfA and 18 characterized dioxygase large subunits found in the Swiss-prot database and listed in 19 Table 3.4...... 304 20 Figure A5.3. Phylogenetic tree of the predict amino acid sequence of ipfB and 21 characterized dioxygase small subunits found in the Swiss-prot database and listed in 22 Table 3.5...... 305 23
xv
1 LIST OF TABLES
2 3 Table 2.1. GC/MS retention times and major ions of isobutylcatechol (B) and two 4 putative isobutylcatechol meta-cleavage metabolites (C and D)...... 74 5 Table 3.1. Strains and plasmids used in this study...... 93 6 Table 3.2. Primers used in this study. 1: pGEMt- Easy vector system, Promega 7 Corporation, Madison, WI. 2: EZ::TN
xvi
1 LIST OF ABBREVIATIONS AND SYMBOLS
2 3 2,4-DNP: 2,4 dinitrophenol 4 2PPA: 2-phenylpropionic acid 5 3FC: 3-fluorocatechol 6 3TAA: 3-tolylacetic acid 7 4TAA: 4-tolylacetic acid 8 ABC: ATP binding cassette 9 ADP: adenosine diphosphate 10 Amp: ampicillin 11 AntDO- anthranilate 1,2-dioxygenase 12 ATP: adenosine triphosphate 13 BMO: bacterial monooxygenase 14 CCIS: copy control induction solution 15 CDD: conserved domain database 16 CE: capillary electrophoresis 17 Chl: chloramphenicol 18 CoA: coenzyme A 19 COX: cyclooxygenase 20 DNA: deoxyribonucleic acid 21 DTT: dithiothreitol 22 DUF35: domain of unknown function 35 23 EF: enantiomeric fraction 24 FeDox: ferredoxin 25 GC/MS: gas chromatography mass spectroscopy 26 GSH: glutathione 27 HPLC: high performance liquid chromatography 28 IPF: ibuprofen 29 IPTG: Isopropyl β-D-1-thiogalactopyranoside 30 Kan: kanamycin 31 Kow : octanol water partitioning coefficient 32 LB: Luria-Bertani broth 33 LOEC: lowest observed effects concentration 34 MFS: major facilitator superfamily 35 mM: milimolar 36 MSM: mineral salts medium 37 NAD+: oxidized nicotinamide adenine dinucleotide 38 NADH: nicotinamide adenine dinucleotide 39 NADP+: oxidized nicotinamide adenine dinucleotide phosphate 40 NADPH: nicotinamide adenine dinucleotide phosphate 41 NCBI: National Center for Biotechnology Information 42 NIH-NIEHS: National Institutes of Health – National Institute of Environmental Health 43 Sciences 44 NSAID: nonsteroidal anti-inflammatory 45 OB-fold: oligosaccharide/oligonucleotide binding fold
xvii
1 OD 600 : optical density at 600 nanometers 2 ORF: open reading frame 3 PAA: phenylacetic acid 4 PCR: polymerase chain reaction 5 PMSF: phenylmethylsulfonyl fluoride 6 Ppb: part per billion 7 Ppm: part per million 8 RBS: ribosomal binding site 9 RHO: Rieske-type non-heme iron aromatic ring-hydroxylating oxygenases 10 RT-PCR: reverse transcriptase PCR 11 SCP-2: sterol carrier protein 2 12 SCPx: sterol carrier protein X 13 SOE: strand overlap extension 14 STP: sewage treatment plant 15 TCA: tricarboxylic acid cycle 16 Tet: tetracycline 17 µM: micromolar 18 WCS: washed cell suspension 19 X-gal: bromo-chloro-indolyl-galactopyranoside 20 ∆: gene deletion mutant
xviii
1 2 CHAPTER 1
3 INTRODUCTION
4
5 Pharmaceuticals in the Environment
6
7 The ecotoxicology community has become increasingly concerned with the issue
8 of human pharmaceuticals as potential environmental contaminants (Halling Sorensen,
9 Nielsen et al. 1998; Daughton and Ternes 1999; Jorgensen and Halling-Sorensen 2000;
10 Dietrich, Webb et al. 2002, Kagle et al. 2009). We have become distinctly aware that the
11 world is not a limitless sink for our wastes (Andonova and Mitchell 2010; Peattie 2010;
12 Schwarzenbach, Egli et al. 2010). At this stage in our cultural evolution, we are quite
13 aware that our waste products can adversely impact the natural environment and
14 ourselves.
15 Within the last decade, an initially puzzling and cataclysmic drop in Central Asian
16 vulture populations was clearly linked to the veterinary use of the anti-inflammatory
17 diclofenac (Oaks, Gilbert et al. 2004). These species of vultures were powerfully
18 impacted due to a combination of very high exposure levels via feeding on deceased,
19 recently-dosed cattle and an extremely severe idiosyncratic nephritic sensitivity that led
20 to death by visceral gout (Oaks, Gilbert et al. 2004). The severe consequences of this
21 entirely unanticipated toxic manifestation will likely serve as a rallying cry against the
22 indiscriminant use of pharmaceuticals in the environment for a long time to come.
23 While on one hand it can be argued that virtually no adverse environmental effect
24 has ever been linked to the environmental presence of human (rather than veterinary)
1
1 pharmaceuticals, that pharmaceuticals are metabolized by the human body and are mobile
2 and readily diluted in the hydrosphere such that they will not reach notable
3 concentrations (Jones, Voulvoulis et al. 2004). On the other hand, unlike many pollutants
4 traditionally given much more attention, pharmaceuticals are specifically designed to be
5 biologically active. Even though they are released in low concentrations, they are
6 released in an overwhelmingly complex diversity of parent compounds and metabolites,
7 raising the possibility of addition and synergistic toxicity (Flaherty and Dodson 2005;
8 Chou 2006; Laetz, Baldwin et al. 2009). Mixture toxicity is a poorly developed, highly
9 controversial field (Chou 2006; Baas, van Houte et al. 2007). Predictions of mixture
10 effects are currently not feasible due in large part to the absence of precise dose-relevant
11 mechanistic data (Borgert, Quill et al. 2004; Cedergreen, Christensen et al. 2008; Crofton
12 2008; Kortenkamp 2008).
13 Given the inadequacy of the knowledge base regarding the potential effects of
14 these compounds at environmentally relevant concentrations, many governments the
15 world over have begun to apply the loosely-defined Precautionary Principle to guide
16 policy development in regards to environmental risks (Ferrari and Mons et al. 2004;
17 Gardiner 2006). Whereas classical risk analysis and cost-benefit analysis depend upon
18 having a handle on the mathematical underpinnings of cause-effect relationships, the
19 Precautionary Principle is invoked in the absence of proof of causation (Raffensberger
20 and Tickner 1999; Gardiner 2006). Given the complexity of the problem, the subtlety of
21 the potential effects involved, and the limited scientific information available, it seems
22 that application of the conservative Precautionary Principle may be the best way to limit
23 the potential environmental risks of pharmaceuticals.
2
1 Pharmaceuticals are released primarily from sewage treatment plants (STPs) into
2 bodies of water. There is potential for untreated waste to be released when STP capacity
3 is exceeded or for effluent to be poorly diluted during periods of drought, during which
4 STP effluent may be the main source of flow, thus leading to concentration spikes
5 (Loraine and Pettigrove 2006). For example, concentrations of pharmaceuticals in STP
6 effluent can vary by over an order of magnitude depending on rainfall levels (Peng, Yu et
7 al. 2008). Additionally, while most pharmaceuticals investigated so far have are
8 effectively removed during wastewater treatment (>90%), many, such as carbemezipine,
9 clofibric acid, and diclofenac, are not (Heberer 2002). Additionally, little work has been
10 done to resolve what amount of apparent removal might be due to sorption to the solid
11 phase followed by land application of the resulting solids (Xia, Bhandari et al. 2005).
12 Ternes et al. suggest that solid phase sorption is likely a very significant fate for
13 pharmaceuticals depending on their charge and K ow (Ternes, Joss et al. 2005). In the few
14 studies that have investigated the capacity of acidic pharmaceuticals to sorb to solid
15 phases, little or no sorbtion capacity has been observed (Heberer, Verstraeten et al. 2001;
16 Jones, Lester et al. 2005), though these studies have not considered biolsolids in
17 particular. This route is generally regarded as more important for lipophiles or for
18 chemicals that have the capacity to sorb to the sludge via other mechanisms (Rabiet,
19 Togola et al. 2006).
20 Release and dilution into the hydrosphere is not the only fate of treated
21 wastewater. Treated wastewater is being increasingly used as irrigation water,
22 particularly in dry western states in the USA (Pedersen, Yeager et al. 2003; Pedersen,
23 Soliman et al. 2005; Kinney, Furlong et al. 2006). Kinney et al. (Kinney, Furlong et al.
3
1 2006) state that “in 1993, it was estimated that 2.4% of wastewater effluent was used for
2 irrigation in the United States.” Only a decade later, it was estimated that the percentage
3 had increased to 7.4% (Xu, Wu et al. 2009). In especially dry, densely populated areas
4 such as Israel, the percentage of reclaimed water used for irrigation can be as much as
5 66% (Kinney, Furlong et al. 2006). The xenobiotics remaining in this treated wastewater
6 are available to directly impact soil ecosystems with little or no dilution. Indeed,
7 common pharmaceuticals have been detected at part-per-billion levels in both treated
8 wastewater used for irrigation and associated agricultural fields in Colorado (Kinney,
9 Furlong et al. 2006), Mexico City (Siemens, Huschek et al. 2008), and southern
10 California (Xu, Wu et al. 2009) and were even found to persist from one growing season
11 to another (Kinney, Furlong et al. 2006). As populations and urbanization increase, this
12 will become a more and more prevalent practice.
13 Of potentially greater importance from a political and public perception
14 viewpoint, is the possibility of contamination of drinking water with pharmaceuticals.
15 Chefetz et al. (Chefetz, Mualem et al. 2008) showed that several common
16 pharmaceuticals are readily mobile in soil and may reach groundwater following
17 application to agricultural fields via sludge or treated wastewater, establishing a mode of
18 transfer between the human waste stream and water reservoirs. A handful of studies have
19 examined to what extent drinking water treatment methods are effective at removal of
20 pharmaceutical residues. An investigation by Stackelberg et al. (Stackelberg, Gibs et al.
21 2007) found that the most common methods such as chlorination and charcoal filtration
22 are partially effective, removing some compounds, but not others, though the authors
23 point out that chlorination could be generating an array of unknown chemically activated
4
1 reaction products. Huerta-Fontella and Fontella (Huerta-Fontela and Ventura 2008)
2 showed that a more thorough drinking water treatment process that includes several
3 oxidative and advanced filtration steps is almost completely effective at removing
4 pharmaceutical residues.
5 Presumably due to constant release into the hydrosphere via wastewater effluent
6 processes, direct land application followed by transport through groundwater systems,
7 and incomplete removal during drinking water treatment processes, a very wide variety
8 of pharmaceuticals have been detected in drinking water supplies at part-per-trillion to
9 part-per-billion levels the world over (see (Fent, Weston et al. 2006; Benotti, Trenholm et
10 al. 2008; Huerta-Fontela and Ventura 2008; Corcoran, Winter et al. 2010; Kummerer
11 2010) for summaries of available data). However, a recent thorough risk analysis of
12 fifteen commonly detected pharmaceuticals with known endpoints including
13 carcinogenic, developmental, and reproductive toxicity predicted no effects on humans at
14 any concentrations that have been detected in drinking water (Bruce, Pleus et al. 2010).
15 A thorough review of available pharmaceutical ecotoxicological data by Fent et al. (Fent,
16 Weston et al. 2006) showed that with few exceptions, available lowest observed effect
17 concentration (LOEC) data suggests virtually no environmental impact of such
18 compounds, though they point out that such data is sorely lacking for individual
19 compounds and almost nonexistent for complex mixtures. Efforts to create a
20 comprehensive database of pharmaceutical ecotoxicological data are currently underway
21 (www.wikipharma.org (Molander, Agerstrand et al. 2009)).
22 The scientific and policy communities are faced with a vastly complex issue
23 where the potential for harm is not understood due to a lack of data regarding the
5
1 toxicological and ecological questions posed. Several comprehensive reviews are
2 available that summarize data regarding the presence and effects of pharmaceuticals.
3 They all come to the same conclusion: that not enough data exists to adequately answer
4 the pertinent questions (Stuer-Lauridsen, Birkved et al. 2000; Heberer 2002; Jones,
5 Voulvoulis et al. 2004; Cunningham, Buzby et al. 2006; DeLange, Noordoven et al.
6 2006; Fent, Weston et al. 2006; Hernando, Mezcua et al. 2006; Schwarzenbach, Escher et
7 al. 2006; Dorne, Skinner et al. 2007; Kummerer 2008; Kummerer 2010; Santos, Araújo et
8 al. 2010).
9
10 Ibuprofen
11
12 Ibuprofen is one of the most commonly consumed pharmaceuticals in the world
13 (see (Fent, Weston et al. 2006) for a partial review of country-by-country consumption).
14 It is also a good candidate for study because it is a member of a diverse class of
15 compounds that share certain structural features in common: the non-steroidal anti-
16 inflammatories (NSAIDs). The NSAIDs themselves are a very significant class of
17 pharmaceuticals from an environmental risk perspective. The dose of most NSAIDs is
18 particularly high, typically hundreds of milligrams to grams per day. Many NSAIDs are
19 purchased over the counter with virtually no regulation, largely because of their very
20 broad margin of safety; for example, ibuprofen is considered one of the safest drugs ever
21 (Rainsford 2009). There are also many NSAIDs that are available by prescription, either
22 because they serve specific needs or because they are young in a regulatory sense. Many
23 of these NSAIDs share a phenylacetic acid moiety which is believed to be responsible for
6
1 their activity (Figure 1.1).
O
COOH COOH
Ibuprofen Ketoprofen
COOH Cl NH COOH
Cl Cl
Naproxen Diclofenac 2
3 Figure 1.1. Common non-steroidal anti-inflammatory pharmaceuticals with a phenylacetic 4 acid moiety (bolded). 5
6 Their typical mode of action is the inhibition of cyclo-oxygenase (COX) enzymes that
7 cleave arachadonic acid in humans to create prostglandins which are potent paracrine
8 signaling compounds (Figure 1.2) (Fent, Weston et al. 2006).
7
COOH
arachadonic acid
COX enzymes
O COOH
HO OH PGE1 and other prostaglandins 1
2 Figure 1.2. General action of cyclooxygenase enzymes (Fent, Weston et al. 2006). 3
4 However, prostglandins are a rather broad class of paracrine compounds that play a
5 variety of roles in animals in addition to inflammation including regulation of gastric
6 mucosa (Vane 1971) renal vascular tissue (Hao and Breyer 2008) and egg shell thickness
7 in birds (Lundholm 1997). Prostglandins also play many roles in fish including control
8 of ovulation (Mercure and Van der Kraak 1996; Sorbera, Asturiano et al. 2001), sex
9 hormone regulation (Lister and Van der Kraak 2008), sexual behavior (Sorensen and
10 Goetz 1993), cortisol regulation (Mommsen, Vijayan et al. 1999) and even function as
11 sex pheromones (Sorensen and Geotz 1988; Moore and Waring 1996). At high doses,
12 NSAIDs can cause liver damage following metabolic activation (reviewed by Fent et al.
13 (Fent, Weston et al. 2006)), although the relevance of this toxic outcome at representative
14 environmental concentrations is unknown.
15 In addition to its wide consumption and structural similarity to a number of other
16 pharmaceuticals, an obvious reason to investigate the fate of ibuprofen is the fact that
8
1 ibuprofen has been detected in bodies of water all over the world. Environmental
2 concentrations of ibuprofen have been found to range from low part-per-trillion (Buser,
3 Poiger et al. 1999; Stumpf, Ternes et al. 1999; Farre, Ferrer et al. 2001; Winkler,
4 Lawrence et al. 2001; Kolpin, Furlong et al. 2002) to low part-per-billion levels (Buser,
5 Poiger et al. 1999; Farre, Ferrer et al. 2001) (See (Santos, Araújo et al. 2010) for a
6 thorough listing of available environmental concentration data). Ibuprofen has been
7 found in trace concentrations in such disparate locations as drinking water (Heberer,
8 Verstraeten et al. 2001; Jones, Lester et al. 2005; Loraine and Pettigrove 2006; Rabiet,
9 Togola et al. 2006) and the middle of the North Sea (Buser and D. 1998).
10
11 The fate of ibuprofen in the environment
12 The primary pathway by which ibuprofen enters the environment is believed to be
13 via human waste and sewage treatment plants, though deposition into landfills is a
14 poorly-understood alternate route (Bound and Voulvoulis 2005). A significant amount of
15 ibuprofen consumed by humans, 8.9% - 14%, is excreted unmodified or as the
16 glucuronide conjugate (Rudy, Knight et al. 1991; Lee, Peart et al. 2005). The primary
17 human metabolite of ibuprofen is hydroxyibuprofen (Figure 1.3), which has been
18 detected in sewage treatment plants but whose fate and effects are otherwise completely
19 unknown (Paxeus 2004). Two other minor human ibuprofen metabolites
20 carboxyibuprofen and carboxyhydratropic acid (Figure 3) are similarly uninvestigated.
9
COOH HO HOOC hydroxyibuprofen COOH
carboxyhydratropic acid COOH COOH
carboxyibuprofen 1
2 Figure 1.3. Human primary metabolites of ibuprofen (Rudy, Knight et al. 1991; Lee, Peart 3 et al. 2005). 4
5 Given all the pathways by which ibuprofen can enter the environment combined
6 with the high therapeutic dose and high rate of consumption, it is not surprising that
7 ibuprofen is one of the most frequently detected pharmaceutical compounds in aquatic
8 systems. The input, while somewhat sporadic, is continuous for the most part, leading to
9 a situation of continual low level exposure for the exposed aquatic communities.
10 Hydroxyibuprofen, the primary human metabolite of ibuprofen, has also been detected
11 though not quantified in surface waters (Winkler, Lawrence et al. 2001).
12 As soon as ibuprofen enters the waste stream, it is likely to be acted upon by
13 microorganisms via two general types of metabolic/enzymatic systems: non-specific co-
14 metabolic processes or assimilative metabolism. The biological transformation of
15 ibuprofen has been investigated in broad strokes using analytical chemistry techniques.
16 Microbe-dependent accumulation of hydroxyibuprofen and carboxyibuprofen (identical
17 to those illustrated in Figure 1.3) have been observed in a handful of studies (Zwiener,
18 Seeger et al. 2002; Quintana, Weiss et al. 2005; Marco-Urrea, Pérez-Trujillo et al. 2009),
10
1 though in each those studies these the side-chain oxidation products that accumulated
2 only accounted for a small amount of ibuprofen disappearance. There is no evidence in
3 the literature that the assimilative metabolism of ibuprofen has been investigated outside
4 of the work presented in this dissertation.
5 Ibuprofen has been found to be biologically degraded in sewage treatment plants
6 and in the aquatic environment with varying efficiency. Buser et al. (Buser, Poiger et al.
7 1999) found ibuprofen to have a half-life of 3 hours in activated sludge and 20 days in
8 lake water. Zweiner et al. (Zwiener, Seeger et al. 2002) found ibuprofen to have a half-
9 life of less than a day in a batch experiment. Sewage treatment plants have for the most
10 part been shown to be at least 90-95% efficient in removal of ibuprofen (96%-99.9%;
11 (Buser, Poiger et al. 1999)) with certain exceptions (22%-75%; (Stumpf, Ternes et al.
12 1999)), particularly in the developing world where STP performance can be highly
13 variable. Whether the ibuprofen is mineralized (degraded all the way to carbon dioxide
14 and water), transformed to stable but as of yet unidentified products, assimilated into
15 biomass, or sorbed by solids, etc. has not been investigated. Buser et al. (Buser, Poiger et
16 al. 1999) did demonstrate, however, that ibuprofen is rather persistent in sterile lake-
17 water, with no degradation occurring within 37 days even when exposed to daylight. An
18 investigation in to the persistence of ibuprofen in a low-nutrient Swiss lake-water found
19 that ibuprofen was surprisingly recalcitrant, with a half-life of 32 days (Tixier, Singer et
20 al. 2003). In this system, ibuprofen did not sorb to sediment to a significant degree, nor
21 did it volatilize.
22 Ibuprofen is non-volatile (Henry’s Law constant = 1.23 x 10 -6 atm m 3/mole),
23 sparingly water soluble (Kow = 3.97 (Jones, Lester et al. 2005)), does not sorb to
11
1 sediment (Jones, Lester et al. 2005), and is little-affected by physical degradative forces.
2 These characteristics are similar to many other pharmaceuticals (Tixier, Singer et al.
3 2003) and can be said to be an adequate, if crude, picture for the fate of pharmaceuticals
4 in general. In fact, pharmaceuticals are designed specifically to have these properties:
5 they must be non-volatile and physically stable so that they have a respectable shelf-life.
6 The fact that ibuprofen is chiral should not be excluded from studies on ibuprofen
7 fate. The formulation of ibuprofen in use across the world is a 50/50 R/S mixture with
8 the exception of some specialty products in use in Europe. This is likely because no
9 method for isolating the active enantiomer is sufficiently cost-effective to justify the
10 marginal benefits provided. The S-enantiomer is the biologically active compound. The
11 R-enantiomer has no known biological activity. Humans readily perform a chiral
12 inversion on ibuprofen, converting the R- to the S-form (Daughton 2002). Therefore,
13 ibuprofen and its byproducts are primarily excreted as the S-enantiomer. Interestingly,
14 Buser et al. (Buser, Poiger et al. 1999) found that the R isomer persisted longer in
15 biologically active lake water spiked with racemic ibuprofen, implying either that a chiral
16 inversion mechanism (S to R) was at work and/or that the S-enantiomer is more readily
17 degraded.
18 While, as mentioned above, some STPs remove up to 99% of the ibuprofen load
19 (Buser, Poiger et al. 1999), these facilities still represent of a source from which
20 ibuprofen can reach the environment. Simply due to the constancy of their outputs, many
21 STPs must also occasionally release untreated waste during periods of high precipitation
22 or equipment failure (Loraine and Pettigrove 2006). Also, during dry periods, the bodies
23 of water that sewage effluent is discharged into can be depleted, thus limiting the effect
12
1 of dilution and thereby effectively increasing the concentration of ibuprofen in the
2 receiving waters (Loraine and Pettigrove 2006).
3 The fate of unused pharmaceuticals is largely unknown. One survey-based
4 British research project was conducted so as to gain some insight on this subject for
5 ibuprofen and three other pharmaceuticals (Bound and Voulvoulis 2005). They might be
6 flushed down the toilet, in which case they would enter the same waste stream as outlined
7 above. Alternatively, people may return them to a pharmacy, a route that was found to be
8 rather uncommon in the case of ibuprofen. The primary mode of ibuprofen disposal was
9 found to be via solid waste disposal, which overall accounted for 8% of total ibuprofen
10 fate in the Bound et al. study (Bound and Voulvoulis 2005).
11
12 Ibuprofen Ecotoxicology
13 A comprehensive body of literature exists that describes the toxicology of
14 ibuprofen in mammalian systems at or near concentrations reached in the body during
15 normal therapeutic use, which are far above what is ever seen in the environment
16 (reviewed in (Rainsford 1999)). Ibuprofen has also been found to inhibit the growth of
17 common pathogenic bacteria and was for a time investigated for use as an antibiotic
18 (Cederlund and Mardh 1993; Elvers and Wright 1995). However, once again these
19 inhibitory effects were found only at concentrations far above those found in the
20 environment. Only in recent years has the toxicology of environmentally relevant
21 concentrations of ibuprofen been investigated (Cleuvers 2004; Pomati, Netting et al.
22 2004; Richards, Wilson et al. 2004; Escher, Bramaz et al. 2005; Pomati, Castiglioni et al.
23 2006; Flippin, Hugget et al. 2007)
13
1 Ecotoxicological data indicate that the potential for acute or sub-acute toxicity is
2 far too low to be of environmental relevance in a variety of species studied (Cleuvers
3 2004; Escher, Bramaz et al. 2005). However, investigations into subtle and mixture
4 toxicity have suggested that some effects can be caused by environmentally relevant
5 concentrations (Pomati, Netting et al. 2004; Richards, Wilson et al. 2004; Flippin, Hugget
6 et al. 2007; Han, Choi et al. 2010)
7 A number of studies have found various effects on fish at concentrations
8 approaching environmental relevance. For example, Flippen et al. (Flippin, Hugget et al.
9 2007) found that environmentally relevant ibuprofen concentrations caused subtle
10 alterations in reproductive function in female Japanese medaka fish including increased
11 clutch size and decresed egg development time, hypothetically due to ibuprofen’s effects
12 on cyclooxygenases and prostaglandin synthesis in vertebrate reproduction. Han et al.
13 found that ibuprofen delayed hatching in Japanese medaka at concentrations as low as 0.1
14 µg/L (Han, Choi et al. 2010). David et al. (David and Pancharatna 2009) found that
15 ibuprofen elicited a wide variety of developmental and behavioral effects in Zebrafish at
16 10ug/L.
17 Schnell et al. (Schnell, Bols et al. 2009) found that while they did not detect
18 effects on trout cells at relevant concentrations of ibuprofen on its own, pharmaceutical
19 mixture results suggested synergistic effects when ibuprofen was used in combination
20 with other classes of drugs.
21 One of the most profound effects was described by Pomati et al. (Pomati, Netting
22 et al. 2004) who found that ibuprofen strongly stimulated the growth of the cyanobacter
23 Synecohcystis, causing a 72% increase in growth rate at an ibuprofen concentration of 10
14
1 ppb. The same study revealed a 25% growth reduction in the growth of the aquatic plant
2 Duckweed ( Lemna minor ) at concentrations of ibuprofen that might be found in sewage
3 treatment plant effluent. Results from both of these species suggest that primary
4 producers, which are of high importance in oligotrophic aquatic ecosystems, may be
5 sensitive to the effects of ibuprofen.
6 Richards et al. (Richards, Wilson et al. 2004) investigated the effects on aquatic
7 community structure caused by mixtures of pharmaceuticals at environmentally relevant
8 concentrations. The mixture that they tested included ibuprofen, fluoxitine (commonly
9 known as prozac), and ciprofloxacin. The complexity and burdensomeness of doing
10 mesocosm studies prohibited a large sample number and thus reduced statistical certainty.
11 However, a mixture of the three compounds including ibuprofen at a concentration of 60
12 parts per billion caused significant effects on the aquatic community structure. There was
13 an overall reduction in microbial diversity and algae became more predominant
14 (Richards, Wilson et al. 2004).
15 Pomati et al. (Pomati, Castiglioni et al. 2006) investigated the effects of a
16 complex mixture of thirteen pharmaceuticals, including ibuprofen, on human embryonic
17 cells. The exposure concentrations were based on existing data regarding the highest
18 reported riverine concentrations of the pharmaceuticals. A 10%-30% decrease in cell
19 proliferation was observed with an acculation of cells at the G2/M phase and associated
20 overexpression of cell cycling proteins p16 and p21 , indicating a disruption of cell
21 cycling systems. The cells exhibited increased activation of stress response kinases and
22 glutathione-S-transferase. While the exposure conditions were two steps removed from
23 environmental relevance (maximum observed toxicant concentrations and raw exposure
15
1 of embryonic cells in the absence of an organismal context), the results indicate the
2 potential for pharmaceutical mixtures to have subtle effects over short exposure times.
3 Other than the Pomati et al. (Pomati, Castiglioni et al. 2006) human embryonic
4 cell study, the studies described above made no attempt to discover the mechanism by
5 which ibuprofen was exerting effects on the target organisms. A number of studies have
6 made the attempt to identify modes of ibuprofen toxicity using traditional toxicological
7 methods aimed at identifying major toxic effects including mutagenesis, GSH induction,
8 baseline toxicity (membrane disruption), and estrogenicity, but have only detected
9 toxicity at concentrations far above those relevant in the environment (Cleuvers 2004;
10 Escher, Bramaz et al. 2005). Therefore, the mechanism by which ibuprofen may have
11 exerted effects upon the aquatic organisms at environmentally relevant concentrations
12 remains completely unknown (Pomati, Netting et al. 2004; Richards, Wilson et al. 2004).
13 The organism which is the subject of this dissertation, Sphinogmonas Ibu-2, was
14 enriched and isolated on media containing a much higher concentration (500 mg/L) of
15 ibuprofen than is found in the environment. The low environmental concentration of
16 pharmaceuticals may make the maintenance and expression of specific metabolic genes
17 unfavorable in a selective sense, although it is not clear what the “cut-off” exposure level
18 is for these compounds. Although this subject is beyond the scope of this dissertation, it
19 is likely to be very important in determining whether and how pharmaceuticals are
20 biologically transformed in the environment. That said, similar enrichment based studies
21 have resulted in the isolation of organisms whose degradation pathway biochemistry has
22 been shown to be relevant to pollutant degradation at environmentally relevant pollutant
23 concentrations (Pumphrey and Madsen 2007). Thus, while more work is needed in order
16
1 to understand if the Ibu-2 metabolic paradigm presented below is relevant to more real
2 world conditions, the research chapters presented here represent the first published effort
3 to describe the biochemistry and genetics of ibuprofen degradation. It serves two general
4 purposes; as an important first step in identifying metabolites to search for under more
5 environmentally relevant conditions and as an investigation into what metabolic
6 strategies might be employed by bacteria in the degradation of structurally related
7 chemicals.
8
9 Bacterial metabolism of aromatic chemicals
10
11 Biosynthesis of the six-carbon aromatic ring is essential for the production of
12 certain amino acids and diverse biological cofactors, though many aromatic compounds
13 in the environment are of pyrogenic or petrogenic origin. A large percentage of lignin,
14 which is the second-most common biological compound on the surface of the planet
15 (Kirk and Farrell 1987) is also aromatic, being primarily composted of phenolic
16 heteropolymers (Masai, Katayama et al. 2007). “Aromatic” refers specifically to circular
17 hydrocarbons and heterocycles with delocalized π-orbital electrons (Vaillancourt, Bolin
18 et al. 2006; Phale, Basu et al. 2007); while these are reduced, energy-rich compounds,
19 they are unusually resistant to chemical attack due to the negative resonance of the
20 delocalized electrons and the inaccessibility of the carbons (Vaillancourt, Bolin et al.
21 2006; Phale, Basu et al. 2007). Presumably because they are energy rich and ubiquitous,
22 bacterial pathways for metabolizing aromatic compounds are common. However, the
17
1 unusual stability of the ring necessitates highly specialized enzymatic machinery for
2 catalysis.
3 The bulk of the scientific work examining the microbial metabolism of aromatic
4 chemicals has tended to point to certain rules or trends common to the metabolic
5 strategies that bacteria employ. A simple, if inaccurate, model for aerobic bacterial
6 degradation of the aromatic group is that of the double-dioxygenation; an initial addition
7 of two oxygens to the aromatic group seems sufficient to destabilize the structure and
8 allow for a second dioxygenation event to cleave the ring, which prepares the resulting
9 non-aromatic metabolite for catabolism and eventual entry into central carbon cycling
10 pathways (Masai, Katayama et al. 2007; Phale, Basu et al. 2007; Fuchs 2008; Zeyaullah,
11 Abdelkafe et al. 2009; Ju and Parales 2010). Presumably the initial double oxygenation,
12 whether it be a single hydroxylation of an already-oxidized ring, sequential
13 monoxygenations, or a single dioxygenation event, is required to destabilize the ring,
14 perhaps by altering the resonance of the delocalized π-orbital electrons or by deshielding
15 the ring-carbons (Parales and Resnick 2006; Phale, Basu et al. 2007). In actuality, the
16 double-dioxygenation paradigm is not by any means universally applicable, though this
17 paradigm holds true for the aerobic degradation of simple aromatic chemicals such as
18 benzene and toluene and remains a likely first hypothesis for predicting the mechanism of
19 an unknown degradative pathway.
20
21 Classical double-dioxygenation metabolism of aromatics
22 While dihydroxylation of the aromatic ring itself is a nearly universal prerequisite
23 for ring cleavage , microbes possess a diverse array of mechanisms for doing this
18
1 (Yamaguchi and Fujisawa 1978; Schweizer, Markus et al. 1987; Butler and Mason 1997)
2 (Figure 1.4). The two hydroxyl groups can be added in adjacent or opposite positions
3 upon the ring. In the case of aromatic chemicals that do not already have hydroxyl
4 groups, simultaneous dioxygenation via multicomponent dioxygenase and subsequent
5 dehydrogenative rearomatization is the typical route of transformation (Butler and Mason
6 1997; Parales and Resnick 2006). Aromatic compounds that have two adjacent hydroxyl
7 groups are commonly termed catechols. There are fewer examples of opposite position,
8 or para -, hydroxylations such as the homogentisate (2,5-dihydroxyphenylacetic acid)
9 pathway that is commonly used by both eukaryotes and prokaryotes in aromatic amino
10 acid metabolism (Arias-Barrau, Olivera et al. 2004) and in the metabolism of
11 alkylphenols via hydroquinone by an environmental isolate Sphingomonas sp. strain
12 TTNP3 (Corvini 2006).
13 The initial dioxygenation is a reductive hydroxylation requiring high-energy
14 electron input (Parales and Resnick 2006; Kweon, Kim et al. 2008). This power is
15 typically supplied by ferredoxins which are in turn powered by NAD(P)H dependent
16 ferredoxin (FeDox) reductases (Mason and Cammack 1992; Kweon, Kim et al. 2008).
17 The typical electron transfer chain is NAD(P)H > FeDox-Reductase > Ferredoxin > O 2 +
18 substrate. Ferredoxins and their reductases are both redox-active iron-sulfur proteins.
19 Most reductases studied to date use NADH, a ubiquitous reduced nucleotide-based
20 cofactor, as an electron source. While an electron source is required for the initial
21 dioxygenation event, NADH is subsequently regenerated in the following
22 dehydrogenation event (Figure 1.4) (Parales and Resnick 2006), making the conversion
23 of the aromatic to a catechol net energy-neutral.
19
upper pathway lower pathway
COOH COOH O2
ortho- HO HO H cleavage OH OH O 2 H TCA
NADH NAD+ NAD+ NADH O2 OH
cis- dihydrodiol catechol meta- COOH cleavage 1 O
2 Figure 1.4. Basic features of the double-dioxygenation paradigm for the aerobic 3 metabolism of aromatic chemicals by bacteria. “Upper pathway” broadly refers to the 4 reactions leading up to initial ring dioxygenation while “lower pathway” refers to all 5 reactions following the initial dioxygenation. 6
7 Catechols are the substrates for further metabolism by other types of
8 dioxygenases (Figure 1.4). These dioxygenases do not require an electron transport
9 chain, presumably because the destabilized ring is vulnerable to attack at this stage. Ring
10 cleavage in the 2,3 position relative to the two hydroxyl groups of a catechol is termed
11 meta - or extradiol- cleavage, to indicate that fission takes place adjacent to the two
12 hydroxy groups. The ring fission product of a meta-cleavage reaction exhibits a
13 diagnostic yellow color that disappears upon acidification. The alternative to meta-
14 cleavage is ortho - or intradiol- cleavage, in which the ring is broken between the two
15 hydroxy groups. In this situation, no yellow product is observed (See (Harayama, Kok et
16 al. 1992) and (Vaillancourt, Bolin et al. 2006) for reviews of ring-cleavage
17 dioxygenases).
18 The most-studied aromatic acid degradation processes so far involve an initial
19 dioxygenation in the 1,2 or 2,3 position. An example of 1,2 dioxygenation is found in the
20
1 TOL pathway of Pseudomonas putida mt-2. The TOL operon encodes for the genes for
2 the degradation of toluene and the xylenes. Toluene is sequentially oxidized at the
3 methyl group to benzoate. Benzoate is then cis -dioxygenated in the 1,2 position to cis-
4 benzoate dihydrodiol, which is decarboxylated and dehydrogenated to form catechol (1,2-
5 dihydroxybenzene). Catechol is then dioxygenated at the 2,3 position, cleaving the ring
6 (Eaton 1996; Eaton 1997).
7 An example of a 2,3-dioxygenation pathway for the degradation of an aromatic
8 acid is the degradation of 4-isopropylbenzoate (cumate). It is the only aromatic acid with
9 a branched aliphatic substituent in the para -positionwhose degradation pathway has been
10 fully described. Cumate is dioxygenated at the 2,3 position by Pseudomonas putida F1
11 via enzymes encoded by the cmt operon. It is then subsequently dehydrogenated to yield
12 2,3-dihydroxy-4-isopropylbenzoate (Figure 1.5). This compound is then cleaved via
13 dioxygenation across the 3,4-bond, a meta -cleavage that yields a diagnostic pH-
14 dependent yellow product (Defrank and Ribbons 1976; Defrank and Ribbons 1977; Eaton
15 1996; Eaton 1997).
16
COOH COOH COOH COOH OH OH OH H OH OH COOH + H + NADH NAD NAD NADH O2 O 17 O2
18 Figure 1.5. General scheme of the 1,2 dioxygenation cmt pathway (Eaton 1996; Eaton 19 1997). 20
21 Alternatively there are a number of aromatic metabolic strategies that seem to not
22 necessarily require the initial double-oxygenation or even that the ring be oxygenated at
21
1 all. A large portion of biological chemistry, from both a current spatial perspective and
2 certainly in regards to the geological time-frame, occurs under anoxic conditions. Even
3 though oxygen is the most potent electron acceptor on the modern earth, nitrate and ferric
4 iron are both useful and reasonably abundant terminal electron acceptors with sufficient
5 oxidizing power to permit their use during the break down aromatic chemicals by
6 bacteria (see .(Foght 2008; Fuchs 2008; Carmona, Zamarro et al. 2009) for current
7 reviews). Clearly in the absence of ubiquitous oxygen gas, the double-dioxygenation
8 paradigm becomes irrelevant, leading to necessarily different strategies for ring-opening.
9
10 Alternative aerobic ring cleavage substrates
11 Some facultative anaerobes actually utilize the same metabolic system under both
12 aerobic and anaerobic conditions. Bacteria couple a benzoate CoA-ligase mediated first
13 step to a variation of the double-dioxygenation paradigm in which an oxygenase adds
14 dioxgyen across the 2,3 bond while simultaneously dearomatizing the ring, followed by a
15 direct, non oxygen-dependent ring cleavage (Gescher, Eisenreich et al. 2005), while the
16 aerobic metabolism of phenylacetic acid further blurs the line between the two paradigms
17 as described below.
18 There are also other examples of aerobic aromatic ring-openings, namely those of
19 salicylate (Hintner, Lechner et al. 2001) and aminoaromatics (Kulkarni and Chaudhari
20 2007; Ju and Parales 2010) that do not require an intial ring dioxygenation. Additionally,
21 there are a number of examples of pathways that actually utilize trihydroxybenzenes as
22 ring cleavage substrates that, while they do not as dramatically challenge the
23 dihydroxylation model, are variations that should be acknowledged (Zeyer and Kearney
22
1 1984; Kluge, Tschech et al. 1990; Hanne, Kirk et al. 1993; Stolz and Knackmuss 1993;
2 Meulenberg, Pepi et al. 1996; Kadiyala and Spain 1998; Haigler 1999; Johnson, Jain et
3 al. 2000; Schink, Phillips et al. 2000; Dayan, Watson et al. 2007; Kulkarni and Chaudhari
4 2007; Carmona, Zamarro et al. 2009).
5
6 Anaerobic metabolism of aromatics
7 As in aerobic aromatic degradative strategies, the anaerobic reactions responsible
8 for aromatic ring cleavage can also be grouped into upper pathways that converge upon a
9 central ring cleavage substrate and a lower pathway that is responsible for ring cleavage
10 and further degradation (Foght 2008; Fuchs 2008; Carmona, Zamarro et al. 2009). While
11 for a variety of reasons anaerobic pathways have not been as extensively studied as
12 aerobic pathways, the existing evidence seems to suggest that a large number of unique
13 strategies are employed in upper pathway metabolism (Carmona, Zamarro et al. 2009).
14 This may be rationalized in part as the result of the fact that the term “anaerobic” is a
15 catch-all term for pathways that utilize a very wide variety of electron acceptors with
16 very different redox potentials and thus different energetic conditions. For example,
17 upper pathways adapted for using nitrate or ferric iron as electron acceptors (Bouwer and
18 Zehnder 1993; Wilson and Bouwer 1997; Chakraborty and Coates 2004), both of which
19 have moderately high redox potentials, are quite different from those adapted to less ideal
20 electron acceptors such as sulfate (Diaz 2000).
23
upper pathway lower pathway
O OH OS OS CoA CoA
TCA
ATP ADP 2 ATP 2 ADP CoA Benzoyl-CoA + 1 2 NADH 2 NAD
2 Figure 1.6. General scheme of the benzoyl-CoA paradigm for the anaerobic metabolism of 3 aromatic chemicals by bacteria (Carmona, Zamarro et al. 2009). 4
5 Through a wide variety of strategies, anaerobic aromatic metabolism is funneled
6 through a single central metabolite regardless of redox conditions; benzoyl-coenzyme-A
7 (Figure 1.6) (Carmona, Zamarro et al. 2009). Though there is scant evidence so far, it
8 appears that even benzene is converted to benzoyl-CoA under anaerobic conditions, so
9 universal is this metabolic intermediate (Ulrich, Beller et al. 2005). Benzoyl-CoA is
10 cleaved via a drastically different mechanism than that observed in classical aerobic
11 examples. Benzoyl-CoA is cleaved by an energy-dependent reductive hydrolytic
12 mechanism that requires reduced ferredoxin and ATP (reviewed by Boll (Boll 2005).)
13 The ATP-dependent CoA-ligation (or non-energy-dependent CoA-transfer in some cases
14 (Muller and Schink 2000; Leutwein and Heider 2001)) is believed to activate the ring for
15 reductive hydrolysis in a manner analogous to the oxidative activation of the ring for
16 oxidative ring cleavage in addition to facilitating downstream metabolism (Vellemur
17 1995). CoA-ligation may also drive uptake and retention by keeping the intracellular
18 concentration of the substrate effectively low, a strategy generally termed vectoral
19 acylation (Black and DiRusso 2003; Zou, F. et al. 2003)
20
24
1 Metabolism of phenylacetate
2 Recently-described pathways for the bacterial catabolism of phenylacetic acid
3 have also been shown to utilize hydrolytic ring cleavage mechanisms similar to those
4 found in anaerobic pathways (Teufel, Mascaraque et al. 2010). Phenylacetic acid is a
5 relatively simple chemical which upon initial inspection does not seem to warrant
6 catabolic strategies vastly different from those required for the other simple aromatics
7 such as BTEX (benzene, toluene, ethylbenzene, xylene) or benzoates. Indeed, early
8 investigations into phenylacetate biodegradation yielded circumstantial evidence for the
9 utilization of pathways involving homoprotocatechuate (3,4-dihydroxyphenylacetic acid)
10 or homogentisate (2,5-phenylacetic acid) (Roof, Lannon et al. 1953; Kunita 1955; Kunita
11 1955)
12 Ibuprofen is similar in nature to phenylacetic acid in that the acid group is two
13 carbons removed from the aromatic ring. As described below, it was believed until fairly
14 recently that bacteria metabolize phenylacetic acid through the same pathways by which
15 they metabolize hydroxyphenylacetic acids, which they convert via monooxygenation to
16 either 3,4-hydroxyphenylacetic acid (homoprotocatechuate) or 2,5-hydroxyphenylacetic
17 acid (homogentisate) (Sparnins, Chapman et al. 1974; Sparnins and Chapman 1976;
18 Wegst, Tittman et al. 1981; Arias-Barrau, Olivera et al. 2004). Homoprotocatechuate and
19 homogentisate intermediates were first identified during early investigations into the
20 biochemistry of fungal (Kluyver and van Zijp 1951; Perrin and Towers 1973), animal
21 (Knox and Edwards 1955; Crandall, Krueger et al. 1960; Flamm and Crandall 1963;
22 McCormick, Young et al. 1965), and bacterial (Roof, Lannon et al. 1953; Kunita 1955;
23 Kunita 1955; Jamaluddin 1977; Lee Y-L and Dagley 1977) metabolism.
25
1 Fungal metabolism of phenylacetate
2 The capacity of fungi to perform assimilative metabolism of phenylacetic acid via
3 homogentisate is well-established. In 1976, Kishore et al. (Kishore, Sugumaran et al.
4 1976) demonstrated that the fungus Aspergillus niger converted phenylalanine to
5 phenylacetic acid, 2- and 4-hydroxyphenylacetic acid, and homogentisate by performing
6 thin layer chromatography on radiolabelled substrate. They additionally demonstrated
7 that both phenylacetic acid hydroxylase and homogentisate oxygenase activites were
8 upregulated in phenylalanine-grown cultures, offering strong support to the assertion that
9 phenylacetate can be metabolized via homogentisate in fungi. This model was further
10 supported by the subsequent characterization of phenylacetate 2-hydroxylase mixed
11 function NADPH-dependent p450 enzyme in phenylacetic acid grown A. niger cultures
12 (Sugumaran and Vaidyanathan 1979) and in other Aspergillus species (Fernandez-Canon
13 and Penalva 1995; Mingot, Penalva et al. 1999; Rodriguez-Saiz, Barredo et al. 2001).
14 The homogentisate ring-cleavage dioxygenase has also been isolated from fungi
15 (Fernandez-Canon and Penalva 1995). A similar pathway involving the sequential mono-
16 oxidation of ethylbenzene to homogentisate via phenylacetic acid has also been described
17 in the fungus Exophiala lecanii-corni (Gunsch, Cheng et al. 2003; Gunsch, Cheng et al.
18 2005).
19
20 Early research into the bacterial metabolism of phenylacetate
21 In two of the earliest reports, Kunita (Kunita 1955; Kunita 1955) reported that
22 Pseudomonads had the capacity to metabolize phenylacetate via both homogentisate and
23 protocatechuate pathways, though later it was reported that Pseudomonas might
26
1 alternatively metabolize phenylacetic acid via a decarboxylative mechanism to catechol
2 via benzoate based on detection of putative metabolites in the media (Sariaslani,
3 Sudmeier et al. 1982). In the 1950’s, Dagley et al. reported that Vibrio also metabolized
4 phenylacetic acid via homogentisate, though the conclusion was based solely upon an
5 apparently high level of oxygen consumption in washed phenylacetate-grown cells
6 exposed to homogentisate (Dagley, Fewster et al. 1953). However, such up-regulation
7 was measured in the absence of any negative controls making the conclusion tenuous.
8 The authors never actually demonstrated the conversion of phenylacetate to
9 homogentisate (Chapman and Dagley 1962). Later, Boer et al. found evidence in support
10 of this general model in Nocardia , though using the same single experimental technique;
11 oxygen consumption of induced washed-cell systems exposed to homogentisate (Boer,
12 Harder et al. 1988). Pometto et al. (Pometto and Crawford 1985) did offer stronger
13 support for the employment of the homogentisate model in Streptomyces sp. by first
14 demonstrating that phenylacetate accumulated in phenylalanine-grown cultures, leading
15 to the conclusion that phenylacetate is an intermediate of phenylalanine in such bacteria.
16 They further demonstrated that homogentisate dioxygenase activity was higher in
17 phenylalanine grown cells than in tyrosine grown cells. Once again however, there were
18 no negative controls utilized and no statistical testing was employed to support the
19 claims, nor was any direct conversion of phenylacetate to homogentisate achieved
20 (Pometto and Crawford 1985). A 1988 study by van den Tweel et al. (van den Tweel,
21 Smits et al. 1988) again claimed to find evidence for aerobic metabolism of phenylacetate
22 via homogentisate based on up-regulation of associated oxygenase activities in the
23 absence of any direct phenylacetate hydroxylase activity by induced cell-free extracts.
27
1 In 1985, Cooper et al. suggested that the then current model of phenylacetate
2 metabolism via homogentisate or protocatechuate was not tenable because associated
3 dioxygenase activities by phenylacetate grown Pseudomonads were negligible. The
4 same group had definitively demonstrated that 3- and 4-hydroxyphenylacetate were
5 metabolized by E. coli via protocatecuate (Cooper and Skinner 1980). However in the
6 case of phenylacetate-grown E. coli they reported an accumulation of 2-
7 hydroxyphenylacetate, which was a dead-end metabolite, and no enzymatic activities or
8 metabolites indicative of either the protocatechuate or homogentisate pathways being
9 utilized (Cooper, Jones et al. 1985). As described below, it would later be repeatedly
10 shown that this enigmatic 2-hydroxy dead-end metabolite accumulates in several
11 phenylacetate-degrading bacteria and is dead-end side product of the true underlying
12 metabolic pathway.
13
14 Phenylacetyl coenzyme A ligase pathway
15 In the early 1990s several studies published out of Dr. Georg Fuchs’s laboratory
16 characterized phenylacetyl-coenzyme A ligases utilized under anaerobic conditions by
17 Pseudomonas (Dangel, Brackmann et al. 1991; Seyfried, Tschech et al. 1991; Mohamed
18 and Fuchs 1993; Mohamed, Seyfried et al. 1993). Induction of phenylacetyl-coenzyme A
19 ligases under anaerobic conditions were also reported in E. coli (Vitovski 1993),
20 Acinetrobacter (Vitovski 1993), Alcaligenes (Vitovski 1993), and later, Rhodococcus
21 (Navarro-Llorens 2005), Silicibaacter (Yan, Kang et al. 2009), and Thermus
22 thermophilus (Erb, Ismail et al. 2008). As described above, coenzyme A (CoA) is a
23 nucleotide-based cofactor utilized in a wide variety of metabolic systems throughout all
28
1 branches of life (Vellemur 1995; Leonardi, Zhang et al. 2005; Spry, Kirk et al. 2008).
2 CoA is utilized by bacteria under anaerobic conditions to activate aromatic substances for
3 ring-cleavage and subsequent catabolism and possibly also to drive uptake (Vellemur
4 1995; Carmona, Zamarro et al. 2009). Dangel et al. (Dangel, Brackmann et al. 1991)
5 demonstrated using radiolabelled substrate that Pseudomomas activates phenylacetic acid
6 under anaerobic conditions using a coenzyme A ligase and then proceeds to
7 decarboxylate it to benzoyl-coenzyme A with equimolar release of radiolabelled CO 2 gas
8 via a phenylglyoxylate intermediate (Figure 1.7), whereas Seyfried et al. (Seyfried,
9 Tschech et al. 1991) showed that several environmental Pseudomonas isolates obtained
10 from an anaerobic phenylacetate enrichment were able to metabolize a variety of putative
11 intermediate compounds that were consistent with the decarboxylative benzoyl-coenzyme
12 A pathway.
O O O
CoA O O S OH S OH CoA
ATP AMP H2O CoA CoA CO2 CoA phenylglyoxylate 13
14 Figure 1.7. The phenylglyoxylate pathway (Dangel, Brackmann et al. 1991), which channels 15 phenylacetate to the central benzoyl-CoA pathway under anaerobic conditions in 16 Pseudomonas (Dangel, Brackmann et al. 1991), Thauera aromatica via (Hirsch, Schagger et 17 al. 1998) and Azoarcus evansii (Hirsch, Schagger et al. 1998) 18 19 In the same Pseudomonas system in the same laboratory, Mohamed et al. (Mohamed and
20 Fuchs 1993) characterized phenylacetyl-CoA ligase activity using chromatographic
21 methods, showing considerable up-regulation of the activity in induced cells versus
22 glucose-grown cells. Curiously, the group reported that the activity was also induced
29
1 under aerobic conditions to a lesser extent, but discarded the information as artifactual.
2 In the following years, several more projects came out of the Fuchs lab describing in
3 detail the biochemistry of metabolism of phenylacetyl-coenzme A to benzoyl-coenzyme
4 A via phenylglyoxylate under anaerobic conditions in Thauera aromatica (Hirsch,
5 Schagger et al. 1998) and Azoarcus evansii (Hirsch, Schagger et al. 1998). It would
6 shortly be reported that aerobically-grown Azoarcus evansii also employs a phenylacetyl-
7 CoA ligase (Mohamed 2000; Rost, Haas et al. 2002) by demonstrating the in vivo CoA-
8 ligase activity of radiolabelled compounds preferentially by phenylacetate-induced cells
9 (Mohamed 2000).
10 A short time previously, a Spanish research group led by Dr. Jose Luengo
11 described the induction of a phenylacetyl-coenzyme A enzyme in aerobically-grown
12 Pseudomonas putida U (Martinez-Blanco, Reglero et al. 1990). The group concentrated
13 the enzymatic fraction of induced cell cultures and demonstrated ATP and Mg 2+
14 dependent conversion of phenylacetate to phenylacetyl-coenzyme A via HPLC. At the
15 time, such an intermediate in an aerobic aromatic pathway was both unprecedented and
16 ran counter to the accepted model of metabolism via homogentisate or protocatechuate;
17 accordingly, the researchers hedged by stating that such enzymatic activity might actually
18 be due to a non-specific activity or a detoxification mechanism (Martinez-Blanco,
19 Reglero et al. 1990). This uncertainty was shortly cleared up with the demonstration that
20 genetic knockout mutants that had lost the ability to grow on phenylacetate accordingly
21 lost the ability to generate phenylacetyl-coenzyme A (Schleissner, Olivera et al. 1994).
22 The 1.3 kilobase phenylacetyl-coenzyme A ligase gene with high similarity to acetyl-
23 CoA ligases and fatty acid-CoA ligases was identified in Pseudomonas putida U via Tn5
30
1 transposon mutagenesis (Minambres, Martinez-Blanco et al. 1996). A nearly identical
2 gene (86% identity) was identified in P. putida Y2 wherein it is necessary for the
3 metabolism of styrene via phenylacetate (Velasco, Alonso et al. 1998).
4 Shortly thereafter, several genes associated with aerobic phenylacetate
5 metabolism in P. putida U (Olivera, Minambres et al. 1998) and the styrene-metabolizer
6 P. putida Y2 (Alonso, Bartolomé-Martín et al. 2003; Bartolomé-Martín, Martínez-García
7 et al. 2004) were identified and sequenced. In addition to the coenzyme A ligase that had
8 already been described ( phaE ), four genes associated with ring hydroxylation ( phaFGHI )
9 and a gene encoding a putative ring-opening enzyme ( phaL ) were identified. The
10 putative ring hydroxylation genes were identified based on conserved oxidase and
11 electron transfer domains and by the fact that when they were knocked out, phenylacetate
12 accumulated during the metabolism of chemicals degraded via a phenyacetyl-coenzyme
13 A intermediate (Olivera, Minambres et al. 1998). When phaL was knocked out, 2-
14 hydroxyphenylacetate accumulated in the media, leading to the tentative conclusion that
15 this monohydroxylated chemical was actually an intermediate (later shown to be wrong
16 as described below) upon which the phaL gene product performed a ring fission though
17 sequence analysis revealed no similarity to any known ring-fission associated genes
18 (Olivera, Minambres et al. 1998).
19 Genes closely related to those of the Pha operon in P. putida U were also
20 discovered in the aerobic phenylacetate-metabolizer E. coli W (Ferrandez, Minambres et
21 al. 1998; Olivera, Minambres et al. 1998); a phenylacetyl-coenzyme A ligase ( paaK ), a
22 multicomponent oxygenase ( paaABCDE ), and putative lower-pathway-encoding genes
23 (paaGJZ). Sequence analysis of the putative paaB and paaE gene products showed
31
1 rieske iron clusters and conserved FAD+ and NAD(P)+ binding domains with similarities
2 to the electron transport components of some toluene and phenol mono-oxygenases,
3 suggesting that these two genes encode an electron transport system. paaACD showed no
4 similarities to any functionally characterized genes and were hypothesized to encode a
5 multicomponent phenylacetyl-coenzyme A oxygenase. Knocking out paaG, paaJ, or
6 paaZ resulted in accumulation of 2-hydroxyphenylacetate which was clearly a dead-end
7 metabolite in E. coli W. paaG and paaJ showed similarities to genes that encode beta-
8 oxidation enzymes, enoyl-CoA hydratase/isomerase and beta-ketoadipyl-CoA thioloase
9 respectively and as such were expected to be involved in metabolizing the product of ring
10 cleavage. By default, PaaZ appeared to be the ring-opening enzyme, though it bore no
11 resemblance to any known ring-opening enzyme; rather, like phaL (Olivera, Minambres
12 et al. 1998), it bore only a conserved aldehyde dehydrogenase domain, and ultimately
13 provided no clear hypothesis regarding how ring-opening might occur (Ferrandez,
14 Minambres et al. 1998). Mohammed et al. would later show that similar genes and
15 metabolites were present in Azoarcus evansii, Escherichia coli, Rhodopseudomonas
16 palustris and Bacillus stearothermophilus, indicating that this coenzyme A mediated
17 pathway is widely distributed in bacteria (Mohamed, Ismail et al. 2002). Again the
18 accumulation of the apparent dead-end metabolite 2-hydroxyphenylacetate was observed,
19 though it was postulated that it might be an abiotic breakdown product of 2-
20 hydroxyphenylacetyl-CoA which might be a true metabolic intermediate (Mohamed,
21 Ismail et al. 2002).
22 Rost et al. attempted to identify the ring-cleavage substrate by knocking out pacL ,
23 the putative gene thought to encode phenylacetate ring-cleavage activity in A. evansii (the
32
1 equivalent of paaZ in E. coli W and phaL in P. putida U) (Rost, Haas et al. 2002). In the
2 knockout mutant, a compound identified via NMR as 2,4,6-cycloheptatriene-1-one was
3 identified, though no attempts to see whether or not this was metabolizable by induced
4 cells was reported. The authors suggested that this unexpected accumulating metabolite
5 was the product of rearrangement of the actual ring-cleavage substrate by housekeeping
6 enzymes or abiotic reaction (Rost, Haas et al. 2002). Ismail et al. attempted to solve the
7 ring-opening question using the same tactics (Ismail, Mohamed et al. 2003); NMR
8 analysis of knockout mutants in E. coli K12. As had been previously reported (Olivera,
9 Minambres et al. 1998; Mohamed, Ismail et al. 2002), paaG and paaZ knockouts
10 accumulated 2-hydroxyphenylacetate. However, an additional metabolite was detected in
11 paaG knockouts; the gamma-lactone of 2-hydroxyphenylacetate (Figure 1.8 compound
12 4). Although PaaG was expected to act upon the ring-opened metabolite due to its
13 similarity to beta-oxidation enoyl-CoA hydratase/isomerase proteins, the accumulation of
14 a unique eight-carbon aromatic compound (Figure 1.8 structure 2) suggested instead that
15 it might actually catalyze ring-opening via an isomerization of an activated aromatic-CoA
16 compound. The structure of the lactone was consistent with the abiotic dehydration of a
17 dearomatized 1,2-dihyroxy metabolite as is classically utilized in aerobic aromatic
18 pathways by bacteria (Gibson 1968), though the postulated 1,2-dihydroxy was not
19 detected directly (Ismail, Mohamed et al. 2003). The 2,4,6-cycloheptene-1-one
20 metabolite detected by Rost et al. (Rost, Haas et al. 2002) was also postulated to be an
21 abiotic breakdown product of the putative 1,2-diol intermediate (Ismail, Mohamed et al.
22 2003).
33
1 Further analysis of the putative multicomponent oxygenase system paaABCDE
2 from E. coli (Fernandez, Ferrandez et al. 2006) expanded upon and supported the putative
3 role that these genes play in phenylacetate metabolism. Sequence and phylogenetic
4 analyses placed this five-gene operon firmly within the bacterial diiron multicomponent
5 oxygnase (BMO) family, which contains such multicomponent oxygenases that
6 monooxygenate methane, ammonia, linear alkanes, tetrahydrofuran, phenol, and toluene.
7 While it had been postulated that this phenylacetyl-CoA oxygenase performed a
8 dioxygenation, the sequence analyses strongly suggested a monooxygenation mechanism
9 (Fernandez, Ferrandez et al. 2006). Indeed, a 2010 report by the Fuchs lab offered strong
10 support for a monooxygenation mechanism of a sort unprecedented in aromatic
11 metabolism (Teufel, Mascaraque et al. 2010). When Teufel et al. exogenously expressed
12 the paaABCDE operon from E. coli K12 and exposed purified enzymatic reaction to
13 radiolabelled phenylacetyl-CoA, an epoxide, 1,2-epoxyphenylacetyl-CoA (Figure 1.8
14 compound 11) was identified via NMR analysis. Enzymatic assay with labelled oxygen
15 gas or labelled water revealed that the source of the epoxy-oxygen was diooxygen rather
16 than water. Sequence analysis supported the previous predictions (Olivera, Minambres et
17 al. 1998; Fernandez, Ferrandez et al. 2006) that PaaACD encodes for a multicomponent
18 BMO while PaaB and PaaE play the electron-transfer role. The facts that the 2-
19 hydroxyphenylacetic acid and lactone metabolites previously observed in ring-cleavage
20 deficient mutants (Rost, Haas et al. 2002; Ismail, Mohamed et al. 2003) were also
21 consistent with abiotic degradation of this epoxy metabolite and that the epoxide was
22 more consistent with the conserved monooxygenase character of the ring cleavage operon
23 paaABCDE (Fernandez, Ferrandez et al. 2006) appeared to have resolved the question of
34
1 how ring hydroxylation proceeds and is summarized in Figure 1.8.
O O O O O CoA CoA CoA CoA OH S S S S O PaaK PaaABCDE PaaGO PaaZ O
+ ATP AMP NADPH NADP + NADP NADPH COOH O2 H2O CoA H2O 1 2 3
O O
OH O OH
4 5 2
3 Figure 1.8. The paa pathway for the aerobic metabolism of phenylacetate (Teufel, 4 Mascaraque et al. 2010). 5
6 The NMR experiments upon E. coli K12 paa clones and mutants by Teufel et al.
7 (Teufel, Mascaraque et al. 2010) also cast light upon the mechanism of ring opening
8 catalyzed by PaaG and PaaZ. In a manner somewhat consistent with it’s conserved
9 enoyl-CoA hydratase/isomerase identity, purified PaaG catalyzed the isomerization of the
10 epoxyphenylacetyl-CoA to 2-oxepin-2-(3H)-ylideneacetyl-CoA(oxepin-CoA) (Figure 1.8
11 compound 2), a reversible reaction. Further addition of purified PaaZ catalyzed the
12 NADP+-dependent conversion of epoxide and oxepin-CoA into 3-oxo-5,6-
13 dehydrosuberyl-CoA (Figure 1.8 compound 3). Previous studies had identified only an
14 aldehyde dehyrdrogenase domain in PaaZ (Ferrandez, Minambres et al. 1998), though
15 further sequence analysis by Teufel et al. (Teufel, Mascaraque et al. 2010) revealed an
16 additional enoyl-CoA-hydratase (MaoC) domain which the authors took to indicate that
17 final ring cleavage was accomplished in a multi-step reaction catalyzed by PaaZ in which
35
1 the MaoC hydratase domain adds water across the C2-C3 bond, the ring is cleaved by the
2 tautomerization of the unstable reaction product, and then the aldehyde dehydrogenase
3 domain futher oxidizes the resulting aldehyde at the C8 position in an NADP+ dependent
4 manner to produce the observed final metabolite. While the multi-step proposed PaaZ
5 mechanism will require much more experimentation to establish firmly, the formation of
6 an epoxy-CoA by PaaABCDE, the oxepin by PaaG, and ultimate ring cleavage by PaaZ
7 was clearly demonstrated (Teufel, Mascaraque et al. 2010).
8 The presence of paa -like genes in 16% of sequenced bacterial genomes suggests
9 that this CoA-ligase hydrolytic ring-cleavage mechanism may be a central paradigm for
10 the aerobic metabolism of aromatic chemicals (Teufel, Mascaraque et al. 2010). Aerobic
11 coenzyme A mediated metabolism of benzoate derivatives has also been described as
12 mentioned above (Fuchs 2008). Such pathways are currently regarded as “hybrid”
13 because they combine aspects of both a classical anaerobic (CoA ligation and hydrolytic
14 ring cleavage) with aspects of typical aerobic (oxygenation of the aromatic ring)
15 pathways (Fuchs 2008). The PAA hybrid pathway was particularly difficult to
16 characterize, likely due to the novelty of the enzymes involved and the ephemeral
17 qualities of the epoxy and oxepin metabolites produced. The true extent of its relevance
18 has therefore been clouded however based on the prevelance of uncharacterized paa
19 homologs in the genomes of sequenced bacteria, it is becoming clear that similar hybrid
20 paradigms for the metabolism of aromatic acids are wide spread and may be as common
21 as the better characterized double-dioxygenation and anaerobic benzoyl-CoA paradigms.
22
23
36
1 Bacterial metabolism of 2-phenylpropionate
2 Even more structurally similar to ibuprofen than phenylacetate is 2-
3 phenylpropionate. However, data regarding the microbial degradation of
4 phenylpropionate is much scarcer than that regarding phenylacetate. Site-specific
5 hydroxylation of phenylpropionate has been observed in the gram-positive bacterium
6 Streptomyces rimosus , which converts phenylpropionate to 4-hydroxyphenylpropionate
7 (Kuge, Mochida et al. 1991). Pseudomonas cepacia has been shown to metabolize
8 phenylpropionate and tropic acid (2-phenyl-3-hydroxypropionic acid) via
9 decarboxylation of the alpha methyl group to phenylacetaldehyde followed by oxidation
10 to phenylacetate (Andreoni, Baggi et al. 1992). Long et al. (Long 1997) studied in detail
11 the steps involved in the oxidation of tropic acid to phenylacetate by Pseudomonas sp.
12 strain AT3. They demonstrated that tropic acid is dehydrogenated to phenylmalonic
13 semialdehyde (2-phenyl-3-oxopropionic acid), decarboxylated to phenylacetaldehyde,
14 and dehydrogenated to phenylacetic acid.
15
16 Metabolism of ibuprofen
17 Little information exists regarding how ibuprofen is oxidatively metabolized by
18 environmental microbes. The fungus Verticillum lecanii has been shown to hydroxylate
19 ibuprofen on the isobutyl group prior to metabolism, forming (S)-2-[4-(2-hydroxy-2-
20 methylpropyl)phenyl]propionic acid (Hanlon, Kooloobandi et al. 1994). In biofilm
21 reactor experiments, Zweiner et al. (Zwiener, Seeger et al. 2002) and Winkler et al.
22 (Winkler, Lawrence et al. 2001) reported a small increase in the concentration of
23 hydroxyibuprofen and carboxyibuprofen concomitant with the decrease in concentration
37
1 of ibuprofen under aerobic conditions. Quintana et al. (Quintana, Weiss et al. 2005) also
2 reported identifying two isomers of hydroxyibprofen in spiked active sewage sludge.
3 Short-lived intermediates such as substituted catechols would have not been detected
4 using the techniques described in any of the studies listed above, nor was the generation
5 of the hydroxylated metabolites linked to assimilative metabolism. This data suggests
6 that microbes could be metabolizing ibuprofen through a mono-oxygenation process
7 similar to that observed in Verticillum lecanii (Hutt, Kooloobandi et al. 1993) . However,
8 in the Zweiner et al. study (Zwiener, Seeger et al. 2002) the hydroxy- and
9 carboxyibuprofen accounted for only 7% of the ibuprofen that was degraded. How that
10 remaining 90+% was metabolized was not determined.
11 Chapter two of this dissertation provides evidence detailing a unique metabolic
12 pathway for the degradation of ibuprofen whereby the acid side chain is removed and a
13 catecholic intermediate is formed. Chapter three details the genes found in
14 Sphinogmonas Ibu-2 which encode the observed metabolic pathway described in chapter
15 two.
16
17 Transport of aromatics and related chemicals by bacteria
18 Most aromatic acid transport systems studied to date have been found to utilize
19 inner membrane porins of the major facilitator superfamily (MFS) that tend to couple
20 transport to the influx of hydrogen ions (Marger and Saier 1993; Pao, Paulsen et al. 1998;
21 Kahng, Byrne et al. 2000; Kasai, Inoue et al. 2001). Such proteins have been found to be
22 required for the uptake of phthalate (Chang, Dennis et al. 2009), benzoate (Clark,
23 Momany et al. 2002; Ledger, Flores-Aceituno et al. 2009; Wang, Xu et al. 2011),
38
1 chlorobenzoate (Ledger, Flores-Aceituno et al. 2009), 4-hydroxybenzoate (Ditty and
2 Harwood 2002), and protocatechuate (Harwood and Parales 1996). Transport of
3 phenylacetic acid has been found to take place via a similar mechanism in Pseudomonas
4 putida U (Schleissner, Olivera et al. 1994) wherein PhaK is a porin that forms an outer
5 membrane channel with a high degree of substrate specificity while PhaJ is an inner
6 membrane MFS permease, also with high specificity (Olivera, Minambres et al. 1998). It
7 was demonstrated that in P. putida U, transport is most likely coupled to electrochemical
8 gradients and that the membrane is impermeable to phenylacetic acid. However, the
9 membrane characteristics of P. putida U are considered highly unusual in that P. putida
10 U is impervious to membrane disruption by fatty acids (Schleissner, Olivera et al. 1994).
11 Therefore it is not clear that this transport system is representative of phenylacetic acid
12 transport in general.
13 Despite its transport through a porin in Pseudomonas putida U, phenylacetate
14 metabolism and regulation have been shown in recent years to perhaps be more akin to
15 that of fatty acids than aromatic compounds. For example, phenylacetate is ligated to
16 coenzyme-A as a prerequisite for further metabolism and the CoA ligate serves as a
17 genetic regulator for downstream metabolic elements (Galan, Garcia et al. 2004).
18 A second type of transport system, the ABC transporter, has been found to be
19 utilized in the uptake of certain aromatic acids. ABC (short for ATP-binding cassette)
20 transporters are multi component active uptake systems unlike the MFS permease
21 symporters. Whereas MFS proteins are porins, ABC systems are not (Busch and Saier
22 2002). ABC systems have been found to be utilized for the uptake of 3-
23 hydroxyphenylacetate (Arias-Barrau, Sandoval et al. 2005) and phthalate (Hara, Stewart
39
1 et al. 2009). It is not clear why drastically different types of uptake strategies should be
2 in use for the transport of such similar chemicals.
3 Because ibuprofen is nominally an aromatic acid, one may hypothesize that it is
4 likely to be transported using an MFS porin or an ABC transporter. However, it is not a
5 foregone conclusion that the aromatic ring is the main feature dictating the type of
6 transport system employed. From a metabolic perspective, the aromatic ring poses a
7 unique challenge. The dispersed electron cloud of the aromatic ring is particularly
8 resistant to chemical modification. Entirely unique systems of catabolic enzymes have
9 evolved specifically to tackle this challenge. On the other hand, from the perspective of
10 transport across biological membranes, the aromatic ring is of little significance since it is
11 simply a slightly bulky hydrophobic group which should have little trouble traversing the
12 membrane. It is therefore possible that other structural characteristics will contribute to
13 the selection of transport systems; while ibuprofen is an aromatic acid, it also has a long
14 hydrophobic tail which includes the aromatic ring and the isobutyl-group, making it
15 drastically more hydrophobic than any of the chemicals listed above.
16 Fatty acids are transported in an entirely different manner than aromatic acids.
17 No inner-membrane spanning pores are required, nor is transport coupled directly to any
18 ion gradients (Black and DiRusso 2003). This is most likely due to the tendency for fatty
19 acids to partition into biological membranes due to their surfactant qualities. This
20 characteristic makes for a chemical that can partition into and out of biological
21 membranes, such that the membrane only slows diffusion to some degree but cannot
22 prevent it. This presents a bacterium seeking to utilize the fatty acid with an entirely
23 different situation from that faced by those seeking to utilize phenylacetate. In the case
40
1 of phenylacetate, the challenge lies in getting the molecule into the cell, whereas with
2 fatty acids which freely diffuse across the membrane in both directions, the challenge lies
3 in keeping the molecule inside the cell.
4 The transport approach that has evolved in the case of fatty acids which is found
5 across all domains of life can be generally termed “vectorial ligation.” (Black and
6 DiRusso 2003; Zou, F. et al. 2003). The fatty acid, which freely passes into the cell down
7 its concentration gradient, is ligated to coenzyme A, a bulky hydrophilic cofactor, a
8 process that serves two transport purposes; the fatty acid is now trapped inside the cell
9 because the CoA group cannot cross the membrane and the intracellular concentration of
10 the fatty acid is effectively kept low, thus maintaining a concentration gradient and
11 insuring continued influx (Weimar, DiRusso et al. 2002). It is actually this fatty acid
12 CoA ligate that then typically serves as an inducer/repressor of regulation and as
13 substrate a for further catabolism (Black, Faegerman et al. 2000; Campbell and Cronan
14 2001; DiRusso and Black 2004).
15 While the fact that vectorial ligation is a driving force in the uptake of fatty acids
16 is at this point uncontestable, there is mounting evidence that there are additional
17 transport mechanisms in place that enhance uptake. FadL is an outer membrane spanning
18 protein that is found in many bacteria (van den Berg, Black et al. 2004; Hearn, Patel et al.
19 2008). FadL has high affinity for fatty acids and is proposed to translocate fatty acids via
20 an accessory protein (TonB) driven by conformational change (Ferguson and Deisenhofer
21 2002; van den Berg, Black et al. 2004). Intriguingly, the transport of toluene by two
22 other TonB-dependent members of the FadL family has been demonstrated in the cases
23 of TbuX (Kahng, Byrne et al. 2000) and TodX (Wang, Rawlings et al. 1995).
41
1 The aliphatic side chain of ibuprofen that renders it much more hydrophobic than
2 phenylacetic acid (K ow 3.9 vs 1.4 (Hansch, Leo et al. 1995; Tixier, Singer et al. 2003)).
3 This hydrophobic nature implies that it will have a much greater capacity to passively
4 diffuse across the membrane, reducing the requirement for channel proteins such as the
5 major facilitators and porins that have been observed to be involved in the transport of
6 more hydrophilic aromatic acids. Chapter four of this dissertation details preliminary
7 evidence suggesting that, like long chain fatty acids, ibuprofen transport proceeds via
8 vectoral ligation, is dependent upon a CoA ligase, and does not rely upon a specific
9 transmembrane transport system, though consideration of fatty acid and toluene transport
10 systems suggest that further unidentified proteins may be involved.
11
12 Conclusions
13
14 The fate of ibuprofen in the environment is a poorly understood phenomenon.
15 Given the overall lack of experimental evidence upon which to solidly base a hypothesis
16 regarding how ibuprofen and related NSAIDs may be metabolized by environmental
17 microbes, a direct experimental approach using classical microbiological methods was
18 undertaken. By isolating an organism capable of growth on ibuprofen and studying the
19 chemistry and genetics of the phenomenon we hoped to arrive at a solid understanding of
20 said mechanism and to therefore make a solid contribution to the environmental sciences
21 and microbiology.
22
42
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1 CHAPTER 2
2
3 FORMATION OF CATECHOLS VIA ACID SIDE CHAIN REMOVAL FROM
4 IBUPROFEN AND RELATED AROMATIC ACIDS 1
5
6
7 Abstract 8 9 Although ibuprofen (2-(4-isobutylphenyl)-propionic acid) is one of the
10 most widely consumed drugs in the world, little is known regarding its degradation
11 by environmental bacteria. Sphingomonas Ibu-2 was isolated from a wastewater
12 treatment plant based on its ability to use ibuprofen as a sole carbon and energy
13 source. A slight preference towards the R-enantiomer was observed, though both
14 ibuprofen enantiomers were metabolized. A yellow color, indicative of meta -
15 cleavage, accumulated in the culture supernatant when Ibu-2 was grown on
16 ibuprofen. When and only when 3-flurocatechol was used to poison the meta -
17 cleavage system, isobutylcatechol was identified in the culture supernatant via
18 GC/MS analysis. Ibuprofen-induced washed cell suspensions also metabolized
19 phenylacetic acid and 2-phenylpropionic acid to catechol, while 3- and 4-
20 tolylacetic acids and 2-(4-tolyl)-propionic acid were metabolized to the
21 corresponding methyl catechols before ring cleavage. This data suggests that in
22 contrast to the widely distributed coenzyme A ligase, homogentisate, or
23 homoprotocatechuate pathways for metabolism of phenylacetic acids, Ibu-2
1This chapter has been published in Applied and Environmental Microbiology October 2005, p. 6121-6125, Vol. 71, No. 10
64
1 removes the acidic side chain of ibuprofen and related compounds prior to ring-
2 cleavage.
3
4 Introduction
5 Ibuprofen (2-(4-isobutylphenyl-propionic acid)) is a pharmaceutical drug used for
6 its analgesic, antipyretic, and anti-inflammatory properties. It is the third most consumed
7 drug in the world, with an estimated annual production of several kilotons (Buser, Poiger
8 et al. 1999). Approximately 10% of the ibuprofen consumed by humans is excreted
9 unmodified or as the glucuronide conjugate (Lee, Williams et al. 1985; Rudy, Knight et
10 al. 1991). Wastewater treatment processes have been found to remove ibuprofen with
11 varying success, which may explain why Kolpin et al. (Kolpin, Furlong et al. 2002)
12 detected ibuprofen in 9.5% of the bodies of water that they surveyed. Environmental
13 concentrations of ibuprofen have been found to range from low part-per-trillion (Buser,
14 Poiger et al. 1999; Stumpf, Ternes et al. 1999; Farre, Ferrer et al. 2001; Winkler,
15 Lawrence et al. 2001; Kolpin, Furlong et al. 2002) to low part-per-billion levels (Buser,
16 Poiger et al. 1999; Farre, Ferrer et al. 2001).
17 Little information exists regarding how ibuprofen is oxidatively metabolized by
18 environmental microbes. Side chain hydroxylation has been reported (Kolpin, Furlong et
19 al. 2002; Zwiener, Seeger et al. 2002), along with the formation of carboxyhydratropic
20 acid (2-(4-carboxyphenyl)propionic acid) (Buser, Poiger et al. 1999; Zwiener, Seeger et
21 al. 2002) and ibuprofenol (2-(4-isobutylphenyl)-propanol) under anaerobic conditions
22 (Chen and Rosazza 1994).
65
1 The most similar compounds whose bacterial metabolism have been described
2 include 4-isopropylbenzoate (cumate), phenylacetic acid (PAA), and 2-phenylpropionic
3 acid (2PPA). Cumate is dioxygenated at the 2,3 position by Pseudomonas putida F1
4 carrying the cmt operon and subsequently meta-cleaved (Defrank and Ribbons 1976;
5 Defrank and Ribbons 1977; Eaton 1996; Eaton 1997). Other routes for the metabolism of
6 phenylacetic acids include the well-characterized homoprotocatechuate (Sparnins and
7 Chapman 1976) and homogentisate pathways (van den Tweel, Smits et al. 1988), as well
8 as the more recently described CoA-ligase pathway (Ismail, Mohamed et al. 2003).
9 Additionally, Streptomyces rimosus has been shown to convert 2PPA to 4-hydroxy-2PPA
10 (Kuge, Mochida et al. 1991). Finally, Pseudomonas cepacia has been shown to
11 metabolize 2PPA (2) and tropic acid (21) (2-phenyl-3-hydroxypropionic acid) via a
12 pathway involving decarboxylation to phenylacetaldehyde followed by oxidation to PAA
13 (Andreoni, Baggi et al. 1992).
14 In this study we report upon the isolation of a bacterium capable of utilizing
15 ibuprofen as a sole carbon and energy source. We also provide evidence of an apparently
16 novel metabolic pathway for the degradation of ibuprofen and related PAAs.
17
18 Methods
19
20 Materials and Strains
21 NAD + was purchased from SigmaAldrich (St. Louis, MO). Dextrin 10 was
22 purchased from Fluka BioChemika (Buchs, Switzerland). All other chemicals were
23 purchased from Acros (Morris Plains, NJ). A 50/50 enantiomeric mixture of R/S -
66
1 ibuprofen was used unless stated otherwise. Mineral salts medium (MSM) was prepared
2 as previously described (McCullar 1994).
3
4 Isolation of Ibu-2 via enrichment of sewage sludge
5 Sewage sludge was enriched with ibuprofen according to standard protocols
6 (Krieg 1981). A single colony was isolated and designated strain Ibu-2. A fragment of
7 the 16S rRNA gene from Ibu-2 was PCR amplified using the universal primers 27F
8 (AGAGTTTGATCMTGGCTCAG) and 1055R (CGGCCATGCACCACC) (Lane 1991)
9 and was sequenced using the 27F and 1055R primers.
10
11 Stereospecificity
12 Ibu-2 was inoculated into 500 mg/L R/S -ibuprofen, 500 mg/L S-ibuprofen, or 250
13 mg/L R/S -ibuprofen. Pure R-ibuprofen was not used because it is not commercially
14 available. Maximal cell density was determined spectrophometrically.
15 Chiral capillary electrophoresis (CE) was performed on the supernatants of
16 ibuprofen grown Ibu-2 cultures in order to determine if a difference existed in the rate at
17 which the enantiomers were metabolized. Supernatants were harvested and filtered as
18 described above. Samples were run on an HP3D CE using a 40 cm x 50 µm fused silica
19 column from Agilent technologies (Palo Alto, CA) and a method adapted from Simo et
20 al. (Simo 2002). Cassette temperature was set at 25 ºC. The running buffer was
21 composed of 6% dextrin 10 and 150 mM sodium borate at pH 9. Prior to each injection,
22 the column was preconditioned for 1 min with water, 1 min with 0.1 M NaOH and 50
23 mM SDS, and finally for 2 min with the running buffer. Injection was performed by
67
1 applying 10 mbar pressure for 9 seconds followed by running buffer injection at 50 mbar
2 for 5 seconds. Voltage was applied at +20 kV. A diode array detector was used with
3 detection and reference wavelengths set at 194 +/- 2 nm and 500 +/- 80 nm respectively.
4
5 Substrate specificity analysis with washed cells
6 Ibu-2 was grown in MSM containing 500 mg/L ibuprofen or 0.1% glucose.
7 Washed cell suspensions (WCS) were prepared according to standard protocols (Focht
8 1994). Test compounds were added to a final concentration of 500 mg/L in 500 µl
9 aliquots of WCS and yellow color generation was monitored for thirty min. The yellow
10 products were further characterized by determining their absorbance maxima via
11 spectrophotometry. Yellow color was assumed to be indicative of meta -cleavage product
12 and was thus taken as positive indication of metabolism of the test compound.
13 Chemicals tested included phenol, 2-, 3-, and 4-methylphenol, catechol, 3-
14 methylcatechol, 4-methylcatechol, 4-tertbutylcatechol, benzoate, 4-methylbenzoate,
15 phenylacetaldehyde, phenylacetic acid, R- and S-2-hydroxy-2-phenylacetic acid
16 (mandelic acid) 2-, 3-, and 4-tolylacetic acid, 2- and 4-(hydroxyphenyl)acetic acid, 2- and
17 3-phenylpropionic acid, 2-(4-tolyl)propionic acid, 2-phenyl-3-hydroxypropionic acid
18 (tropic acid), 3-phenyl-2-propenoic acid (cinnamic acid), 2-phenylbutyric acid, and 2,2-
19 diphenylacetic acid.
20
21 Growth substrate analysis
22 Growth of Ibu-2 was tested on compounds that gave a positive result in the
23 substrate specificity analysis. Tests were performed in triplicate in test tubes. 5 ml of an
68
1 overnight Ibu-2 culture grown on ibuprofen was used to inoculate MSM containing 250
2 mg/L or 500 mg/L of the chemical of interest. The tubes were placed on a vertical rotor
3 and monitored over the course of one week for growth via changes in OD at 600 nm
4 (OD 600 ).
5 Compounds that did not support growth on their own were assayed for their
6 ability to support growth in the presence of ibuprofen as an inducer. Ibu-2 was
7 inoculated into mixtures of 250 mg/L of these compounds plus 250 mg/L ibuprofen in
8 MSM.
9
10 Analysis of culture supernatants by GC/MS
11 Ibu-2 was inoculated into 1 L of MSM containing 500 mg of ibuprofen. When
12 grown on ibuprofen, the culture accumulated a yellow color. The culture was allowed to
13 continue growing until that color reached an apparent maximum level (48-60 hours). At
14 this point, the supernatant was harvested via centrifugation and filtered through a 0.22 µm
15 filter. The supernatant was then acidified to pH 3 using 1 M HCl and extracted with 50
16 ml of ethyl acetate. The extract was concentrated to a volume of 2 ml under nitrogen at
17 35 °C. The samples were then methylated with diazomethane by standard protocols
18 (Hecht and Kozarich 1972). After thirty min at room temperature, the samples were
19 evaporated to a minimal volume under a nitrogen stream and analyzed via GC/MS using
20 an HP 6890 GC equipped with an HP-5MS column (5% phenyl methyl siloxane 30 m x
21 0.25 mm, 0.25 µm film thickness) using helium as the carrier gas with a flow rate of 1
22 ml/min. The injector temperature was 250°C. The initial oven temperature of 40°C was
23 held for 1 min, then ramped at a rate of 10°C/min to 250°C. The temperature was held at
69
1 250°C for 7 min and then ramped up at 30°C/min until 300°C. The detector was an HP
2 5973 MSD with the quadrupole and source set at 150ºC and 230ºC respectively.
3
4 Analysis of catecholic ibuprofen intermediates
5 3-fluorocatechol, a meta -cleavage inhibitor, was added to a final concentration of
6 50 mg/L in 100 ml of mid-log-phase Ibu-2 culture. After 30 min, the supernatant was
7 removed and filtered. Potassium carbonate and acetic anhydride were added to final
8 concentrations of 1.5% and 0.5% respectively. This aqueous acetylation was performed
9 to selectively acetylate aromatic hydroxyl groups (Mars 1997). After thirty min, the
10 samples were acidified to pH 3, extracted with ethyl acetate, and dried over a sodium
11 sulfate column. The extracts were then methylated with diazomethane and analyzed as
12 described above. A culture with ibuprofen but without 3-fluorocatechol was also
13 analyzed.
14
15 Characterization of catechols produced from ibuprofen analogs
16 Assays were performed with WCS and the 2-arylpropionic acids and phenylacetic
17 acids that produced yellow metabolites. The assays were performed with and without the
18 addition of 3-fluorocatechol. The supernatants were passed through a 0.22 µm filter and
19 then analyzed via HPLC for accumulation of catechols. The HPLC retention times of the
20 metabolites were compared to those of catechol and methylcatechol standards. HPLC
21 was performed using 70% methanol, 30% 40 mM acetic acid as the eluent. The sample
22 was pumped at a rate of 1 ml/min using a Waters Model 590 pump through a Varian
23 Microsorb-MV C18 column (250 mm by 4.6 mm). Samples were injected by a Shimadzu
70
1 SIL-10AD AP autoinjector and detected with a Shimadzu SPD-10A VP UV-Vis detector
2 by monitoring absorbance at 285 nm.
3
4 Cell-free extract assays
5 Ibuprofen-grown Ibu-2 was washed with 0.9% NaCl to remove the
6 exopolysaccharide matrix (Richau 2000) and then resuspended in a minimal volume of
7 sonication buffer (100 mM tris, 2 µm phenylmethylsulfonyl fluoride, and 1 µm
8 dithiothreitol at pH 8 (Long 1997)). The cells were then lysed via sonication using a
9 Branson Sonifer 450 set at 100% duty cycle using three cycles of 30 seconds each
10 separated by 1 min rest time . The cells were kept on ice at all times. The samples were
11 then centrifuged at 21000 x g at 4 °C for 15 min and the supernatant retained. Extracts
12 were assayed for activity by adding catechol to a small aliquot and monitoring for yellow
13 product generation.
14
15 Assaying sidechain oxidation
16 Cell extracts from Pseudomonas AT3 grown on tropic acid, prepared using the
17 method described above, were used as a positive control for side chain oxidation (Long
18 1997). The assay, which monitors NAD +/NADP + reduction, was performed with cell free
19 extracts from Ibu-2 or AT3 in sonication buffer with either 0.1 µM tropic acid or
20 ibuprofen and 0.1 µM of either NAD + or NADP +. The reactions were incubated at room
21 temperature and monitored for reduction of NAD + or NADP + at 340 nm.
22
23
71
1 Results and Discussion
2
3 Ibu-2 grew on ibuprofen as a sole source of carbon and energy (data not shown).
4 When grown on ibuprofen in liquid media, a yellow color appeared in the supernatant.
5 This yellow color disappeared upon acidification and reappeared upon neutralization, a
6 phenomenon diagnostic of meta-cleavage products ( mcp ). Sequencing and BLAST
7 analysis (Altschul 1990) of the 16S rRNA gene fragment revealed that Ibu-2 was 98%
8 identical to Sphingomonas species over 967 bp. Ibu-2 had yellow pigmentation and
9 tended to develop an exopolysaccharide matrix, especially when grown on glucose. Both
10 of these characteristics are common to Sphingomonas species (Pollock 1993).
11
12 Stereospecificity
13 Ibu-2 grew to the same maximum cell density on 500 mg/L R/S -ibuprofen as it
14 did on 500 mg/L S-ibuprofen. Both of these values were approximately twice that
15 obtained using 250 mg/L S ibuprofen or R/S -ibuprofen. During growth on R/S -ibuprofen
16 the entantiomeric fraction dropped to less than 35% R-ibuprofen before both isomers
17 where completely removed, suggesting that Ibu-2 may preferentially degrade the R
18 enantiomer (Figure 2.1).
19
72
0.6 600 0.5 500 0.4 400 300 0.3 200 0.2 mg ibuprofen/L
100 0.1 EF of R ibuprofen 0 0.0 0 20 40 60 80 100 Time (hrs) 1 2 Figure 2.1. The enantiomeric fraction (EF) of R-ibuprofen (-○-) and overall ibuprofen 3 concentration (-♦♦♦-) in a growing Ibu-2 culture in which ibuprofen is the sole carbon and 4 energy source. The concentrations of both ibuprofen enantiomers were determined by 5 chiral CE analysis. 6
7 GC/MS analysis of culture supernatant extracts
8 When ibuprofen-grown cultures were poisoned with 3-fluorocatechol, a
9 metabolite accumulated in the supernatant whose mass spectrum was consistent with
10 isobutylcatechol (metabolite B, Table 2.1). The mass spectrum of this compound
11 revealed a molecular ion at m/z 250, which is consistent with diacetylated
12 isobutylcatechol. The two acetyl groups, which were added during aqueous acetylation,
13 are diagnostic of the presence of two aromatic hydroxyl groups (Mars 1997). Acetyl
14 groups give predictable losses of m/z 42, which in this case accounted for the peaks at m/z
15 208 and 166. The other large peak at m/z 123 represents a loss of 43 which is consistent
16 with the loss of the isopropyl group from the base ion fragment. The combination of this
17 mass spectral fragmentation pattern, the derivatizable nature of metabolite during
73
1 aqueous acetylation, and its accumulation only in the presence of 3-fluorocatechol are
2 strong evidence that the peak detected via GC-MS was indeed isobutylcatechol.
3 Mass spectra were also obtained for two other compounds from the extract of a
4 culture that was not poisoned with 3-fluorocatechol and which had accumulated high
5 levels of mcp (metabolites C and D, Table 2.1).
6
7 Table 2.1. GC/MS retention times and major ions of isobutylcatechol (B) and two putative 8 isobutylcatechol meta-cleavage metabolites (C and D). MS ret. time (min.) mass (relative abundance)
b 15.7 123(99) 166(100) 208(17) 250(4)
c 14.7 226(2) 167(100) 151(6) 137(1) 123(8)
d 15.8 256(10) 225(10) 197(100) 139(20)
9
10 These spectra were not detected in the extract of cultures poisoned with 3-
11 fluorocatechol. One spectrum was consistent with the mcp of isbutylcatechol: 5-formyl-
12 2-hydroxy-7-methylocta-2,4-dienoic acid (metabolite C, figure 2.2), and the other was
13 consistent with its formyl-oxidized derivative, 2-hydroxy-5-isobutylhexa-2,4-dienedioic
14 acid (metabolite D, figure 2.2) .
15
74
COOH OH OH OH OH COOH COOH COOH O
1 a b c d 2 Figure 2.2. Proposed pathway for the metabolism of ibuprofen by Ibu-2. Metabolites b-d 3 were all detected via GC/MS. b: isobutylcatechol; c: 5-formyl-2-hydroxy-7-methylocta- 4 2,4-dienoic acid; d: 2-hydroxy-5-isobutylhexa-2,4-dienedioic acid 5
6 As expected, treatment with diazomethane methylated both the acidic hydroxyl
7 and the alpha carbon hydroxyl groups of metabolites C, giving a molecular ion of m/z
8 226. The m/z 167 fragment represents a loss of 59 from the parent ion, which is
9 consistent with the loss of a methylated carboxyl group and is a common loss from
10 aliphatic esters. The m/z 137 fragment is consistent with the loss of CH 2O from the m/z
11 167 fragment.
12 The major fragments of metabolite D ( m/z 256 / 225 / 197 / 139) are consistent
13 with the expected transformation product of metabolite C. After derivatization this
14 would be expected to have three additional methyl groups, one on each acidic hydroxyl
15 group and one on the alpha hydroxyl group. A loss of 31 to give m/z 225 is consistent
16 with loss of CH 2OH from the parent ion. An alternative loss from the parent ion (m/z
17 256) yielded a fragment with an m/z 197 and is consistent with the loss of a methylated
18 carboxylic acid group (-59). Further impact of this fragment would be expected to result
19 in a loss of 58 which would correspond to removal of the second methylated carboxylic
20 acid group and yield the fragment with an m/z 139.
21
75
1 Substrate specificity analysis
2 Ibu-2 washed cell suspension was also able to metabolize phenylacetic acid, 3-
3 and 4-tolylacetic acids, 2-phenylpropionic acid, and 2-(4-tolyl)-propionic acid. However,
4 it was not able to metabolize 2-phenylbutyric acid or 2,2-diphenylacetic acid, which
5 implies that the nature of the substitution on the alpha carbon is important. Neither
6 phenol nor any methylphenol was metabolized, making it less likely that a phenolic
7 metabolite was involved. Washed cell suspension could not metabolize either mandelic
8 acids or tropic acid (2-phenyl-3-hydroxypropionic acid), implying that hydroxylation of
9 the acid side chain was not an intermediate step in side-chain removal. Furthermore,
10 washed cell suspension was not able to metabolize benzoate or 4-methylbenzoate,
11 suggesting that benzoic acids, which have been shown to be intermediates in the
12 anaerobic degradation of phenylacetic acid by a Pseudomonas sp. and by Azoarcus
13 evansii (Mohamed, Ismail et al. 2002), were not likely to be intermediates in ibuprofen
14 degradation. Other compounds that were not metabolized include 4-tertbutylcatechol,
15 phenylacetaldehyde, 2-tolylacetic acid, 2- and 4-(hydroxyphenyl)acetic acid, 3-
16 phenylpropionic acid, 3-phenyl-2-propenoic acid (cinnamic acid). In all cases, it is
17 possible that lack of metabolism was due to lack of transport into the cell.
18
19 Growth substrate analysis
20 The only ibuprofen analogs that supported growth of Ibu-2 without the presence
21 of ibuprofen as an inducer were 3-tolylacetic acid and 2-(4-tolyl)propionic acid.
22 However, 4-tolylacetic acid was able to support growth when ibuprofen was also present
23 in the media. Phenylacetic acid and 2-phenylpropionic acid did not support growth under
76
1 any conditions (Figure 2.3). Surprisingly, 3-tolylacetic acid supported less than half of
2 the growth permitted by ibuprofen or 4-tolylacetic and 4-tolylpropionic acids (p < 0.02).
3 This suggests the possibility of toxicity or incomplete metabolism. If the latter were true,
4 accumulation of a metabolite might have been predicted. However, no dead-end
5 metabolites were detected when the culture supernatant from 3-tolylacetic acid grown
6 cells was subjected to HPLC or GC/MS analysis (data not shown).
77
500 2(4t)pa 250 2(4t)pa 500 4taa 250 4taa 500 3taa 250 3taa 500 2ppa 250 2ppa 500 paa 250 paa 500 ipf 250 ipf 250 ipf / 250 3taa 250 ipf / 250 4taa 250 ipf / 250 2ppa 250 ipf / 250 paa
0 0.2 0.4 0.6 0.8 OD 1
2 Figure 2.3. Average final culture density (n=3) as measured by OD 600 when Ibu-2 was 3 inoculated in liquid MSM culture containing ibuprofen or its analogs (ipf ibuprofen, paa 4 phenylacetic acid, 2ppa 2-phenylpropionic acid, 3taa 3-tolylacetic acid, 4taa 4-tolylacetic 5 acid, 2(4t)pa 2-(4-tolyl)propionic acid). 6
7
78
1 Characterization of catechols produced from ibuprofen analogs
2 When the supernatants of WCS that had been incubated with ibuprofen analogs
3 and 3-fluorocatechol were examined via HPLC, each exhibited a novel peak. These
4 peaks were only present after the addition of 3-fluorocatechol. In the phenylacetic acid
5 and 2-phenylpropionic acid samples, a peak appeared whose retention time matched that
6 of catechol (6.4 min). Within 2 hrs, almost 60% of the phenylacetic acid added was
7 converted to catechol and no other intermediates were detected, suggesting that the
8 observed deacylation was not merely an unproductive side reaction. In the 3-tolylacetic
9 acid sample, a peak appeared whose retention time matched that of 3-methylcatechol
10 (13.5 min). In the 4-tolylacetic acid and 2-(4-tolyl)-propionic acid samples, a peak
11 appeared whose retention time matched that of 4-methylcatechol (11.6 min).
12
13 Characterization of meta-cleavage products of ibuprofen analogs.
14 The mcps of phenylacetic acid, 2-phenylpropionic acid, and catechol all had the
15 same maximum absorbance wavelength (378 nm). The mcps of 3-tolylacetic acid and 3-
16 methylcatechol also had the same maximum absorbance wavelength (380 nm), as did 4-
17 tolylacetic acid, 2-(4-tolyl)-propionic acid, and 4-methylcatechol (384 nm). These
18 observations all suggest that deacylation of the acidic side chain occurred before ring
19 cleavage.
20
21 Cell-free extract activities upon ibuprofen
22 Ibu-2 cell-free extracts readily produced mcp from catechol, 3-methylcatechol,
23 and 4-methylcatechol (data not shown). Ibu-2 cell-free extracts did not generate any
79
1 yellow color when incubated with ibuprofen with or without the addition of
2 NADH/ferrous iron, nor was any ibuprofen disappearance detected via HPLC under any
3 conditions. The Ibu-2 cell free extracts did not reduce NAD + or NADP + in the presence
4 of either ibuprofen or tropic acid, although the positive control reduced NAD + in the
5 presence of tropic acid. Cofactor reduction woµld have been expected if Ibu-2 used a
6 Pseudomonas AT3-like mechanism to oxidize the sidechain of ibuprofen.
7
8 Conclusions
9
10 Unlike Variovorax sp Ibu-1, which has been suggested to dioxygenate the
11 aromatic ring of ibuprofen in the 2 and 3 positions (Murdoch 2002), Ibu-2 appears to
12 metabolize ibuprofen through a novel mechanism resµlting in removal of the propionic
13 acid moiety and dioxygenation of the ring at the 1,2 position, giving rise to
14 isobutylcatechol. The accumµlation of this compound when and only when a meta-
15 cleavage inhibitor was added suggests that isobutylcatechol is further metabolized via
16 meta-cleavage. The identification of compounds consistent with the meta-cleavage of
17 isobutylcatechol only when a meta-cleavage inhibitor was not added lends further support
18 to this conclusion.
19 As a similar side chain removal of related 2-phenylpropionic acids or
20 phenylacetic acids has not been previously reported, the ability of Ibu-2 to metabolize
21 other aromatic acids was determined. The production of catechols and their respective
22 mcps from these compounds provides additional evidence for this unique pathway.
80
1 The exact mechanism whereby Ibu-2 accomplished this acid side chain removal
2 remains to be determined. Although it is likely that other steps were required to activate
3 the acid moiety prior to removal, we could not detect any evidence suggesting the
4 involvement of other intermediates prior to the formation of catechols. Further work will
5 be required before the mechanism whereby this is accomplished can be elucidated.
6
7 Acknowledgements
8
9 RWM was supported in part by NIH-NIEHS Environmental and Molecµlar Toxicology
10 training grant ES 07052-27. Pseudomonas AT3 was a kind gift from Dr. David J.
11 Hopper.
12
81
1 REFERENCES 2 3 Altschul, S. F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990). "Basic local 4 alignment search tool." Journal of Molecular Biology 215 : 403-410. 5 Andreoni, V., G. Baggi, et al. (1992). "Metabolism of phenylpropanoid compounds by 6 Pseudomonas cepacia ." Annali di Microbiologia ed Enzimologia 42 (2): 261-266. 7 Buser, H. R., T. Poiger, et al. (1999). "Occurrence and environmental behavior of the 8 chiral pharmaceutical drug ibuprofen in surface waters and in wastewater." 9 Environmental Science and Technology 33 : 2529-2535. 10 Chen, Y. and J. P. N. Rosazza (1994). "Microbial transformation of ibuprofen by a 11 Nocardia species." Applied and Environmental Microbiology 60 (4): 1292-1296. 12 Defrank, J. J. and D. W. Ribbons (1976). "The p-cymene pathway in Pseudomonas 13 putida strain Pl isolation of a dihydrodiol accumulated by a mutant." Biochemical 14 and Biophysical Research Communications 70 (4): 1129-1135. 15 Defrank, J. J. and D. W. Ribbons (1977). "P-cymene pathway in Pseudomonas putida 16 ring cleavage of 2,3 dihydroxy p-cumate and subsequent reactions." Journal of 17 Bacteriology 129 (3): 1365-1374. 18 Eaton, R. (1996). "p-Cumate catabolic pathway in Pseudomonas putida Fl: cloning and 19 characterization of DNA carrying the cmt operon." J Bacteriol 178 : 1351 - 1362. 20 Eaton, R., W. (1997). "P-Cymene catabolic pathway in Pseudomonas putida F1: Cloning 21 and characterization of DNA encoding conversion of p-cymene to p-cumate." 22 Journal of Bacteriology 179 (10): 3171-3180. 23 Farre, M., I. Ferrer, et al. (2001). "Determination of drugs in surface water and 24 wastewater samples by liquid chromatography-mass spectrometry: methods and 25 preliminary result including toxicity studies with Vibrio fischeri ." Journal of 26 Chormatography A 938 : 187-197. 27 Focht, D. D. (1994). Chapter 20: Microbiological procedures for biodegradation research. 28 Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties . R. 29 W. Weaver, J. S. Angle and P. S. Bottomley. Madison, WI, Soil Science Society 30 of America : 407-426.
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1 Hecht, S. M. and J. W. Kozarich (1972). "A convenient method for the production of 2 diazomethane." Tetrahedron Letters 13 (15): 1501-1502. 3 Ismail, W., M. Mohamed, et al. (2003). "Functional genomics by NMR spectroscopy. 4 Phenylacetate catabolism in Escherichia coli." European Journal of Biochemistry 5 270 (14): 3047-3054. 6 Kolpin, D. W., E. T. Furlong, et al. (2002). "Pharmaceuticals, hormones, and other 7 organic wastewater contaminants in U.S. streams, 1999-2000: A national 8 reconnaissance." Environmental Science and Technology 36 : 1202-1211. 9 Krieg, N. R. (1981). Enrichment and Isolation. Manual of Methods for General 10 Bacteriology . P. Gerhardt. Washington, D.C., American Society for 11 Microbiology : 112-142. 12 Kuge, Y., K. Mochida, et al. (1991). "Microbial hydroxylation of 2 phenylpropionic 13 acid." Agricultural and Biological Chemistry 55 (4): 1099-1104. 14 Lane, D. J. (1991). 16S/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial 15 Systematic . E. S. M. Goodfellow. Chichester, Wiley : 115–175. 16 Lee, E. J. D., K. Williams, et al. (1985). "Stereoselective disposition of ibuprofen 17 enantiomers in man." British Journal of Clinical Pharmacology 19 : 669-674. 18 Long, M. T., Bartholomew, B. A., Smith, M. J., Trudgill, P. W., and D. J. Hopper (1997). 19 "Enzymology of oxidation of tropic acid to phenylacetic acid in metabolism of 20 atropine by Pseudomonas sp. strain AT3." Journal of Bacteriology 179 (4): 1044- 21 1050. 22 Mars, A. E. K., T.; Kaschabek, S. R.; Van Agteren, M.H.; Janssen, D.B. {a}; Reineke, 23 W, (1997). "Microbial degradation of chloroaromatics: Use of the meta-cleavage 24 pathway for mineralization of chlorobenzene." Journal of Bacteriology 179 (14): 25 4530-4537. 26 McCullar, M., Brenner, R., Adams, R., Focht, D. (1994). "Construction of a novel 27 polychlorinated biphenyl-degrading bacterium; utilization of 3,4'- 28 dichlorobiphenyl by Pseudomonas acidovorans M3GY." Applied and 29 Environmental Microbiology 60 : 3833-3839.
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1 Mohamed, M., W. Ismail, et al. (2002). Aerobic metabolism of phenylacetic acids in 2 Azoarcus evansii . Archives of Microbiology , Springer Berlin / Heidelberg. 178: 3 180-192. 4 Murdoch, R. W., Hay, A.G. (2002). Isolation of a bacterium capable of using S-ibuprofen 5 as a sole carbon source . 102nd General Meeting of the American Society for 6 Microbiology, Salt Lake City, UT, USA. 7 Pollock, T. (1993). "Gellan-related polysaccharides and the genus Sphingomonas ." 8 Journal of General Microbiology 139 : 1939-1945. 9 Richau, J., Leitao, J., and Sa-Correia, I. (2000). "Enzymes leading to the nucleotide sugar 10 precursors for exopolysaccharide synthesis in Burkholderia cepacia ." 11 Biochemical and Biophysical Research Communications 276 : 71-76. 12 Rudy, A. C., P. M. Knight, et al. (1991). "Stereoselective metabolism of ibuprofen in 13 humans: administration of R-, S- and racemic ibuprofen." Journal of 14 Pharmacology and Experimental Therapeutics 259 : 1133-1139. 15 Simo, C., Gallardo, A., San Roman, J., Barbas, C., and Cifuentes, A. (2002). "Fast and 16 sensitive capillary electrophoresis method to quantitatively monitor ibuprofen 17 enantiomers released from polymeric drug delivery systems." Journal of 18 Chromotography B 767 : 35-43. 19 Sparnins, V. L. and P. J. Chapman (1976). "Catabolism of L-tyrosine by the 20 homoprotocatechuate pathway in gram positive bacteria." Journal of Bacteriology 21 127 (1): 362-366. 22 Stumpf, M., T. A. Ternes, et al. (1999). "Polar drug residues in sewage and natural waters 23 in the state of Rio de Janeiro, Brazil." The Science of the Total Environment 225 : 24 135-141. 25 van den Tweel, W. J. J., J. P. Smits, et al. (1988). "Catabolism of DL-alpha 26 phenylhydracrylic, phenylacetic, and 3 and 4-hydroxyphenylacetic acid via 27 homogentisic acid in a Flavobacterium Sp." Archives of Microbiology 149 (3): 28 207-213. 29 Winkler, M., J. R. Lawrence, et al. (2001). "Selective degradation of ibuprofen and 30 clofibric acid in two model river biofilm systems." Water Research 35 (13): 3197- 31 3205.
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1 Zwiener, C., S. Seeger, et al. (2002). "Metabolites from the biodegradation of 2 pharmaceutical residues of ibuprofen in biofilm reactors and batch experiments." 3 Analytical and Bioanalytical Chemistry 372 (4): 569-575. 4
85
1 CHAPTER 3
2
3 GENETIC AND CHEMICAL CHARACTERIZATION OF IBUPROFEN
4 AND PHENYLACETIC ACID METABOLISM BY SPHINGOMONAS
5 IBU-2
6
7 Abstract
8
9 Sphingomonas Ibu-2 has the unique ability to cleave the acid side chain from the
10 non-steroidal anti-inflammatory drug ibuprofen and related phenylacetic acid
11 derivatives to yield corresponding catechols under aerobic conditions. To identify
12 the genes involved in this unusual pathway, we constructed a chromosomal
13 library of Ibu-2 DNA in Escherichia coli epi300. Fosmid clones capable of
14 metabolizing ibuprofen to isobutylcatechol were identified. Transposon
15 mutagenesis of one of the fosmids (pFOS3G7) allowed identification of thirteen
16 mutants that failed to produce detectatable catechols. DNA sequencing revealed
17 insertions in five open reading frames ipfABDEF whose predicted amino acid
18 sequences bore similarity to the large and small units of aromatic dioxygenases,
19 an SCPx thiolase, a domain of unknown function 35 (DUF35), and an aromatic
20 coenzyme A ligase respectively. A knockout of ipfH which putatively encodes a
21 ferredoxin reductase component of an aromatic dioxygenase system showed
22 reduced catecholic metabolite production. An open reading frame ipfI which
23 putatively encodes the ferredoxin component of an aromatic dioxygenase was also
86
1 identified on pFOS3G7. Complementation of a markerless loss-of-function ipfD
2 deletion mutant with ipfD restored catechol-generation ability. Subcloned
3 ipfABDEF produced catecholic metabolites, but only in trace amounts. The co-
4 expression of ipfABDEF with ipfH and ipfI greatly enhanced the production of
5 catecholic metabolites. The existence of the isobutylcatechol metabolite and the
6 novel involvement of an SCPx thiolase (IpfD) and a DUF35 protein (IpfE) in the
7 generation of the catechol suggest that this pathway is distinct from others
8 described to date and represents a novel aerobic paradigm employed for the
9 metabolism of alkyl aromatic acids.
10
11 Introduction
12
13 Ibuprofen (2-(4-isobutylphenyl-propionic acid)) is a pharmaceutical with
14 analgesic, antipyretic, and anti-inflammatory properties. With an annual production of
15 several kilotons (Buser, Poiger et al. 1999), it is the most widely used member of a
16 diverse class of pharmaceuticals termed the non-steroidal anti-inflammatory drugs
17 (NSAIDs), many of which share a phenylacetic acid (PAA) core such as diclofenac,
18 naproxen, ketoprofen, and flurbiprofen (Figure 1.1). Approximately 10% of the
19 ibuprofen consumed by humans is excreted unmodified or as the glucuronide conjugate
20 (Lee, Williams et al. 1985; Rudy, Knight et al. 1991). The drug’s popularity, large
21 therapeutic dose, and incomplete human metabolism lead to a large amount of ibuprofen
22 entering the environment with human waste. Wastewater treatment processes have been
23 found to remove ibuprofen to varying degrees (Buser, Poiger et al. 1999; Stumpf, Ternes
87
1 et al. 1999; Farre, Ferrer et al. 2001; Winkler, Lawrence et al. 2001; Kolpin, Furlong et
2 al. 2002; Fent, Weston et al. 2006; Benotti, Trenholm et al. 2008; Huerta-Fontela and
3 Ventura 2008; Corcoran, Winter et al. 2010; Kummerer 2010; Santos, Araújo et al. 2010)
4 which may explain why ibuprofen has been detected in bodies of water all over the world
5 (Buser, Poiger et al. 1999; Stumpf, Ternes et al. 1999; Farre, Ferrer et al. 2001; Winkler,
6 Lawrence et al. 2001; Kolpin, Furlong et al. 2002). Ibuprofen has also been detected in
7 water used for irrigation (Pedersen, Yeager et al. 2003; Pedersen, Soliman et al. 2005;
8 Kinney, Furlong et al. 2006; Siemens, Huschek et al. 2008; Xu, Wu et al. 2009) and
9 municipal drinking supplies (Jones, Lester et al. 2005). Environmental concentrations of
10 ibuprofen have been found to range from low part-per-trillion (Buser, Poiger et al. 1999)
11 to low part-per-billion levels (Buser, Poiger et al. 1999; Farre, Ferrer et al. 2001; Santos,
12 Araújo et al. 2010)
13 Investigations into subtle effects and mixture toxicity have found ibuprofen
14 induced changes on the timing of spawning by medaka (Flippin, Hugget et al. 2007; Han,
15 Choi et al. 2010), growth/predominance of algae and duckweed (Pomati, Netting et al.
16 2004; Richards, Wilson et al. 2004), microbial diversity in aquatic mesocosms (Richards,
17 Wilson et al. 2004), and riverine biofilm communities (Lawrence, Swerhone et al. 2005)
18 at environmentally relevant concentrations.
19 Little information exists regarding how ibuprofen is oxidatively metabolized by
20 environmental microbes. The fungus Verticillum lecanii has been shown to hydroxylate
21 ibuprofen on the isobutyl group (Hanlon 1994) and similar microbially-generated
22 hydroxyibuprofen metabolites along with carboxylated ibuprofen have been detected
23 (Zwiener, Seeger et al. 2002; Quintana, Weiss et al. 2005; Marco-Urrea, Pérez-Trujillo et
88
1 al. 2009). However, these metabolites have not been linked directly with the ability to
2 use ibuprofen as a growth or energy source nor did they account for the majority of the
3 added ibuprofen.
4 Sphingomonas Ibu-2 is able to use racemic ibuprofen as a sole carbon and energy
5 source (Murdoch and Hay 2005). Ibu-2 utilizes a classical catechol meta -cleavage type
6 pathway. However, the catechol that is generated from ibuprofen is isobutylcatechol, the
7 creation of which requires the removal of the propionic acid side-chain from the aromatic
8 ring (Figure 3.1). It was also demonstrated that Ibu-2 performs similar reactions with
9 other phenylacetic acids including phenylacetic acid, 2-phenylpropionic acid, 3- and 4-
10 tolylacetic acids, and 2-(4-tolyl)propionic acid, converting them to the corresponding
11 catechols (catechol or methylcatechol).
89
COOH
OH phenylacetic acid OH COOH 378 nm
catechol
2-phenylpropionic acid
COOH
OH
p-tolylacetic acid OH 384 nm COOH
4-methylcatechol
2(p-tolyl)propionic acid
COOH OH OH 380 nm
m-tolylacetic acid 3-methylcatechol
COOH OH OH 382 nm
1 ibuprofen 4-isobutylcatechol
2 Figure 3.1. Catechols and corresponding meta-cleavage products absorbance maxima and 3 the substrate chemicals as detected in ibuprofen-induced resting Sphingomonas Ibu-2 4 (Chapter 2). 5 6 There is to this author’s knowledge no precedent for the ejection of an acyl group from an
7 aromatic ring in such a manner, though it is reminiscent of 1,2- benzoate dioxygenases
8 that remove the carboxyl moiety from benzoate and which have been extensively
90
1 described (Reiner 1971; Fetzner, Muller et al. 1992; Jeffrey, Cuskey et al. 1992; Eaton
2 1996). This side chain removal phenomenon also differs from published reports of the
3 metabolism by other bacteria of phenylacetic acid which has been shown to take place via
4 coenzyme A ligation followed by epoxidation and non-oxygenolytic ring cleavage but
5 does not involve catecholic intermediates (Martinez-Blanco, Reglero et al. 1990;
6 Mohamed 2000; Rost, Haas et al. 2002; Ismail, Mohamed et al. 2003; Fernandez,
7 Ferrandez et al. 2006; Teufel, Mascaraque et al. 2010).
8 The metabolism of ibuprofen and related phenylacetic acids by Sphingomonas
9 Ibu-2 appears to represent a new paradigm for the metabolism of phenylacetic acids. In
10 order to identify the genes responsible for the conversion of these aromatic acids to the
11 corresponding catechols, a fosmid library was constructed of Ibu-2 total DNA. A fosmid
12 that conferred upon E. coli the ability to generate isobutylcatechol from ibuprofen was
13 subjected to transposon mutagenesis. We report here on the results of these efforts and a
14 description of the steps involved in ibuprofen and phenylacetic acid degradation by
15 Sphingomonas Ibu-2.
16
17 Material and Methods
18
19 Materials
20 Unless otherwise noted, chemicals were purchased from Acros (Morris Plains,
21 NJ). Luria-Bertani broth (LB) was prepared as previously described (Sambrook, Fritsch
22 et al. 1989). Mineral salts medium (MSM) was prepared as previously described
23 (McCullar 1994).
91
1
2 Creation and screening of Ibu-2 fosmid library
3 Ibu-2 chromosomal DNA was extracted from cells harvested from 100 mL of
4 liquid MSM culture containing 500 mg/L ibuprofen (ipf) by the standard alkaline lysis
5 procedure (Sambrook, Fritsch et al. 1989). The harvested DNA was quantified using the
6 Ribogreen Kit (Molecular Probes, Eugene, OR). The DNA was prepared and the fosmid
7 library was created according to the instructions in the CopyControl™ Fosmid Library
8 Production Kit instructions (Epicentre Biotechnologies, Madison, WI). Briefly, the DNA
9 was sheared and size selected for 35kb-45kb. The DNA was then end-repaired, ligated
10 into the fosmid vector, and packaged in phage particles. These particles were then used
11 to transfect E. coli epi300 and successful transformants were selected on LB 25 mg/L
12 chloramphenicol (chl). A 900 clone library (approximately 7X expected genome
13 coverage) was constructed in 96-well plates and stored with the addition of 20% glycerol
14 at -80 deg C. The fosmid library was screened for the accumulation of dark brown
15 catecholic polymers when grown in LB with 50 mg/L ibuprofen in 96-well plates. 10mM
16 arabinose was used to induce the fosmid to high copy number in all metabolic assays.
17
18 Creation and metabolic screening of fosmid clone transposon libraries
19 The EZ::TN
20 used to create transposon insertion mutants of chromosomal library fosmid pFOS3G7
21 which was positive for isobutyl catechol accumulation. The mutagenesis was performed
22 as described in the kit instructions. The reaction was packaged in phage extract
23 (MaxPlax Lambda Packaging Extract, Epicentre Technologies, Madison, WI), transfected
92
1 into E. coli epi300, and selected on LB plates containing 25 mg/L chl and 12 mg/L
2 tetracycline (tet). A 96-clone library of transposon mutants was then screened for loss of
3 the ability to accumulate the brown color associated with catechol production and
4 polymerization.
5 The loss-of-function mutants were characterized by sequencing DNA surrounding
6 the site of transposon insertion using transposon-specific primers FP and RP (Table 3.2).
7 The sequence information was compiled using the SeqManager program (DNAStar, Inc.,
8 Madison, WI) and searched for open reading frames using the GeneQuest program
9 (DNAStar, Inc., Madison, WI). Similarity to known and putative proteins was assessed
10 using BLAST-P (Altschul 1990).
11
12 Table 3.1. Strains and plasmids used in this study. 13 Strains Sphingomonas Ibu- isolated from Ithaca, NY sewage treatment 2 plant via enrichment for growth on ibuprofen this study λpir; hsdR pro recA ; RP4 2-Tc::Mu-Km::Tn7, pro, res-, mod+, streptomycin resistance, (Simon, Priefer et al. E. coli S17 trimethoprim resistance 1983) recA1 subE44 endA1 hsdR17 gyrA96 relA1 thi ∆(lac-proAB ) F' ( traD36 proAB+ lacIq lacZ (Sambrook, Fritsch et E. coli JM109 ∆M15 ) al. 1989) F- mcrA ∆(mrr-hsdRMS-mcrBC) Φ80dlacZ∆M15 ∆lacX74 recA1 endA1 araD139 ∆(ara, leu)7697 galU galK λ - rpsL E. coli epi300 (Str R) nupG trfA tonA Epicentre, Madison, WI
Plasmids contains the template for generating FLP recombinase target lambda red kanamycin (Datsenko and Wanner pKD4 resistance PCR fragment; kanamycin resistant 2000). arabinose-inducible lambda red recombinase (Datsenko and Wanner epi300 3G7 pKD46 expression plasmid; ampicillin resistant 2000).
93
heat inducible FLP recombinase expression (Datsenko and Wanner pCP20 plasmid 2000). copy control fosmid vector, inducible to high copy number with arabinose when hosted in E. pCC1FOS coli epi300, chloramphenicol resistance Epicentre, Madison, WI pCC1FOS Sphingomonas Ibu-2 chromosomal pFOS3G7 library clone 3G7 this study pCC1FOS Sphingomonas Ibu-2 chromosomal pFOS4F6 library clone 4F6 this study broad host range plasmid, chloramphenicol (Kovach, Elzer et al. pBBR1mcs resistance 1995) pBBR1mcs with BamHI/NsiI fragment from pJ25 pCC1FOS 3G7 bearing ipfABDEF this study pFOS3G7 with Tn5 insertion in ipfA , transposon library clone F1, chloramphenicol pFOS3G7Tn: ipfA and tetracycline resistant this study pFOS3G7 with Tn5 insertion in ipfA , transposon library clone H6, chloramphenicol pFOS3G7Tn: ipfB and tetracycline resistant this study pFOS3G7 with Tn5 insertion in ipfD , transposon library clone A2, chloramphenicol pFOS3G7Tn: ipfD and tetracycline resistant this study pFOS3G7 with Tn5 insertion in ipfE , transposon library clone E6, chloramphenicol pFOS3G7Tn: ipfE and tetracycline resistant this study pFOS3G7 with Tn5 insertion in ipfF , transposon library clone F10, chloramphenicol pFOS3G7Tn: ipfF and tetracycline resistant this study pFOS3G7 with markerless deletion of ipfA, pFOS3G7∆ipfA chloramphenicol resistant this study pFOS3G7 with markerless deletion of ipfB, pFOS3G7∆ipfB chloramphenicol resistant this study pFOS3G7 with markerless deletion of ipfD, pFOS3G7∆ipfD chloramphenicol resistant this study pFOS3G7 with markerless deletion of ipfF, pFOS3G7∆ipfE chloramphenicol resistant this study pGEMt-easy ampicillin resistance Promega, Madison, WI pGEMt-easy with ipfArbsF/ipfAR PCR product ipfA including a three frame stop codon and a pGEM: ipfA rbs ribosomal binding site, this study pGEMt-easy with ipfBrbsF/ipfBR PCR product ipfB including a three frame stop codon and a pGEM: ipfB rbs ribosomal binding site, this study pGEMt-easy with ipfDrbsF/ipfER PCR product ipfDE including a three frame stop codon and a pGEM: ipfDE rbs ribosomal binding site, this study pGEMt-easy with ipfDrbsF/ipfDR PCR product ipfD including a three frame stop codon and a pGEM: ipfD rbs ribosomal binding site, this study
94
pGEMt-easy with ipfErbsF/ipfER PCR product ipfE including a three frame stop codon and a pGEM: ipfE rbs ribosomal binding site, this study pGEMt-easy with ipfAF and ipfBR PCR product pGEM: ipfAB ipfAB this study pGEMt-easy with ipfFF/ipfFR PCR amplicon pGEM: ipfF ipfF this study pGEMt-easy with ipfHI PCR amplicon created pGEM: ipfHI by strand overlap extension this study 1 2
3 Functional analysis of pFOS3G7 clones via HPLC
4 Overnight cultures of E. coli epi300 harboring pFOS3G7 or different transposon
5 mutants of pFOS3G7 were inoculated (10% v/v) into five ml of LB containing 50 mg/L
6 ibuprofen, the appropriate antibiotics, and 10mM arabinose. The cultures were incubated
7 at 37°C in a rotary shaker. One ml samples were taken at the initiation of the experiment
8 and at four days and analyzed for ibuprofen concentration via HPLC. The HPLC solvent,
9 80% methanol 20% 40mM acetic acid, was pumped at a rate of 1 ml/min using a Waters
10 Model 590 pump through a Varian Microsorb-MV C18 column (250 mm by 4.6 mm).
11 Samples and standards were injected by a Shimadzu SIL-10AD AP autoinjector and
12 detected with a Shimadzu SPD-10A VP UV-Vis detector. The UV-Vis signal was fed
13 into a PC computer where it was collected and analyzed using Peaksimple (SRI
14 Instruments, Torrance, CA). Ibuprofen was quantified by comparison to a standard
15 curve.
16 GC/MS was performed on overnight cultures to characterize catecholic
17 metabolites or other detectable metabolites that might accumulate. Aqueous acetylation
18 was performed by previously described methods (Murdoch and Hay 2005) in order to
19 selectively derivatize aromatic hydroxyl groups. The sample was then extracted with
20 ethyl acetate and dried over a sodium sulfate column. The ethyl acetate extracts were
95
1 methylated using diazomethane and dried once more over a sodium sulfate column.
2 GC/MS was performed using previously described methods (Murdoch and Hay 2005).
3 Presence or absence of catechols was determined by HPLC analysis of washed
4 cell suspensions that had been concentrated 20-fold and was performed as previously
5 destribed (Murdoch and Hay 2005).
6
7 Fosmid subcloning
8 Analysis of sequence from the transposon mutants implicated an approximately
9 5kb region of fosmid pFOS3G7 as being involved in the production of isobutylcatechol
10 (Figure 3.4). This region was gel purified away from the rest of the fosmid after
11 digestion with BamHI and NsiI. The 5kb fragment was then ligated into pBBR1mcs
12 (Kovach, Elzer et al. 1995) that had been digested with BamHI and PstI. The ligation
13 was transformed into JM109 via electroporation and selected on LB chl 25 mg/L plates.
14 The transformants were screened using primers specific for the desired insert. The
15 resulting plasmid, pJ25, was then harvested and transformed into E. coli epi300 in order
16 to afford comparisons with the fosmid in the same genetic background.
17 PCR strand overlap extension (SOE) strategy was employed to co-express ipfH, a
18 putative ferredoxin reductase gene, and ipfI, a ferredoxin gene, both of which were also
19 located on pFOS3G7. SOE employed PCR primers with additional 5’ homology
20 overlaps between the two PCR products that allowed for artifical joining of the two PCR
21 products. An initial round of PCR using a 50C anneal and 1.5 minute extension with a
22 pFOS3G7 template and the SOE primers (Table 3.2) was used. The products, which
23 were of the predicted size, were gel purified and used as template for three cycles of
96
1 primer-less PCR under the same conditions. IpfFeDoxFsew and ipfFeDoxRedRsew were
2 then added and PCR performed under the same conditions, yielding a combined product
3 of the predicted size. The SOE product was t-cloned into pGEMt-easy, sequence
4 verified, and transformed into E. coli epi300 pJ25.
5 6 Table 3.2. Primers used in this study. 1: pGEMt- Easy vector system, Promega 7 Corporation, Madison, WI. 2: EZ::TN
ipfFeDoxFsew TACCGCCGAGCAGGAATATTACAGCCGCGACA this study ipfFeDoxRsew2 GAAGCGGGTACATTCCTCCAGACGGTCCTC this study ipfFeDoxRedFsew GGAGGAATGTACCCGCTTCACGCACACAATCTA this study ipfFeDoxRedRsew TTTCACCGCAGGCCTATGCCGC this study ipfDlambdaF CATGGGGACGAGGCTGTTCCCAGTCTGGCATTCATCATCTGACGCGCACCGTGTA this study GGCTGGAGCTGCTTC ipfDlambdaR AATGCCTCGTCGTAGCGGTGAAAAAAGGTGCTATCCATCAATGGCTCCAGATGGG this study AATTAGCCATGGTCC ipfAlambdaF GGAAATACTTTCAGCGCCAGTTTTTACACTATTTTTGTGTAGGCTGGAGCTGCTTC this study ipfAlambdaR CCCGAAGGGGGGCGGATGAGGTGGTGATCAATACGCATGGGAATTAGCCATGGTCC this study ipfBlambdaF AGCGTCCTGCGAGCAGGACGCGCAAGGAGATACATCGTGTAGGCTGGAGCTGCTTC this study ipfBlambdaR TGGTCTTTTTCTTCACTCGCGTGACCATGGTGCGCGATGGGAATTAGCCATGGTCC this study ipfElambdaF AGCCCACACCGCACCCTCGCCTGACTGGAGCCATTGGTGTAGGCTGGAGCTGCTTC this study 9 ipfElambdaR TCTTGCCAACATAACTTGTCTCCTTGGTTTTAAGTTATGGGAATTAGCCATGGTCC this study 10 11
12 Metabolic analyses of subclones
13 The metabolic capabilities of E. coli epi300 pJ25 pGEM: ipfHI (J25HI) were
14 studied by adding substrates to mature LB-grown cultures. J25HI was compared directly
97
1 to E. coli epi300 pJ25, E. coli epi300 pGEM: ipfHI , and E. coli epi300 with no vector.
2 Initial expression assays utilizing 25ppm chloramphenicol with the addition of substrate
3 at the time of inoculation failed to demonstrate activity. To promote higher copy
4 numbers of the plasmids prior the introduction of substrate, cultures were grown into
5 stationary phase before introduction of substrate. In order to avoid negative selection
6 effects exerted by the putative toxic catcholic metabolites, substrate addition was delayed
7 until 24 hours after inoculation. At 24 hours 1mM of test substrate added as a 1M
8 solution in ethanol was spiked into the cultures. Test substrates were ibuprofen and
9 phenylacetate both of which have been previously shown to be converted to
10 corresponding deacylated catechols by Sphingomonas Ibu-2 (Chapter 2). The media
11 consisted of LB and 100ppm chloramphenicol and/or 150ppm ampicillin. Following the
12 addition of test substrate, cultures were incubated at 37C for 18 hours.
13 For direct visualization of catechols, ferric choride was added to 150ul of culture
14 to a final concentration of 1.5mM in 96-well plate format. Additionally, HPLC was used
15 to directly quantify substrate and catechols. 40:60 methanol:40mM acetic acid running
16 buffer was used for separation of phenylacetate (10.6 minutes) and catechol (4.3
17 minutes). 70:30 methanol:40mM acetic acid was used to separate ibuprofen (13.2
18 minutes) and a peak at 3 minutes presumed to be isobutylcatechol due to its high
19 absorbance at 280nm. A detection wavelength of 220nm was used for the aromatic acids
20 while a detection wavelength of 280nm was used for the catechols. Standard curves were
21 used to quantify the analytes. Because no standard exists for isobutylcatechol, the
22 catechol standard curve was used to approximate isobutylcatechol concentration.
23
98
1 Complementation of pFOS3G7 ipfABDEF mutants
2 pFOS3G7Tn: ipfF was complemented by the cloning ipfF into pGEMt-easy using
3 the primers ipfFF/ipfFR that had been designed so as to include the native ribosomal
4 binding site (Table 3.2). Attempts to complement other four genes (ipfABDE ) using the
5 same approach described above were unsuccessful (results not shown). In order to
6 reduce the influence of possible polar effects introduced by the Tn5 cassette, markerless
7 mutants were created using the lambda red system using a protocol modified from
8 Datsenko and Wanner (Datsenko and Wanner 2000). pKD4 insertion cassette primers
9 with 36-50bp overlap homology to target gene-flanking regions (Table 3.2) were used to
10 generate insertion cassettes and transformed into E. coli epi300 pFOS3G7 pKD46 with
11 arabinose induction as described previously (Datsenko and Wanner 2000). However, the
12 pCC1 fosmid is induced to high copy number by arabinose which led to difficulty in
13 isolating insertion mutants presumably due to the loss of mutated fosmid during recovery
14 following transformation with the insertion cassette. In order to achieve acceptable
15 recovery of insertion mutants, 10mM arabinose was therefore added during both the
16 recovery and initial plating which increased recovery of insertion mutants by more than
17 an order of magnitude (data not shown). The cassettes were removed by electroporation
18 and induction of pCP20 as previously described (Datsenko and Wanner 2000). Insertions
19 and deletions were confirmed by PCR analysis.
20 Except in the case of ipfF, initial attempts to complement the loss of function
21 knockouts using expression constructs that included the native ribosomal binding site
22 regions were unsuccessful (data not shown). In order to address the possibility that the
23 lack of complementation was due lack of efficient translation, complements were created
99
1 using primers with artificial stop codons and ribosomal binding sites added to the 5’ end
2 of the forward primers (Figure 3.2). PCR was performed using proof-reading polymerase
3 followed by monoadenylation of the resulting blunt-ended product by adding taq
4 polymerase and ATP following purification. Resulting plasmids were sequenced and
5 given the suffix rbs, then transformed into pFOS3G7 deletion mutants or transposon
6 mutants and screened for catechol accumulation. Because pGEM: ipfE rbs failed to
7 complement pFOS3G7∆ipfE , an attempt to complement using pGEM: ipf DErbs was
8 undertaken.
5’-TAG CTAG C TAG C AAGGAG ATATACAT(N 18 -24 )-3’
three-frame stop RBS spacer Gene-specific primer 9
10 Figure 3.2 expression vector forward primer strategy. RBS; ribosomal binding site 11
12 In silico analyses
13 Fosmid transposon library clone sequences were assembled into contiguous units
14 using SeqMan (DNAStar, Inc., Madison, WI) and searched for open reading frames
15 (ORFs) using GeneQuest (DNAStar, Inc., Madison, WI). Translated ORFs were
16 subjected to Blastp analysis against the Swiss-prot database at the National Center for
17 Biotechnology Institute (NCBI) website (Altschul, Madden et al. 1997). Conserved
18 domain analysis was conducted using the CDD tool at NCBI (Marchler-Bauer, Anderson
19 et al. 2003; Marchler-Bauer and Bryant 2004). Phylogenetic trees were built using the
20 COBALT multiple alignment tool (Papadopoulos and Agarwala 2007 ) using Fast
100
1 Minimum Evolution tree building method with Grishin distance. Three-dimensional
2 structure modeling was performed using Swiss-model and visualized using Swiss-PDB
3 viewer (Peitsch 1995; Arnold, Bordoli et al. 2006; Kiefer, Arnold et al. 2009). Protein
4 amino acid sequence alignments were performed using MegAlign (DNAStar, Inc.,
5 Madison, WI).
6
7 Expression of ipfD and ipfE in E. coli K12 paa operon mutants
8 In order to investigate the interaction between E.coli K-12 phenylacetate
9 metabolites (Chapter One, Figure 1.8) and IpfDE, the ipfDE pGEMt-easy expression
10 vector described above was electroporated into E.coli K-12 ∆ paaG , ∆ paaZ , and ∆ paaJ
11 mutants and screened for effects on growth rate, catechol generation, and for effects on
12 the rate of phenylacetate metabolism and 2-hydroxyphenylacetate accumulation. The
13 growth medium was 0.2% glycerol with 7mM phenylacetate, 1mM IPTG, and 150ppm
14 ampicillin adjusted to pH 7. Strains with pGEM: ipfDE rbs were tested against control
15 strains harboring empty pGEMt-easy vector. Phenylaceteate and 2-hydroxyphenylacetate
16 were assayed via HPLC using the same equipment described above with a 40:60
17 methanol:40mM acetic acid eluent. LB medium with 1.5 mM ferric chloride and
18 100ppm p-toluidine along with 1mM IPTG and 150 ppm ampicillin was employed for the
19 detection of catechol.
20
21 Metabolism of Phenylacetyl Coenzyme A in cell-free extracts
22 Further metabolism of phenylacetyl CoA was measured in reactions containing 50
23 µl 2mM phenylacetyl CoA (Sigma, St. Louis, MO), 50 µl 2mM NADH, and 100 µl cell-
101
1 free extract. Crude cell-free extracts of E. coli clones were prepared by centrifuging
2 100ml of mid-exponential growth phase 1mM IPTG or 10mM arabinose induced culture,
3 washing, and resuspending in a minimal volume of sonication buffer (40 mM potassium
4 phosphate, 20% glycerol, 1mM PMSF, 1mM DTT, pH 7.4). Approximately 0.1g of 0.1
5 mm glass beads was then added and the pellets were bead-beaten for three minutes
6 (MiniBeadbeater-8, Biospec Products, Bartlesville, OK). The pellets were then spun
7 down as described above. In both cases, the protein content of the supernatant containing
8 the crude extract was quantified using the Bio-Rad Protein Assay (Bio-Rad Laboratories,
9 Hercules, CA). The reactions were left at room temperature for one hour, whereupon the
10 reaction was terminated by the addition of 20 ul 4M trichloroacetic acid. In order to
11 quantify phenylacetyl CoA, the samples were analyzed via HPLC with UV-Vis detection
12 as described above.
13 GC/MS was used to test for the appearance of putative metabolites of
14 phenylacetyl-CoA. After acidification of the samples to terminate the reaction, the pH
15 was raised to 10 by the addition of sodium hydroxide in order to promote alkaline
16 hydrolysis of the coenzyme group (Mangino, Zografakis et al. 1992). The effectiveness
17 of this procedure was confirmed by noting the disappearance of phenylacetyl-CoA by
18 HPLC in a separate assay. After 20 minutes, aqueous acetylation was performed by
19 previously described methods (Murdoch and Hay 2005) in order to selectively conjugate
20 aromatic hydroxyl groups. The sample was then acidified to pH 2.5 and precipitates were
21 removed via centrifugation. The sample was then extracted with ethyl acetate and dried
22 over a sodium sulfate column. The ethyl acetate extracts were methylated using
23 diazomethane and dried once more over a sodium sulfate column. GC/MS was
102
1 performed using the methods described above. A standard of 2-hydroxyacetic acid, a
2 hypothetical metabolic byproduct, was also subjected to the same treatment and analysis.
3
4
5 Results
6
7 Ibu-2 fosmid library
8 E. coli epi300 pFOS3G7 was the only clone of the 900 clone Ibu-2 fosmid library
9 that accumulated visible brown pigment when exposed to ibuprofen in liquid media. The
10 presence of isobutylcatechol (Murdoch and Hay 2005) in ethyl acetate extracts of
11 acetylated culture supernatant from E. coli epi300 pFOS3G7 grown in LB and exposed to
12 500 mg/L ibuprofen was confirmed via GC/MS (retention time 15.7 minutes with major
13 peaks (relative abundance) of 123(99), 166(100), 208(17), 250 (4)) (Figure 3.3).
103
OH OH
5000000 4500000 4000000 166 3500000 123 3000000 OH O 2500000 O O O 2000000 O abundance 1500000 O
1000000 208 500000 250 0 1 12 23 34 45 56 67 78 89 100 111 122 133 144 155 166 177 188 199 m/z 1
2 Figure 3.3. Mass-spectrum of acetylated isobutylcatechol detected in extracts of E. coli 3 epi300 pFOS3G7 supernatant.
4
5 Screening and Characterization of E. coli epi300 pFOS3G7 transposon library
6 When the 96-clone transposon library of E. coli epi300 pFOS3G7 was screened
7 for catechol accumulation in the presence of ibuprofen, seventeen clones no longer
8 accumulated the characteristic dark catecholic polymerization product, indicating a loss-
9 of-function. Sequencing of the DNA surrounding the transposon insertion and analysis
10 of the sequence information revealed that thirteen of the E. coli epi300 pFOS3G7
11 transposon insertion clones harbored a transposon in a 5kb region of Ibu-2 DNA, while
12 the other four clones had the transposon in the vector. Sequence analysis (DNAStar, Inc.,
13 Madison, WI) revealed that this 5kb region contained five open reading frames (ORFs)
14 (Figure 3.4) with sequence similarities to aromatic and other metabolic enzyme-encoding
15 genes described in more detail below. All five ORFs had representative knockouts
104
1 amongst the loss-of-function mutants. Only ipfB and ipfF had recognizable conserved
2 ribosomal binding sites (Table 3.3).
3 In addition to ipfABDEF, two open reading frames with sequence similarities to a
4 ferredoxin reductase ( ipfH ) and a ferredoxin ( ipfI ) were identified (Figure 3.4). ipfH was
5 identified by observing that a particular transposon library mutant (pFOS3G7Tn: ipfH )
6 accumulated dark catecholic coloring at a slightly slower rate. ipfI was fortuitously
7 identified during random sequencing of pFOS3G7 transposon library clones.
8
9 Table 3.3. DNA immediately upstream of ipf open reading frames. Purine-rich regions 10 with homology to conserved ribosomal binding site are underlined . Only ipfB and ipfF had 11 strong RBSs. 12 ipfA CAGCGCCAGTTTTTACACTATTTTT start
ipfB AGCAGGACGCGCAAGGAGA TACATC start
ipfD TGGCATTCATCATCTGACGCGCACC start
ipfE CACCCTCGCCTGACTGGAG CCATTG start
ipfF AAACTTAAAACCAAGGAGA CAAGTT start 13
14
15
16
105
ipfI ipfH
ipfA ipfB ipfD ipfE ipfF C1 F9 D4 E7 E6 E8 B8 F1 H6 F10 C9 A2 B1 1
2 Figure 3.4. Open reading frames found on pFOS3G7. Large black arrows represent ORFs 3 with high similarity to conserved transposase genes. Small black arrows represent the 4 location and orientation of loss-of-function transposon insertions. ORFs described in this 5 study are named. 6
7 Metabolic analyses of clones and constructs
8 Deletions of genes following application of the lambda-red system was successful
9 as indicated by PCR analysis (Figure 3.5). Ibuprofen disappearance assays revealed
10 several trends. First of all, they clearly demonstrated that expression of pFOS3G7 in E.
11 coli epi300 caused the disappearance of ibuprofen from the culture supernatant (Figure
12 3.6). E. coli epi300 pFOS3G7 loss-of-function mutants representing each putative
13 metabolic gene caused partial or complete loss of ibuprofen disappearance (Figure 3.6)
14 while not producing visible catecholic polymers (Figure 3.7) or any isobutylcatechol
15 detectable by GC/MS (data not shown). Notably, pFOS3G7Tn: ipfF did not degrade
16 ibuprofen at all.
17
106
ipfA ∆ipfA ipfB ∆ipfB ipfD ∆ipfD ipfE ∆ipfE
2kb- 1.6kb-
1kb-
0.5kb-
1
2 Figure 3.5. PCR of indicated ipf genes using either intact pFOS3G7 or makerless mutants. 3 All products were of the predicted size for targeted gene deletion. 4
120%
100%
80%
60%
40%
ibuprofen remaining ibuprofen 20%
0%
l F o fF pf r p 3G7 i nt :i o Tn: c M pGE F :ipf Tn 5 6
7 Figure 3.6. Percentage of 50ppm ibuprofen remaining after two days of incubation in E. 8 coli epi300 pFOS3G7, loss-of-function mutants, and the two successful complementation
107
1 constructs as determined by HPLC analysis of the cultures shown in Figure 3.17. n = 3, 2 standard deviations were too small to be visualized effectively.
3G7 ∆ipfA ∆ipfB ∆ipfD ∆ipfE Tn :ipfF epi300
∆ipfD Tn:ipfF pGEM:ipfD rbs pGEM:ipfF 3
4 Figure 3.7. Catecholic polymer accumulation in E. coli epi300 pFOS3G7, loss-of-function 5 mutants, and the two successful complement constructs grown in LB broth with 50 ppm 6 ibuprofen and 1.5mM ferric chloride. 7
3G7TnF 3G7 3G7TnF pGEM:ipfF
3G7 3G7∆D 3G7∆D pGEM:ipfD rbs 8
9 Figure 3.8. Centrifuged knockout and complement strains showing the tendency of 10 catecholic polymers created in the presence of 1.5mM ferric chloride to pellet along with the 11 cell mass.
108
1 Markerless deletion mutants of the individual ipf genes ( ∆ipfA, ∆ipfB, ∆ ipfD, and
2 ∆ipfE) also failed to produce catecholic metabolites (Figures 3.7 and 3.8) and eliminated
3 less ibuprofen than intact pFOS3G7 (Figure 3.6). Complementation of pFOS3G7 ∆ipfD
4 with pGEM: ipfD rbs and pFOS3G7Tn: ipfF with pGEM: ipfF restored catechol generation
5 and ibuprofen disappearance (3.6-3.8), although we were unable to complement the other
6 mutants.
7 Results of HPLC analysis of E. coli epi300 pFOS3G7Tn: ipfH were consistent
8 with the observation that dark catecholic metabolites accumulate more slowly in this
9 clone (Figure 3.9).
100% 90% 80% 70% 60% 50% 40% 30%
Ibuprofen remaining Ibuprofen 20% 10% 0% 3G7 3G7C3 Tn:ipfH epi300 10
11 Figure 3.9. % of 50ppm ibuprofen metabolizied by E. coli epi300 pFOS3G7, E. coli epi300 12 pFOS3G7Tn: ipfH, and epi300 negative control after four days as determined by HPLC 13 analysis. 14
15 When ipfABDEF were subcloned into pBBR1mcs to create pJ25, E. coli
16 harboring this plasmid produced trace amounts of isobutylcatechol when grown with
17 ibuprofen as detected by GC/MS (data not shown), but no polymerization products were
18 visible nor were levels detectable via HPLC analysis. E. coli epi300 pJ25 pGEM: ipfHI
109
1 cultures showed dark brown coloration in response to ferric chloride addition (Figure
2 3.10) while single vector or vectorless controls contained no detectable dark coloration.
3 HPLC analysis revealed that E. coli epi300 pJ25 pGEM: ipfHI degraded at least 50%
4 more of the ibuprofen and phenylacetate test substrates than single vector or vectorless
5 controls. Additionally, HPLC analysis detected catechol in phenylacetate only in dual-
6 vector cultures. The disappearance of phenylacetate in the dual vector culture (0.58 mM)
7 corresponded closely with the accumulation of catechol (0.37 mM) (Figure 3.11). The
8 putative isobutylcatechol peak was quantified using the catechol standard curve because
9 no standard for isobutylcatechol is available. 0.42 mM ibuprofen disappeared from the
10 pJ25 pGEM: ipfHI culture and approximately 0.25 mM isobutylcatechol appeared (Figure
11 3.12).
110
IPF PAA
pJ25 + pGEM:HI
pJ25
pGEM:HI
Vectorless control
1 75 .5 .2 .1 0 0. 0 0 0 Catechol standard
1 curve (mM)
2 Figure 3.10. 150ul of E. coli epi300 harboring the indicated plasmids following 18 hours of 3 incubation with 1mM ibuprofen (IPF) or phenylacetate (PAA) with 1.5 mM ferric chloride 4 for catecholic metabolite visualization. 5 6
111
0.9 0.8 0.7 0.6
0.5 paa
mM 0.4 cat 0.3 0.2 0.1 0 pJ25 + pHI pJ25 pHI negative 1
2 Figure 3.11. Phenylacetate and catechol concentration in E coli epi300 cultures harboring 3 pJ25 and/or pGEM: ipfHI (pHI) following 18 hours of incubation 1 mM phenylacetate. 4
0.9 0.8 0.7 0.6
0.5 ipf
mM 0.4 ibc 0.3 0.2 0.1 0 pJ25 + pHI pJ25 pHI negative 5
6 Figure 3.12. Ibuprofen and isobutylcatechol concentration in E coli epi300 cultures 7 harboring pJ25 and/or pGEM: ipfHI (pHI) following 18 hours of incubation 1 mM 8 ibuprofen. 9 10
11 Phenylacetyl coenzyme A disappearance in E. coli pGEM:ipfAB cell-free extract
12 The rate of phenylacetyl coenzyme A disappearance in both E. coli pFOS3G7
13 (172 nmol/(mg protein*hr)) and JM109 pGEM: ipfAB (161 nmol/(mg protein*hr)) cell-
112
1 free extracts were significantly higher than in cell-free extract of JM109 (123 nmol/(mg
2 protein*hr)) (p<0.05) (Figure 3.13). While no novel peaks were detected via GC/MS
3 analysis, the peak area of phenylacetic acid was approximately 90% less in the JM109
4 pGEM: ipfAB sample than it was in the JM109 control.
200
150
100 protein * hour * protein 50
nmol PAA-CoA disappearance / mg mg PAA-CoA disappearance/ nmol 0 3G7 pGEM AB JM109 Strain 5 6
7 Figure 3.13. Disappearance of phenylacetyl CoA (PAA-CoA) in cell-free extract 8 experiments. PAA-CoA was quantified via HPLC. n=3. All data points are significantly 9 different from one another as calculated by student’s t-test (p<.05). 10
11 Expression of ipfDE in E. coli K12 paa mutants
12 In order to gain a greater understanding of the role of IpfD and IpfE, we expressed
13 them in E.coli K12 mutants that had lost the ability to cleave the ring of phenylacetic acid
14 (paaG, paaZ, and paaJ mutants). The genes were co-expressed due to the co-dependency
15 of bacterial SCPx thiolases and DUF35 proteins that has been previously observed in the
16 Phl (Bangera and Thomashow 1999) and Bbs (Leuthner and Heider 2000; Kube, Heider
17 et al. 2004; Kuhner, Wohlbrand et al. 2005) pathways. This work was undertaken prior
18 to the recent publication by Teufel et al (Teufel, Mascaraque et al. 2010) with the
113
1 hypothesis that Ibu-2 and K12 might share a common hydroxylated, CoA-ligated
2 intermediate which could be acted upon by IpfD and IpfE, resulting in isobutylcatechol
3 production. While no isobutylcaechol was detected, expression of ipfDE caused a 57%
4 increase in the amount of PAA disappearance (p<0.05) and a 67% increase (p<0.05) in
5 the amount of 2-hydroxyphenylacetate detected in the supernatant of the E. coli K12
6 ∆paaG mutant, but not in the wild type or either of the other two mutants implicated in
7 ring fission (Figure 3.14, also see Figure 1.8 for the paa pathway). The 2-
8 hydroxyphenylacetate accounted for the majority of metabolized phenylacetate in strain
9 K12 ∆ paaG.
10
2 * 1.8 1.6 1.4 1.2 paa metabolized 1 * mM 2-OHpaa accumulated 0.8 0.6 0.4 0.2 0 WT WT G G DE J J DE Z Z DE DE 11
12 Figure 3.14. A; Concentration of phenylacetate remaining after three days in E. coli K12 13 cultures (7mM starting concentration). The strains are WT, wild type; G, ∆ paaG , J, ∆ paaJ ; 14 Z, ∆ paaZ. “DE” indicates presence of pGEM: ipfDE rbs. All strains without 15 pGEM: ipfDE rbs harbored the empty pGEMt-easy vector. All strains were grown in 0.2% 16 glycerol MSM with 1mM IPTG induction at 37C. Asterics indicate significantly higher 2- 17 hydroxyphenylacetate (p<0.05) 18
19
114
1 Discussion
2
3 Previously (Murdoch and Hay 2005), isobutylcatechol was detected in ibuprofen-
4 grown Sphingomonas Ibu-2 cultures when and only when catechol-meta-cleavage
5 activity had been poisoned by the addition of 3-fluorocatechol. The removal of the acid
6 side-chain and consequent creation of the corresponding catechol was demonstrated for a
7 variety of phenylacetic acids by ibuprofen-induced cultures (Figure 3.1). GC/MS spectra
8 corresponding to isobutylcatechol meta-cleavage products were also observed (Table
9 2.1).
10 In order to further study this pathway, a fosmid library was constructed in E. coli
11 using Sphingomonas Ibu-2 chromosomal DNA. E. coli epi300 pFOS3G7 turned brown
12 in the presence of ibuprofen, indicative of accumulation of a catecholic metabolite. The
13 disappearance of ibuprofen from E. coli epi300 pFOS3G7 and accumulation of
14 isobutylcatechol was confirmed via HPLC and GC/MS respectively. Creation and
15 screening of a transposon library of pFOS3G7 led to the discovery of five genes
16 (ipfABDEF) that, when knocked out, prevented isobutylcatehcol accumulation and
17 slowed the disappearance of ibuprofen.
18
19 ipfA
20 Sequence analysis of ipfA revealed that its predicted protein product contained
21 two conserved domains an N-terminal Rieske domain and a C-terminal catalytic domain
22 similar to those of the alpha subunit of anthranilate 1,2-dioxygenase (AntDO, CD03538)
23 conserved domain and related Rieske-type non-heme iron aromatic ring-hydroxylating
115
1 oxygenase (RHO, CD00680) conserved domain. The general RHO domain is typified by
2 having a large hydrophobic pocket that affords aromatic substrate specificity while
3 proteins containing the AntDO subfamily domain are specific for aromatic acids (Gibson
4 and Parales 2000; Ferraro, Gakhar et al. 2005). The multi-domain architecture is similar
5 to those of 1,2-benzoate dioxygenase, 1,2-anthranilate dioxygenase, and 3-
6 phenylpropionate dioxygenases. There was no detectable similarity at any level to the
7 monooxygenase encoding genes of the E. coli paa operon paaABCDE. Thus,
8 phylogenetic analysis clearly places IpfA in the of the RHO superfamily (Table 3.4).
9
10
116
1 Table 3.4. The substrates, similarity measures, and confidence levels of comparisons 2 between the predicted amino acid sequence of ipfA and characterized large subunits of 3 dioxygenases found in the Swiss-prot database.
% positive Query Accession Description (alpha subunit of) Organism % identity E value similarity coverage ABY62759.1 indole 3-acetic acid dioxygenase IacC 34 51 94% 1.00E-62 P. putida P37333.3 biphenyl 2,3-dioxygenase 29 45 93% 6.00E-45 Burkholderia xenovorans AAB62285.1 p-cumate 2,3-dioxygenase 29 43 92% 2.00E-44 P. putida BAB78521.1 2,4-dichlorophenoxyacetic acid dioxygenase 27 44 84% 4.00E-44 Bradyrhizobium sp. HW13 Q7N4W0.1 3-phenylpropionate dioxygenase 28 46 95% 8.00E-44 Photorhabdus luminescens Q52028.1 biphenyl 2,3-dioxygenase 28 45 93% 1.00E-43 P. pseudoalcaligenes Q07944.1 benzene 1,2-dioxygenase 29 45 92% 2.00E-43 P. putida Q51601.3 2-halo benzoate 1,2-dioxygenase 29 46 83% 2.00E-43 Burkholderia cepacia A5W4F2.1 benzene 1,2-dioxygenase 28 45 92% 3.00E-43 P. putida F1 A8A344.1 3-phenylpropionate dioxygenase 28 46 94% 3.00E-43 Escerichia coli HS Q83K39.1 3-phenylpropionate dioxygenase 23 46 94% 4.00E-43 Shigella flexneri Q52438.1 biphenyl 2,3-dioxygenase 28 44 96% 9.00E-43 P. sp. KKS102 P07769.2 benzoate 1,2-dioxygenase 29 47 78% 2.00E-42 Acinetobacter sp. ADP1 ZP06690405.1 anthranilate 1,2-dioxgyenase 28 45 83% 2.00E-41 Acinetobacter sp. SH024 Q51494.1 napthalene 1,2-dioxygenase 28 47 94% 3.00E-41 P. aeruginosa BAH86807.1 2,4-dichlorophenoxyacetic acid dioxygenase 27 45 81% 3.00E-41 Sphingomonas sp. 58-1 P0A110.1 napthalene 1,2-dioxygenase 29 46 93% 4.00E-41 P. putida P23099.1 toluate 1,2-dioxygenase 29 47 70% 1.00E-40 P. putida Q46372.1 biphenyl 2,3-dioxygenase 27 44 95% 7.00E-40 Comomonas testosteroni O07824.1 napthalene 1,2-dioxygenase 28 45 93% 2.00E-39 4 P. fluorescens
5
6 The predicted protein product of ipfA was most closely related to iacC from P. putida ,
7 though the relationship was still distant (34% identity and 51% similar residues over 94%
8 of its length). iacC is predicted to encode the large subunit of an indole-3-acetic acid
117
1 dioxygenase (Leveau and Lindow 2005; Leveau and Gerards 2008), though the specifics
2 of the metabolic pathway have not been described. A sequence analysis of available iac -
3 region from P. putida revealed no genes bearing any similarity to ipfD, ipfE, or ipfF ,
4
5 ipfB
6 Sequence analysis of ipfB reveals strong homology to the beta or small subunits
7 of aromatic dioxygenases. The function of beta-subunits of RHO heterodimers is not
8 well known, but they are believed to serve a largely structural, non-catalytic role (Parales
9 and Resnick 2006). BLAST-P similarity analysis to a variety of dioxygenase beta
10 subunits show 20%+ identity and 40%+ similiarity (Table 3.5).
11
118
1 Table 3.5. The substrates, similarity measures, and confidence levels of comparisons 2 between the predicted amino acid sequence of ipfB and characterized small subunits of 3 dioxygenases found in the Swiss-prot database.
Description (beta subunit of) % Positive Query Accession % Identity E-value Organism similarity coverage 42151 p-cumate 2,3 dioxygenase 33 47 84% 1.00E-16 P. putida P37334.3 biphenyl 2,3 dioxygenase 31 50 85% 4.00E-15 Burholderia xenovorans Q52439.1 biphenyl 2,3 dioxygenase 32 46 79% 7.00E-14 P. sp. KKS102 Q46373.1 biphenyl 2,3 dioxygenase 33 46 83% 1.00E-13 Comamonas testosteroni A5W4F1.1 toluene 2,3 dioxygenase 25 48 86% 6.00E-13 P. putida F1 Q07945.1 benzene 1,2-dioxygenase 25 48 83% 8.00E-13 P. putida Q7N4V9.1 3-phenylpropionate dioxygenase 29 49 87% 1.00E-12 Photorhabdus luminescens P23100.1 toluate 1,2-dioxygenase 30 50 58% 5.00E-12 P. putida Q31XV1.1 3-phenylpropionate dioxygenase 28 48 87% 1.00E-11 Shigella boydii Q8XA73.1 3-phenylpropionate dioxygenase 28 47 87% 1.00E-11 E. coli P07770.1 benzoate 1,2-dioxygenase 26 49 53% 2.00E-09 Acinetobacter sp. ADP1 BAB78522.1 2,4-dichlorophenoxyacetic acid dioxygenase 26 44 83% 2.00E-08 Bradyrhizobium sp. HW13 ABY62760.1 indole-3-acetic acid dioxygenase 23 45 91% 3.00E-08 P. putida BAH86808.1 2,4-dichlorophenoxyacetic acid dioxygenase 24 43 83% 8.00E-07 Sphingomonas sp. 58-1 ADG20790.1 anthranilate 1,2-dioxygenase 25 44 74% 9.00E-07 Burkholderia sp. CCGE1002 1NDO_F napthalene dioxygenase 23 45 73% 3.00E-06 4 Pseudomonas
5
6 ipfD
7 Analysis of the predicted amino acid sequence of ipfD (Marchler-Bauer,
8 Anderson et al. 2003; Marchler-Bauer and Bryant 2004), revealed the presence of a
9 highly conserved domain similar to those found within the condensing enzyme family of
10 proteins related to sterol carrier protein X (SCPx) (E-value = 1.02x10-51 ) (Stolowich,
11 Petrescu et al. 2002). Condensing enzymes generally catalyze the condensation and/or
12 thiolytic cleavage of acetyl groups to or from beta-keto acids.
119
1 There are two main types of condensing enzymes; decarboxylating and non-
2 decarboxylating. Decarboxylating enzymes, which perform Claisen-like, CO 2-evolving
3 condensations, are largely represented by the many variants of polyketide synthesis
4 proteins found within bacteria which are integrally involved in the synthesis of polyketide
5 polymers (Ferrer, Jez et al. 1999; Das and Khosla 2009; Crawford and Townsend 2010).
6 Non-decarboxylating enzymes, also called beta-keto thiolases are separated into
7 two main groups by sequence phylogeny; thiolases and SCPx thiolases (Heath and Rock
8 2002). The general thiolase group is typified by the 3-keto thiolases involved in fatty
9 acid synthesis and catabolism such as FadA (Yang, He Yang et al. 1991). ”SCP”-x
10 thiolases are termed “sterol carrier proteins” due to early observations that disruption of
11 the gene led to steroid metabolism disfunction in mammals (Gallegos, Atshaves et al.
12 2001; Stolowich, Petrescu et al. 2002). While eukaryotic SCPx was originally suspected
13 to be involved in transport of cholesterols and fatty acids (Puglielli, Rigotti et al. 1995;
14 Gallegos, Atshaves et al. 2001), more detailed molecular analyses have demonstrated it’s
15 involvement in two specific metabolic reactions in eukaryotes; bile-acid synthesis
16 (Takeuchi, Chen et al. 2004)(Figure 3.15a) and the beta-oxidation of pristanoyl-CoA, a
17 branched-chain fatty acid (Figure 3.15b) (Wanders, Denis et al. 1997; Verhoeven and
18 Jakobs 2001; Westin, Hunt et al. 2007 ),.
120
O A. B. OH COSCoA
CH3 CH3 CH3 CH3
H3C COSCoA HO OH O
CoA CoA SCPx SCPx propionyl CoA propionyl CoA
COSCoA OH CH3 CH3 CH3
H3C COSCoA 1 HO OH
2 Figure 3.15. Reactions catalyzed by sterol carrier protein X (SCPx) in animals. A; the 3 thiolytic depropionation of 24-oxo-3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA (Takeuchi, 4 Chen et al. 2004): B; the thiolytic depropionation of 3-ketopristanoyl-CoA (Westin, Hunt et 5 al. 2007 ). 6
7 In both cases, SCPx is specifically involved in the beta-oxidation of an alpha-methyl
8 beta-keto fatty acid. While there are over two thousand SCPx-type bacterial genes in the
9 NCBI database, only four have been characterized to any degree. Phylogenetic analysis
10 of IpfD places it within the somewhat divergent bacterial SCPx group which, however, is
11 more closely related to eukaryotic SCPx proteins than it is to other bacterial FadA-type
12 traditional thiolases (Figure 3.16 and Table 3.6).
121
Bacterial FadA thiolase Pseudomomasfragi
Pseudomonas putida
Pseudomas putida
Escherichia coli
Enterobactercloacae
Chicken, Gallus gallus Eukaryotic SCPx
Cow, Bos taurus
Rabbit, Oryctolagus cuniculus
Mouse, Mus musculus
Moth, Bombyx morii Bacterial SCPx
Benzylsuccinyl-CoA thiolase bbsB, Aromatoleumaromaticum EbN1
PhlC, Pseudomonas fluorescens
SCPx ORF2, Rhodococcus sp.
IpfD
1 DitF, Pseudomonas abietaniphila
2 Figure 3.16. Phylogenetic tree of characterized thiolase-type proteins found in the Swiss- 3 prot database and listed in Table 3.5. 4
5 Table 3.6. The similarity measures and confidence levels of comparisons between the 6 predicted amino acid product of ipfD and and characterized thiolase-type proteins found in 7 the Swiss-prot database.
Query Accession Description Organism % Identity % Positive E-value coverage AAD21068.1 DitF P. abietaniphila 34 45 89% 1.00E-42 AAK50624.1 Camphor ORF2 Rhodococcus sp 33 49 91% 1.00E-37 YP_158078.1 BbsB Aromatoleum aromaticum EbN1 34 50 53% 5.00E-26 Q07598.1 SCPx Gallus gallus 30 44 64% 8.00E-23 NP_001075504.1 SCPx Oryctolagus cuniculus 28 44 68% 1.00E-22 P07857.2 SCPx Bos taurus 27 42 68% 3.00E-21 P32020.3 SCPx Mus musculus 26 43 63% 4.00E-20 NP_001037378.1 SCPx Bombyx morii 28 43 53% 3.00E-17 AAB48108.1 PhlC P. putida 36 52 39% 3.00E-14 P28790.2 FadA P. fragi 30 46 49% 2.00E-06 P21151.3 FadA E. coli 27 40 71% 3.00E-06 Q9R9W0.1 FadA P. putida 30 46 49% 6.00E-06 Q93Q11.1 FadA P. putida 30 46 49% 1.00E-05 8 Q9F0Y6.1 FadA Enterobacter cloacae 23 36 45% 3.00E-05
9
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1 Four bacterial SCPx genes have been described at some level. ditF
2 (Pseudomonas abietaniphila ) is a part of a catabolic cluster for abietic acid (Martin and
3 W. 1999; Martin and Mohn 2000)(Figure 3.17a). Knocking out the gene leads to
4 complete loss of growth on abietic acid, though neither the gene product nor the reactions
5 have been described further. PhlC (Pseudomonas putida ) is required for a polyketide
6 synthase-like action in conjunction with an acyl carrier protein and a domain of unknown
7 function 35 protein in the synthesis of a polyketide antibiotic from acetyl-CoA and
8 malonyl-CoA building blocks (Bangera and Thomashow 1999). The Phl proteins are
9 also able to catalyze the addition of an acyl group directy to an aromatic ring (Figure
10 3.17b). Rhodococcus sp. NCIMB 9784 camphor catabolic cluster ORF-2 (Grogan,
11 Roberts et al. 2001; Roberts, Grodan et al. 2004) may be involved in the metabolism of
12 camphor to alpha-campholinic acid by camK , a p450 hydroxylase, though the pathway
13 again has not been characterized nor has the gene’s involvement been confirmed. It has
14 however been noted that the pathway involves a Claisen-type reaction in the thiolytic
15 cleavage reaction between the 1,3-ketone carbons (Figure 3.17c) (Grogan, Roberts et al.
16 2001). While that work provides a hypothetical role for the involvement of an SCPx
17 thiolase, it is fully possible that ORF2 is involved in another aspect of the poorly-
18 described pathway. BbsB from Aromatoleum aromaticum EbN1 (Kube, Heider et al.
19 2004; Kuhner, Wohlbrand et al. 2005) and Thauera aromatica (Leuthner and Heider
20 2000) are part of a relatively better described pathway for the metabolism of toluene to
21 benzoyl-CoA under anaerobic denitrifying conditions. BbsB in conjunction with BbsA
22 performs the decondensation of an alpha-aliphatic beta-keto coenzyme A adduct in a
123
1 manner that mirrors the role of eukaryotic SCPx in the metabolism of bile acids and
2 branched fatty acids (Figure 3.17d).
3
ABCD COSCoA OH O O
COOH
COOH HO OH O O
acylCoA CoA camK + DitF PhlABC BbsAB ORF2? CoA succinylCoA
O OH O O COSCoA meta-cleavage
HO OH 4 COOH
5 Figure 3.17. General schema of bacterial metabolic pathways that require the action of 6 SCPx thiolases: A; DitF, B; PhlC, C; camphor ORF2, D; BbsB. 7
8 Although the branched nature of several of these substrates suggests a similar
9 mechianism may be catalyzed by IpfD, further consideration of ibuprofen and the
10 isobutylcatechol intermediates (Figure 3.1) reveals that a classical thiolase type reaction
11 is not possible due to the fact that there is no way for the beta-carbon, presumably part of
12 the aromatic ring, to be bonded to the sulfur atom of a coenzyme A residue. The thiolase
13 reaction when performed in a reverse catabolic role is defined by the use of the sulfur
14 residue to break the alpha-beta carbon bond; because such a reaction is not apparently
15 possible based on what is known of the ipf reaction, no such thiolase reaction can be
16 performed. Both the thiolase and eukaryotic SCPx type proteins share two primary
124
1 catalytic residues that have been shown to be crucial to thiolase activity, a cysteine in the
2 80-100 aa region and a histidine in the 350-400 region (Bangera and Thomashow 1999).
3 Alignment of IpfD and other bacterial SCPx thiolases against well-characterized SPCx
4 and FadA genes shows that IpfD lacks these crucial residues (Figure 3.18); in IpfD, the
5 N-terminal catalytic cysteine is replaced by glycine, a substitution that would be expected
6 to remove catalytic ability. The C-terminal catalytic histidine, a positively charged
7 residue, is replaced by tyrosine, a weakly negatively charged residue, a substitution that is
8 generally associated with changes in catalytic properties (Paganl, Zagato et al. 1994 ; Liu,
9 Moanne-Loccoz et al. 1999). Interestingly, the two bacterial SCPx proteins known to
10 catalyze classical thiolase reactions, PhlC and BbsB, have both of these catalytic
11 residues. More work needs to be done before a mechanistic explanation for the role of
12 IpfD in ibuprofen metabolism can be established, however, given its similarity to SCPx
13 proteins it may interact with the branched sidechain of ibuprofen.
125
ipfI
G Y G R C H G H C H C H C H C H C H C H C H C H C H C H C H 1
2 Figure 3.18. Alignment of conserved catalytic residues of IpfD, bacterial and eukaryotic 3 SCPx thiolases, and FadA fatty acid thiolases. Conserved catalytic residues are highlighted 4 in light grey, variations from the conserved catalytic residues are highlighted in dark grey. 5 Created using MegAlign (DNAStar, Inc., Madison, WI). 6
7 ipfE
8 Conserved domain analysis of the predicted amino acid sequence of ipfF revealed
9 that it contains a Domain of Unknown Function 35 (DUF35) motif (E-value = 3.71x10 -
10 11 ). Over 600 DUF35 proteins have been found in bacteria and archaea, but none have
11 been identified in eukaryotes. Interestingly, there are 43 cases in the NCBI database
12 when a DUF35 gene is found directly adjacent to nondecarboxylating condensing
13 enzymes, a protein family of which IpfD is predicted to be part. The role played by only
14 two DUF35 proteins have been characterized to any degree. PhlB (Pseudomonas putida )
15 plays a role in the polyketide synthase pathway described above. PhlB acts in
126
1 conjunction with the SCPx protein PhlC, though its role is unclear. BbsA, which has
2 been characterized in Thauera aromatica (Leuthner and Heider 2000) and Aromatoleum
3 aromaticum EbN1(Kube, Heider et al. 2004; Kuhner, Wohlbrand et al. 2005) is required
4 for the decondensation reaction catalyzed by BbsB described above (Figure 3.14d)
5 though its role is also unclear. An uncharacterized representative of the DUF35 family
6 from Sulfolobus solfataricus was recently crystallized (Krishna, Aravind et al. 2010),
7 revealing a three domain structure (Figure 3.19). At the N-terminal is a conserved but
8 entirely uncharacterized two-helix motif termed the 14-3-3 interacting domain. Next is a
9 zinc-finger motif; zinc-fingers are often associated with nucleotide binding proteins and
10 have been clearly shown to interact directly with nucleotides. Based on this general
11 property and a genome context analysis, it has been hypothesized that in the case of
12 DUF35, this zinc-finger motif interacts with the adenosine moiety of coenzyme A
13 (Krishna, Aravind et al. 2010). At the C-terminal is an oligosaccharide/oligonucleotide
14 binding fold (OB-fold), a double-barreled motif often associated with binding small
15 molecules, which in the case of DUF35 is hypothesized to interact with the hydrophobic
16 end of acyl-CoA molecules based on the presence of hydrophobic amino acid functional
17 groups in the putative binding pocket (Krishna, Aravind et al. 2010). None of the
18 domains is predicted to have any catalytic role.
19 An alignment and structural modeling of IpfE using the Sulfolobus DUF35
20 template using the Swiss model program (Peitsch 1995; Arnold, Bordoli et al. 2006;
21 Kiefer, Arnold et al. 2009) reveals a strong homology and a stable model (E-value =
22 1.40e-31) though only for the zinc-finger and OB-fold domains (Figure 3.19). The
23 crystallographers hypothesized that the close proximity of the zinc-finger and OB-fold
127
1 motifs, taking into consideration their genomic contexts, suggests that DUF35 interacts
2 specifically with acyl-CoA molecules, perhaps serving a similar role as that played by
3 acyl carrier protein (SCP-2) in eukaryotes. SCP-2 in eukaryotes is tightly associated with
4 SCPx proteins in that it is actually transcribed and translated as a single polypeptide only
5 to be proteolytically cleaved into two proteins following translation. (Gallegos, Atshaves
6 et al. 2001; Takeuchi, Chen et al. 2004; Westin, Hunt et al. 2007 ). No homology could
7 be detected, however, between SCP2 and DUF35 using BLAST-P.
8
OB-Fold OB-Fold 14-3-3 interacting 14-3-3
Zinc finger Zinc finger Sulfolobus IpfE 9 DUF35
10 Figure 3.19. Three dimensional model of the predictied amino acid sequence of ipfE 11 modelled on the structure of Sulfolobus DUF35 (Krishna, Aravind et al. 2010) with 12 conserved domains labeled. Image created using Pov-Ray (Persistence of Vision Raytracer 13 Pty. Ltd., Victoria, Australia). 14 15
16
128
1 ipfF
2 Analysis of ipfF is described in Chapter 4. Briefly, the putative gene product of
3 ipfF has similarity to coenzyme A ligases of the luxE superfamily.
4
5 ipfH
6 During initial screening of pFOS3G7 transposon mutants, a clone designated
7 pFOS3G7TnC3 that appeared to have stunted accumulation of catecholic polymer was
8 targeted for sequencing analysis. The transposon insertion in clone 3G7C3 was within an
9 ORF that appears to encode for a ferredoxin reductase ipfH .
10 Ibprofen disappearance analysis of E. coli epi300 pFOS3G7Tn: ipfH via HPLC
11 was in accordance with the observation of lower catechol accumulation (Figure 3.9).
12 TnH metabolized ibuprofen at roughly half the rate as E. coli epi300 pFOS3G7.
13 However, the fact that TnH still produces isobutylcatechol from ibuprofen is curious; if
14 IpfH is actually part of an electron transport chain for IpfAB, then it would be expected
15 that knockout out ipfH would lead to total loss of function. The fact that the pathway still
16 functions suggests that there is perhaps an additional copy of the gene on pFOS3G7 or
17 that the IpfAB electron transport chain is able to make use of a compatible system in E.
18 coli.
19 Blast-X analysis against the Swissprot protein database revealed strong
20 similarities to a number of bacterial ferredoxin reductase components from aromatic
21 dioxygenase systems (Table 3.7) in addition to several other electron transport chain
22 reductases. This information in combination with the two pieces of metabolic generation
23 support a role for IpfH in the ipf pathway as part of the IpfAB electron transport chain.
129
1 Table 3.7. Most similar proteins in the Swiss-prot database to IpfH as determined by 2 BlastP. Accession Identity % coverage E value Max ident P43494.2 Rhodocoxin reductase 89% 4.00E-43 48% P37337.2 Biphenyl dioxygenase system ferredoxin reductase 87% 2.00E-37 53% Q07946.1 Benzene 1,2-dioxygenase system ferredoxin reductase 79% 3.00E-37 71% P16640.1 Putidaredoxin reductase 89% 7.00E-35 46% A5W4E9.1 Toluene 1,2-dioxygenase system ferredoxin reductase component 88% 2.00E-34 64% B7N6C9.1 3-phenylpropionate dioxygenase ferredoxin reductase component 81% 2.00E-33 47% P08087.3 Benzene 1,2-dioxygenase system ferredoxin reductase component 88% 2.00E-29 64% Q9L4M8.1 Rubredoxin-NAD(+) reductase 75% 9.00E-28 43% 3 P33009.1 Terpredoxin reductase 72% 2.00E-24 55%
4 ipfI
5 Blast-X analysis against the Swissprot database revealed significant homology to
6 a single characterized plant-like 2Fe-2S ferredoxin protein (P23263.1), part of a salicylate
7 hydrogenase complex and a member of the plant-like ferredoxins, so called because such
8 proteins play a role in chloroplast electron transport chains and are also commonly parts
9 of aromatic dioxygenase complexes.
10
11 Ipf pathway overview
12
13 While complementation of ipfF was accomplished by cloning in the gene with the
14 native ribosomal binding site (Table 3.2), attempts to complement the other transposon
15 mutants were unsuccessful. In order to minimize the possibility of polar effects,
16 markerless gene deletions were introduced into pFOS3G7 using a modified lambda red
17 protocol. Each of the deletions failed to produce catechols (Figures 3.7 and 3.8).
18 Because an initial attempt to complement pFOS3G7∆ ipfD using the native ribosomal
19 binding site failed, complements were constructed using a forward primer that introduced
20 a three frame stop codon followed by a strong conserved ribosomal binding site (Figure
21 3.2) in a high copy number vector under the lacZ promoter of the high copy number
130
1 vector pGEMt-easy. Complementation of pFOS3G7∆ ipfD with pGEM: ipfD rbs restored
2 activity.
3 The cooperation of an SCPx thiolase (IpfD) and a DUF35 protein (IpfE) is not
4 without precedent. In the benzosuccinyl-CoA pathway of Thauera aromatica , an SCPx
5 and a DUF35 coordinate together to perform a beta-ketothiolytic decondsation (Figure
6 3.17d). Two similar genes are also involved in a polyketide aromatic acetylation in P.
7 putida . While these two situations represent the only well-characterized reactions
8 catalyzed by either type of protein in bacteria, examination of the NCBI database clearly
9 shows that such genes are present in many bacteria; over 2000 putative SCPx thiolases
10 and over 1500 DUF35 encoding genes have been identified by sequence analyses. There
11 are also at least 43 cases in which these two gene types are located directly adjacent to
12 one another in bacteria. In animals, SCPx proteins have been shown to catalyze the
13 removal of propionyl-CoA from alpha-methyl-beta-keto coenzyme A fatty acids via a
14 classical thiolytic reaction mechanism (Figure 3.15)(Wanders, Denis et al. 1997;
15 Verhoeven and Jakobs 2001; Takeuchi, Chen et al. 2004; Westin, Hunt et al. 2007 ). The
16 metabolite that IpfDE is predicted to act upon is dearomatized 1,2-diol-ibuprofen-CoA,
17 an alpha-methyl-beta-hydroxyl CoA fatty acid. Similarly, IpfDE is predicted to remove a
18 propionyl-CoA group (Figure 3.20).
19
131
COOH COSCoA COSCoA OH OH IpfF IpfABHIOH IpfDE OH H
ATP, CoA ADP O2, NADH ATP, CoA priopionyl-CoA
I II III IV 1
2 Figure 3.20. Proposed pathway by which Sphingomonas Ibu-2 metabolizes ibuprofen to 3 isobutylcatechol. I: Ibuprofen, II: Ibuprofen-CoA, III: 1,2-cis-diol-2-hydroibuprofen- 4 CoA, IV: 4-isobutylcatechol. The creation of II is suggested by the fact that IpfF was 5 shown to CoA-ligate ibuprofen in an enzyme assay and by the analagous CoA ligation of 6 phenylacetic acid as confirmed by HPLC analysis (discussed in Chapter 4). Additionally, 7 the creation of phenylacetyl-coenzyme A, catalyzed by the same enzyme, was confirmed via 8 HPLC. III has not been detected and is hypothesized based upon the putative function of 9 IpfAB. The identity of IV was confirmed via GC/MS. 10
11 However, the predicted reaction mechanism does not allow for a thiolytic decondensation
12 due to the mechanistic constraint caused by bond limitations of the ring beta carbon.
13 Consistent with the requirement for a unique mechanism, IpfD does not have the amino
14 acid residues associated with thiolase activity (Figure 3.18). The DUF35 proteins are
15 predicted to have an affinity for acyl-CoA groups based on crystal structure analysis
16 (Krishna, Aravind et al. 2010) and may serve a function similar to that played by sterol
17 carrier protein 2 (SCP-2) in eukaryotes. SCP2 is an acyl carrier closely associated in
18 function with SCPx proteins (Gallegos, Atshaves et al. 2001; Stolowich, Petrescu et al.
19 2002). SCP2 domains are not found in bacteria while DUF35 domains are not found in
20 eukaryotes, a fact which suggests the possbility of convergent evolution.
21 Expressing ipfDE in E. coli K12 ∆ paaG caused an increase in the amount of the
22 dead-end metabolite 2-hydroxyphenylacetate that was produced (Figure 3.14). E. coli
23 K12 ∆ paaG , which lacks an isomerase capable of dearomatizing the phenylacetyl ring,
24 accumulates 1,2-epoxyphenylacetyl-CoA which is rapidly abiotically degraded primarily
132
1 to 2-hydroxyphenylacetate (Figure 1.8)(Teufel, Mascaraque et al. 2010). Since
2 expression of ipfDE did not increase 2-hydroxyphenlacetate levels in the wildtype or in
3 the knockouts of the two downstream steps paaZ and paaJ, it is likely that the epoxy
4 metabolite which is the substrate of PaaG is interacting with IpfDE in some manner. For
5 many years, it was suspected that the bacterial phenylacetyl coenzyme A pathway
6 involved a 1,2-dihydroxy intermediate which IpfDE would be hypothesized to convert to
7 catechol by analogy to the ipf pathway (Ismail, Mohamed et al. 2003). The fact that we
8 could not detect catechol via ferric chloride assay or by HPLC in the paaG mutant
9 expressing ipfDE is consistent with recent evidence of the involvement of an epoxide
10 intermediate in the paa pathway. The increased product of 2-hydroxyphenylacetate,
11 however, suggests that there may be some interaction between IpfDE and the putative
12 epoxyphenylacetyl-CoA; perhaps the epoxide is similar enough to the predicted 1,2-
13 dihydroxyl intermediate that IpfDE is hypothesized to act upon that an interaction occurs
14 in such a way as to either speed the hydrolysis of the epoxide or alter reaction kinetics in
15 such a way that further production of the labile epoxide is favored. While inconclusive,
16 this data is consistent with the involvement of IpfDE in the ipf and PAA degradation
17 pathways in Ibu-2.
18 As discussed in more detail in Chapter 4, the predicted amino acid sequence of
19 ipfF has limited similarity to a wide variety of coenzyme A ligases, including that from
20 the Fad operon of E. coli, and to several ligases used in the anaerobic metabolism of
21 benzoic acid (Table 4.2). IpfF showed no similarity to human xenobiotic/medium-chain
22 fatty acid:CoA ligase which has been shown to be capable of performing ibuprofen CoA
23 ligation (Vessey, Hu et al. 1996). That ipfF encodes for ligase activity of ibuprofen and
133
1 phenylacetate but not an oxidized intermediate is strongly supported by the complete and
2 complementable lack of ibuprofen disappearance in the HPLC assay of E. coli epi300
3 pFOS3G7Tn: ipfF while all other loss-of-function clones retained some initial metabolic
4 activity (Figure 3.6). Indeed, an initial coenzyme A ligation is the case in characterized
5 fatty acid metabolic systems and in the phenylacetic acid catabolic pathway of P. putida
6 U (Garcia, Olivera Elias et al. 2000; Teufel, Mascaraque et al. 2010).
7 Ibuprofen degradation in E. coli harboring pJ25 was very poor when compared
8 with E. coli epi300 pFOS3G7, even though ipfABDEF expression was under the control
9 of the lac promoter. Only trace amounts of catecholic metabolites were detected. One
10 explanation for the reduced activity encoded by pJ25 is the need for other gene products
11 encoded by pFOS3G7 that facilitate metabolism. Indeed, preliminary sequence analysis
12 of non-loss-of-function pFOS3G7 transposon mutants revealed a putative ferredoxin
13 (ipfI ) and a ferredoxin reductase ( ipfH ). HPLC analysis from a knockout of the putative
14 ferredoxin reductase (ipfH ) in E. coli epi300 pFOS3G7 showed decreased ibuprofen
15 disappearance though catechols are still generated (Appendix 2, Figure A2.2). Cloning
16 and expressing these genes along with the ipfABDEF cassette found on pJ25 resulted in
17 successful reconstruction of the ipf upper pathway, with 0.37 mM phenylacetate being
18 converted to catechol within 18 hours (Figure 3.12) and approximately 0.25 mM
19 ibuprofen being converted to isobutylcatechol (Figure 3.13). The dependency of the
20 ipfABDEF pJ25 cassette upon the co-expression of ipfHI is consistent with the fact that
21 many ring-hydroxylating aromatic dioxygenases require a reductase component for
22 activity (Mason and Cammack 1992; Butler and Mason 1997) and offers circumstantial
134
1 support for the involvement of ipfAB, a putative aromatic ring dioxygenase, in the
2 pathway.
3 While the initial data suggest that ipfAB are required for metabolism, we were
4 unable to elucidate the mechanisms by which they function or any further metabolic
5 intermediates. When ipfAB was cloned and expressed in E. coli and concentrated cell-
6 free extract was exposed to phenylacetyl-CoA, the disappearance of phenylacetyl-CoA
7 was significantly higher than in the control extract (161 vs 123 nM phenylacetyl-CoA /
8 mg protein * hr, Figure 3.10, p<0.05). The backround disappearance of the phenylacetyl-
9 CoA in the control, possibly due to abiotic hydrolysis or non-specific enzymatic activityr,
10 makes it difficult to draw any firm conclusions about the role of IpfAB, especially given
11 that GC/MS analysis failed to identify any reaction products in crude-extracts incubated
12 with phenylacetyl-CoA.
13 It is not clear what roles IpfD and IpfE are playing. pFOS3G7-harboring clones
14 with deletions of either of these genes were clearly unable to generate isobutylcatechol
15 (Figure 3.12). However, attempts to identify accumulated intermediates via HPLC or
16 GC/MS proved fruitless. Chromatographic analysis of these mutants with the addition of
17 14 C-ring-labelled ibuprofen or phenylacetic acid might allow for identification of any
18 metabolite or byproduct that may be accumulating. While preliminary biochemical
19 evidence and putative function implied from sequence similarity suggest that IpfAB
20 dioxygenates ipfCoA and that IpfDE catalyze removal of the acyl-CoA group (Figure
21 3.20), these reactions warrant more investigation.
22 The removal of the acidic side-chain and associated oxidation of the 1-position of
23 the aromatic rings of ibuprofen and phenylacetic acid are reminiscent of the removal of
135
1 an acyl group during beta -oxidation of fatty acids (Eaton 1996; Trotter 2001) such as is
2 performed by the well-characterized Fad operon of E. coli (Black, DiRusso et al. 1992;
3 Kunau, Dommes et al. 1995; Campbell and Cronan Jr. 2002). This work reveals that
4 ibuprofen and other phenylacetic acids, a class of chemical that includes many of the
5 non-steroidal anti-inflammatory pharmaceuticals, may be metabolized by a novel type of
6 pathway involving coenzyme A ligation, removal of the acidic side-chain, and generation
7 of a catechol through the acitivty of a thiolase/acyl-transferase and an aromatic
8 dioxygenase.
9
10 All sequences can be found in the National Center for Biotechnology Information (NCBI,
11 http://www.ncbi.nlm.nih.gov) nucleotide sequence database under accession number
12 EF090268.
13 Acknowledgements
14
15 R.W.M was supported in part by NIH-NIEHS Environmental and Molecular Toxicology
16 training grant ES 07052-27 and the SUNY Graduate Diversity Fellowship Program.
17 Anastasia Kazimirova provided invaluable techinical support.
18
136
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1 CHAPTER 4
2
3 UPTAKE OF PHENYLACETIC ACID BY SPHINGOMONAS IBU-2 FOSMID
4 LIBRARY CLONE 3G7
5
6 Abstract
7
8 Sphingomonas Ibu-2 chromosomal DNA fosmid clone 3G7 ( E. coli epi300
9 pFOS3G7) exhibits the novel ability to metabolize ibuprofen to isobutylcatechol
10 and phenylacetate to catechol. The mechanism by which phenylacetate enters E.
11 coli epi300 pFOS3G7 was investigated by radiolabelled uptake assays with
12 special attention paid to the putative ibuprofen and phenylacetate coenzyme A
13 ligase ipfF. Sphingomonas Ibu-2 exhibited constituitive phenylacetyl coenzyme
14 A ligation activity. pFOS3G7 was also shown to confer phenylacetate coenzyme
15 A ligation. When cloned into E. coli a high-copy number ipfF expression
16 construct conferred a 68-fold increase in the rate of phenylacetate coenzyme A
17 ligation and also catalyzed the coenzyme A ligation of ibuprofen. Uptake of
18 phenylacetic acid by E. coli harboring pFOS3G7 was found to be enhanced by
19 ipfF ; when ipfF was knocked out, uptake was decreased two- to eight-foldUptake
20 by E. coli epi300 pFOS3G7 was reduced to ipfF knockout levels following
21 incubation with the metabolic poisons potassium cyanide or 2,4-dinitrophenol,
22 although residual uptake by the ipfF knockout was unaffected by metabolic
23 poisons, indicating that no other energy-dependent process was involved in
146
1 phenylacetic acid uptake in this fosmid clone Uptake of phenylacetic acid was
2 greater at pH 5.5 than at pH 7. In silico analyses of the putative product of ipfF
3 predicted the presence of a small membrane-associating helix, suggesting that the
4 coenzyme A ligase might transiently bind to the cell membrane in a manner
5 similar to that shown by the fatty acid coenzyme A ligase FadD which drives the
6 uptake of fatty acids via vectorial acylation. These results are consistent with a
7 coenzyme A ligation driven vectorial acylation mechanism for uptake of
8 phenylacetic acid and ibuprofen which is more typical of fatty acid uptake and
9 differs markedly from other recently described mechanisms for phenylacetic acid
10 uptake which rely on symporters.
11
12 Introduction
13 Ibuprofen (2-(4-isobutylphenyl)-propionic acid) is a very widely used over the
14 counter pharmaceutical compound which is self-administered primarily as an anti-
15 inflammatory and pain reliever. It is consumed and excreted in sufficiently large
16 quantities to be routinely detected in sewage treatment effluents and surface waters
17 world-wide (Buser, Poiger et al. 1999; Stumpf, Ternes et al. 1999; Farre, Ferrer et al.
18 2001; Winkler, Lawrence et al. 2001; Kolpin, Furlong et al. 2002). Compounds like
19 ibuprofen that are not technically recalcitrant but which are continuously released into the
20 environment are considered to be “pseudopersistent (Daughton 2002)”, like many other
21 pharmaceuticals that enter the environment regularly from municipal sewage treatment
22 plants. The environmental implications of this extremely diverse soup of biologically
23 active compounds flowing into surface waters are not completely understood (Stuer-
147
1 Lauridsen, Birkved et al. 2000; Heberer 2002; Jones, Voulvoulis et al. 2004;
2 Cunningham, Buzby et al. 2006; DeLange, Noordoven et al. 2006; Fent, Weston et al.
3 2006; Hernando, Mezcua et al. 2006; Schwarzenbach, Escher et al. 2006; Dorne, Skinner
4 et al. 2007; Kummerer 2008; Kummerer 2010; Santos, Araújo et al. 2010). In order to
5 begin to characterize the hazards associated with these pseudopersistent compounds more
6 data is needed regarding their possible biological transformations and other aspects of
7 their environmental fate.
8 This chapter describes attempts to elucidate the mechanism by which Ibu-2
9 transports ibuprofen and phenylacetic acid into the cell. Uptake is an essential aspect of
10 any catabolic system as it is an obvious prerequisite for induction and further metabolism
11 of the chemical in question (constitutive expression is not typically used in the case of
12 most catabolic systems (Madigan and Martinko 2006)). Below a certain threshold, a
13 chemical will tend to persist due to a combination of physical mass transfer (Bosma,
14 Middledorp et al. 1997) and biological factors (Schmidt, Alexander et al. 1985). As
15 concentration increases, the threshold is eventually reached where the chemical is
16 transported into the cell where it will accumulate until the concentration is sufficient to
17 trigger induction (Louis and Becskei 2002). Induction, somewhat non-intuitively, is
18 seldom an analog process; it has been demonstrated that induction systems tend to be
19 functionally binary in that below a particular threshold, no transcription takes place and
20 when the threshold is crossed, transcription springs into action. It is partially due to the
21 binary nature of the induction process that chemicals can persist to a surprising degree at
22 low concentrations. For example, it has been demonstrated that metabolism of 3-
23 phenylpropionic acid, an aromatic acid somewhat similar to ibuprofen, by E. coli is not
148
1 induced at concentrations below 3 mg/L (Kovar, Chaloupka et al. 2002). On the other
2 hand, it has been demonstrated that phylogenetically undefined sewage and oligotrophic
3 lake samples can mineralize aromatic acids at concentrations as low as a few ng/L
4 (Rubin, Subba-Rao et al. 1982). In part because transport is an integral part of the
5 induction process, understanding transport may be of assistance in predicting the
6 environmental fate characteristics of a particular chemical.
7 Aromatic acids, including phenylacetic acid (Olivera, Minambres et al. 1998),
8 phthalate (Chang, Dennis et al. 2009), benzoate (Clark, Momany et al. 2002; Ledger,
9 Flores-Aceituno et al. 2009; Wang, Xu et al. 2011), chlorobenzoate (Ledger, Flores-
10 Aceituno et al. 2009), and 4-hydroxybenzoate (Ditty and Harwood 2002), tend to be
11 taken up by major facilitator systems that couple uptake to chemiosmotic gradients
12 (Marger and Saier 1993; Pao, Paulsen et al. 1998; Kahng, Byrne et al. 2000; Kasai, Inoue
13 et al. 2001). There is also evidence for the involvement of ABC type transporter systems
14 required for the uptake of phthalate (Hara, Stewart et al. 2009) and 3-
15 hydroxyphenylacetate (Arias-Barrau, Sandoval et al. 2005). On the other hand, fatty acid
16 uptake is at least partially driven by vectorial acylation processes that require no specific
17 membrane channels but instead relies on diffusion of the substrate into the cytoplasm
18 where it is then ligated to coenzyme A and thereby trapped inside the cell (Weimar,
19 DiRusso et al. 2002; Black and DiRusso 2003; Zou, F. et al. 2003). Additionally, there is
20 mounting evidence that the vectorial acylation process works in concert with the action of
21 a TonB-dependent outer membrane transporter FadL in the case of fatty acid transport
22 (Ferguson and Deisenhofer 2002; van den Berg, Black et al. 2004) and similar proteins
149
1 TodX and TbuX in the case of toluene (Wang, Rawlings et al. 1995; Kahng, Byrne et al.
2 2000).
3 Although an aromatic acid, ibuprofen’s four-carbon aliphatic side-chain and alpha
4 methyl group also make it similar to fatty acids. In fact, analysis of the octanol water
5 portioning coefficient of ibuprofen using the structure activity software KowWin in the
6 program Episuite (Agency 2000) suggests that the majority of the hydrophobicity of
7 ibuprofen comes from the aliphatic carbons. That Ibu-2 treats ibuprofen as a fatty acid is
8 suggested by the catabolic processes that have been demonstrated to be involved or are
9 predicted based on the gene sequences of the ipf operon; coenzyme A ligation and
10 oxidation of the beta carbon followed by the action of a thiolase-type protein. Given that
11 ibuprofen is metabolized in a manner similar to that by which fatty acids are metabolized
12 and given the hydrophobicity of the ibuprofen molecule, I hypothesized that it is
13 transported into the cell using a fatty acid-like mechanism driven by vectorial acylation.
14 In this study, transport assays using E.coli heterologously expressing ipfF was employed
15 to examine the roles played by these different genes in the uptake of ibuprofen and
16 phenylacetic acid by Ibu-2.
17
18 Methods
19 Unless otherwise noted, chemicals were purchased from Acros (Morris Plains,
20 NJ). Luria-Bertani broth (LB) was prepared as previously described (Sambrook, Fritsch
21 et al. 1989). Phenylacetyl CoA was purchased from Sigma (St. Louis, MO) and
22 coenzyme A was purchased from Roche Diagnostics (Indianapolis, IN).
23
150
1 Strains and Clones: Construction and Identity
2 ipfF was PCR amplified using the primers ipfFF and ipfFR (Table 3.2). The
3 resulting PCR product was cloned into pGEMt-easy (Promega Corp., Madison, WI) using
4 the manufacturers instructions, creating pGEM: ipfF. pGEM: ipfF was transformed into E.
5 coli JM109 via electroporation. Successful clones were selected on LB ampicillin with
6 blue/white screening via X-gal and 1mM IPTG then sequenced.
7
8 Table 4.1. Strains and plasmids used in this study. Strains Sphingomonas Ibu- isolated from Ithaca, NY sewage treatment 2 plant via enrichment for growth on ibuprofen this study recA1 subE44 endA1 hsdR17 gyrA96 relA1 thi ∆(lac-proAB ) F' ( traD36 proAB+ lacIq lacZ (Sambrook, Fritsch et E. coli JM109 ∆M15 ) al. 1989) F- mcrA ∆(mrr-hsdRMS-mcrBC) Φ80dlacZ∆M15 ∆lacX74 recA1 endA1 araD139 ∆(ara, leu)7697 galU galK λ - rpsL E. coli epi300 (Str R) nupG trfA tonA Epicentre, Madison, WI
Plasmids copy control fosmid vector, inducible to high copy number with arabinose when hosted in E. pCC1FOS coli epi300, chloramphenicol resistance Epicentre, Madison, WI pCC1FOS Sphingomonas Ibu-2 chromosomal pFOS3G7 library clone 3G7 this study pFOS3G7 with Tn5 insertion in ipfA , transposon library clone F1, chloramphenicol pFOS3G7Tn: ipfA and tetracycline resistant this study pFOS3G7 with Tn5 insertion in ipfF , transposon library clone F10, chloramphenicol pFOS3G7Tn: ipfF and tetracycline resistant this study
pGEMt-easy ampicillin resistance Promega, Madison, WI pGEMt-easy with ipfFF/ipfFR PCR amplicon pGEM: ipfF ipfF this study 9
10
151
1 Coenzyme A ligase activity
2 Wild type Ibu-2 and E. coli harboring either pFOS3G7 or various subclones were
3 assayed for phenylacetyl coenzyme A ligase and ibuprofen coenzyme A ligase activity.
4 P. putida U was also used as a positive control because it is known to perform
5 phenylacetyl coenzyme A ligation (Martinez-Blanco, Reglero et al. 1990). The
6 inducibility of phenylacetyl coenzyme A ligase activity in Ibu-2 was tested by growing
7 two batches of Ibu-2 in LB and adding 50ppm ibuprofen to one for one hour prior to
8 preparation of cell-free extract.
9 The method described by Martinez-Blanco et al. (Martinez-Blanco, Reglero et al.
10 1990) was used with minor modifications for measuring phenylacetyl coenzyme A ligase
11 activity and ibuprofen coenzyme A ligase activity. In the case of Ibu-2 and P.putida U,
12 crude extract was prepared by first concentrating 100mL of E. coli or a 1000mL of P
13 .putida or Ibu-2 culture via centrifugation, followed by two washes with 10 mM
14 phosphate buffer (pH 7.4) and resuspension in 1-2 ml of sonication buffer (40 mM
15 potassium phosphate, 20% glycerol, 1mM PMSF, 1mM DTT, pH 7.4). Larger volumes
16 of P. putida and Ibu-2 cultures were used due to the lower overall culture density yielded
17 by growth on minimal media. E. coli cultures were grown in LB media with IPTG or
18 arabinose for induction, P. putida was grown in MSM with 5mM PAA, and Ibu-2 was
19 grown in MSM with 500 mg/L ibuprofen. The cell pellet was then sonicated (Branson
20 Sonifier 450, Branson Ultrasonics, Danbury, CT) using three one minute cycles at
21 maximum output with one minute rest time on ice in between each cycle. The cell lysate
22 was then spun for twenty minutes at 15,000 x g at 4° C. In the case of E. coli clones,
23 crude extracts were prepared by centrifuging 100ml to 200ml of mid-exponential growth
152
1 phase of either IPTG or 10mM arabinose induced culture, washing, and resuspending in
2 1-2 ml of sonication buffer. Approximately 0.1g of 0.1 mm glass beads was then added
3 and the pellets were bead-beaten for three minutes (MiniBeadbeater-8, Biospec Products,
4 Bartlesville, OK). The pellets were then spun down as described above. In both cases,
5 the protein content of the supernatant containing the crude extract was quantified using
6 the Bio-Rad Protein Assay Kit with bovine serum albumin as a standard (Bio-Rad
7 Laboratories, Hercules, CA).
8 The reaction mixture for Co-A ligase assays included 4.2 µl 0.2 M MgCl 2, 17 µl
9 0.1 M ATP, 10 µl 20 mM coenzyme A, 10 µl 0.1 M phenylacetic acid or ibuprofen, 20 µl
10 cell-free extract (~0.1 mg of crude protein), and 17 µl neutral hydroxylamine solution.
11 Neutral hydroxylamine solution was prepared freshly by mixing 1 ml of 5 M
12 hydroxylamine hydrochloride, 1.25 ml 4 M KOH, and 250 µl of water. 150 µl ferric
13 chloride reagent (0.37 M ferric chloride, 20 mM trichloroacetic acid, and 0.66 M
14 hydrochloric acid) was added to terminate the assay at the prescribed time. Samples were
15 spun down to remove precipitate, and the OD540nm was measured using a µ-Quant
16 spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT). Concentrations of the
17 phenylacetate ferric chloride complex were calculated using the known extinction
18 coefficient of 0.9 /(mM)(cm) (Martinez-Blanco, Reglero et al. 1990). Because the
19 extinction coefficient of the ibuprofen ferric chloride complex (putatively derived from
20 ibuprofen-CoA) is unknown, the phenylacetate ferric chloride value was used.
21 The disappearance of phenylacetic acid and concomitant appearance of
22 phenylacetyl-CoA was also measured via HPLC using the methods described by
23 Mohamed et al. (Mohamed and Fuchs 1993). The reactions were performed as listed
153
1 above except for the exclusion of neutral hydroxylamine and the addition of an equimolar
2 amount of coenzyme A. For the detection of phenylacetyl-CoA (retention time 45
3 minutes), the running solvent was 30mM potassium phosphate buffer pH7 with 9%
4 methanol and 7% acetonitrile. Phenylacetic acid was analyzed using 50% 40mM acetic
5 acid and 50% methanol and eluted from the column at 10 minutes. Phenylacetic acid was
6 monitored at 210 nm and the appearance of phenylacetyl CoA was monitored at 254 nm.
7
8 Uptake in Ibu-2 and E. coli constructs
9 To examine the contribution of certain genes to uptake rates of Ibu-2, E. coli
10 epi300 pFOS3G7, and E. coli epi300 pFOS3G7 with a transposon insertion in ipfF were
11 selected for uptake assays. To determine if the impact of this gene product was
12 concentration dependent, a range of phenylacetic acid concentrations was used; 0.5µM,
13 2.5µM, 25µM, 100µM, 500µM, and 1mM.
14
15 Effect of metabolic poisons on phenylacetic acid uptake by E. coli epi300 pFOS3G7 , and
16 E. coli epi300 pFOS3G7Tn:ipfF
17 Metabolic poisons were utilized to examine the energy-requirement of the uptake
18 system. Two poisons with different modes of action were used; potassium cyanide and
19 2,4-dinitrophenol (2,4-DNP). Poisoning assays were performed on E. coli epi300
20 pFOS3G7 and E. coli epi300 pFOS3G7Tn: ipfF using a phenylacetate concentration of
21 25µM. The assays were performed as described above, except that after the initial wash
22 step, the cells were suspended in 0.5 mM KCN, 0.5 mM DNP, or no poison, all in MSM
154
1 for 10 minutes. The cells were then centrifuged again, resuspended with the radiolabeled
2 substrate, and the assay proceeded as described above.
3
4 Effect of pH on uptake by E. coli epi300 pFOS3G7
5 The impact of pH on phenylacetic acid uptake was examined in E. coli epi300
6 pFOS3G7. If the solute was merely diffusing across the membrane, then lowering pH
7 should increase the degree of uptake due to the protonation of the acid group, thereby
8 eliminating its negative charge and facilitating passage of the neutral molecule into the
9 biological membrane. A wide range of phenylacetic acid concentrations was examined,
10 from 0.5 uM to 1 mM. A low pH (5.5) and neutral pH (7) were used. The assay was
11 performed as described above except that the solution of radiolabelled phenylacetic acid
12 MSM was adjusted to the desired pH with 1M HCl.
13
14 Uptake Assays
15 Techniques for uptake assays were adapted from those described by Leveau et al.
16 (Leveau, Zehnder et al. 1998). Ibu-2 was inoculated (5% V/V) in MSM with 500 ppm
17 ibuprofen using a late exponential growth phase culture and grown for approximately 24
18 hours, which allowed for harvest of cells in mid-exponential growth phase. All the E.
19 coli strains were similarly inoculated (5% V/V) into LB with appropriate antibiotics using
20 overnight cultures, then induced to high-copy number using 10mM arabinose and grown
21 for 5 hours. Cultures were centrifuged and washed with MSM pH 7.4 once before being
22 centrifuged again and then resuspended to an approximate optical density (600nm) of one
23 in MSM.
155
1 For each data set, 50,000 dpm of 14 C phenylacetic acid was added to 3mL MSM
2 containing twice the desired concentration of unlabeled phenylacetic acid at pH 7.4. To
3 initiate the uptake assay, 3mL of washed cell suspension was added to the 3mL
4 phenylacetic acid solution. The resulting 6mL solution was well-mixed and then filtered
5 in 2mL aliquots at the appropriate time. The filter apparatus consisted of a 6mL plastic
6 syringe attached to a reusable filter cartridge preloaded with a 0.45 micron filter
7 membrane. At one minute, the cell suspensions were forced through the filter. Each
8 membrane was then washed with 2mL of 50 uM phenylacetic acid MSM solution to
9 displace any labeled surface-bound phenylacetic acid. The filters were then removed,
10 placed in scintillation tubes containing 5mL scintillation cocktail, and measured in a
11 scintillation counter. The fraction of 14 C phenylacetic acid retained on the membranes
12 was assumed to equal the fraction of total phenylacetic acid taken up by the cells.
13 Phenylacetic acid uptake was standardized to protein content of the cell
14 suspensions. Each sample was centrifuged and the protein quantified via the Bradford
15 assay (Bio-Rad Laboratories, Hercules, CA) after they had been boiled in 0.1 M NaOH
16 for ten minutes.
17
18 In silico analyses of ipfF
19 Phylogenetic analysis of the putative gene product of ipfF was performed with the
20 COBALT multiple alignment tool (Papadopoulos and Agarwala 2007 ) using Fast
21 Minimum Evolution tree building method with Grishin distance. The putative protein
22 product of ipfF was aligned with paaK from E. coli (NCBI BAE76428.1), the recently
23 crystallized paaK1 and paaK2 from Burkholderia cenocepacia (PDB 2Y27 and 2Y4O),
156
1 and FadD fatty acid coenzyme A ligase from E. coli (NCBI P69451.1) in the MegAlign
2 program (DNAStar, Inc.) using clustalW distance measure. Initial screening of proteins
3 for transmembrane helices was performed using the PHDhtm algorithm (Rost, Casiado et
4 al. 1995) provided by the PSIPRED server for definitive prediction of treansmembrane
5 helices at 95% confidence level (Jones 2007 ) and using the TMHMM algorithm to
6 provide redundancy of transmembrane helix detection and also to provide visual output
7 (Krogh, Larsson et al. 2001).
8
9 Results
10
11 Assaying coenzyme A ligase activity
12 CoA ligase activity in Ibu-2 cell free extracts was determined using both
13 phenylacetic acid and ibuprofen as substrates. Ibu-2 crude extract catalyzed phenylacetyl
14 CoA ligation (Figure 4.1) at a rate of approximately 10 nmol/mg/minute, which was
15 similar to that of P. putida U (data not shown). Ibu-2 extracts also catalyzed the CoA
16 ligation of ibuprofen when compared to P. putida U (p=0.007), which did not yield
17 detectable CoA-ligation product. However, the rate of ibuprofen CoA ligation by Ibu-2
18 was much lower; while almost all phenylacetic acid was ligated within two hours, only
19 33% (+/- 4%) of the ibuprofen was ligated in 63 hours. Prior exposure of Ibu-2 to
20 Ibuprofen had no affect on CoA ligase activity (p=0.36) (Figure 4.1).
21 Crude extract from E. coli epi300 pFOS3G7 or E. coli JM109 pGEM: ipfF both
22 contained detectable phenylacetyl CoA activity, 34 and 19.6 nmoles / mg protein / minute
23 compared to 0.5 in vectorless epi300 (Figure 4.1). HPLC analyses confirmed that the
157
1 accumulation of phenylacetyl CoA was concomitant with the disappearance of
2 phenylacetic acid (data not shown). Both phenylacetic acid and ibuprofen CoA-ligase
3 activities were completely dependent upon the presence of ATP and Mg ++ (data not
4 shown).
5
PAA
50
40
30
20
10
0 Ibu-2 Ibu-2 F 3G7 AB epi300
protein/minute (induced) (uninduced)
Strain 6 formedCoA /mg nmol PAA
7 Figure 4.1. Phenylacetic acid coenzyme A ligase activity in the cell-free extracts of different 8 strains. “F” represents the pGEMt-easy ipfF clone while “AB” is the respective ipfAB 9 clone. (n=3). 10
11 Uptake of phenylacetic acid in all strains, clones, and knockouts
12 The results detailed in chapter 3 demonstrated that E. coli epi300
13 pFOS3G7Tn :ipfF was not able to produce isobutylcatechol from ibuprofen. At all
14 concentrations studied, E. coli epi300 pFOS3G7Tn :ipfF displayed less phenylacetic acid
15 uptake than E. coli epi300 pFOS3G7 (Figure 4.2).
158
100
10
1 3G7
0.1 TnF
0.01
0.001 nmolpaa uptake / (mg protein *min) 0.1 1 10 100 1000 uM phenylacetate 1
2 Figure 4.2. Uptake of phenylacetic acid by strains E. coli epi300 pFOS3G7, E. coli epi300 E. 3 coli and epi300 pFOS3G7Tn :ipfF over a range of phenylacetic acid concentrations. Uptake 4 was standardized to total cell protein. E. coli epi300 pFOS3G7Tn :ipfF was significantly less 5 (n=3, p < 0.05) than E. coli epi300 pFOS3G7 at all concentrations tested. 6
7 Effect of metabolic poisons on E. coli epi300 pFOS3G7, and E. coli epi300
8 pFOS3G7Tn:ipfF
9 Uptake assays using Ibu-2 proved to be infeasible. Ibu-2 clogged up the filter
10 membranes rapidly, prohibiting prompt filtration to any degree. This is likely due to its
11 extensive exopolysaccharide layer which has been frequently observed in other
12 Sphingomonads (Pollock 1993). The metabolic poisons significantly reduced
13 phenylacetic acid uptake by E. coli epi300 pFOS3G7 (Figure 4.3). Uptake was reduced
14 by approximately 23-32% by both poisons (p<0.05). Metabolic poisons did not affect
15 uptake by E. coli epi300 pFOS3G7Tn :ipfF to any statistically detectable degree (Figure
16 4.3).
159
0.18 0.16 0.14 0.12 control 0.1 KCN 0.08 2,4-DNP 0.06 0.04 0.02 0 3G7 TnF
1 nmol / uptake PAA (mg protein * min)
2 Figure 4.3. Uptake of 25 µM phenylacetic acid by E. coli epi300 pFOS3G7 and E. coli 3 epi300 pFOS3G7Tn: ipfF exposed to 0.5mM of the metabolic poisons 2,4 dinitrophenol or 4 potassium cyanide. Both pFOS3G7 poisoning treatments exhibited significantly less uptake 5 than the non-poisoned control (n=3, p < 0.05) while no other differences were significant. 6
7 Effect of pH on uptake
8 The impact on uptake of phenylacetic acid by E. coli epi300 pFOS3G7 at a low
9 vs. high pH was quantified by calculating the ratio of uptake at pH 5.5 vs. pH 7. At all
10 concentrations other than the lowest, uptake was higher at low pH, indicating that
11 diffusion across the membrane is a significant driving force in uptake.
12 The ratio of low/high pH uptake showing a trend of being proportional to the
13 logarithm of phenylacetic acid concentration (best fit line R=0.8941, Figure 4.4). In
14 other words, low pH had more of an impact on higher concentrations of phenylacetate,
15 suggesting that pH drives transport to some degree but that there is also an underlying
16 non-pH based transport mechanism at work. If no such mechanism existed, one would
17 expect pH to alter uptake equivalently at all solute concentrations.
18
160
8 7 6 R2 = 0.8941 5 4 pH7 3 2 1 0
nmol uptake / mg protein : pH 5.5 / / pH5.5: protein mg uptake / nmol 0.1 1 10 100 1000 10000 mM PAA 1
2 Figure 4.4. Phenylacetic acid uptake by E. coli epi300 pFOS3G7at pH 5.5 divided by 3 uptake at pH 7. All data points other than for 0.5mM phenylacetate demonstrate that 4 uptake was higher at pH 5.5 vs pH 7 as is consistent with the vectorial acylation mechanism 5 (n=3, p < 0.05). 6
7 Discussion
8
9 ipfF was identified by transposon mutagenesis of pFOS3G7 and metabolic
10 screening for loss of ability to convert ibuprofen to a catecholic metabolite as described
11 in Chapter 3. ipfF had modest similarity to a number genes encoding coenzyme A
12 ligases, particularly to those involved in degradation of aromatic acids with three
13 (Achterholt, Priefert et al. 2000) and four (Veith, Herzberg et al. 2004) carbon acidic
14 sidechains.
15 Conserved domain analysis of the predict amino acid sequence of ipfF revealed it
16 to be a member of the LuxE superfamily (E-value = 1.64e-33), which harbor a domain
17 associated with the formation of thioester bonds with representatives amongst all
18 domains of life. The superfamily includes the coenzyme A ligases. A BlastP search of
161
1 IpfF against the Swissprot database reavealed similarities to a wide variety of coenzyme
2 A ligases including those for aromatic acids and for fatty acids (Table 4.2). IpfF does not
3 clearly group with any particular type of coenzyme A ligase; curiously, amongst the
4 proteins with significant homology, it is most distantly related to the phenylacetyl CoA
5 ligases (Figure 4.5) with which it shares only 40% similarity over 50-60% of the
6 sequence, while sharing 45+% similar residues across 95+% of the sequence in the case
7 of the long-chain fatty acid coenzyme A ligases from bacteria (Matsuoka, Hirooka et al.
8 2007 ) and even with 4-coumarate CoA ligase from the plant Arabidopsis (Ehlting,
9 Battner et al. 1999) (Table 4.2).
10
11 Table 4.2. The substrates, similarity measures, and confidence levels between IpfF and 12 characterized coenzyme-A ligases found in the Swiss-prot database.
Description (alpha subunit of) Query Accession % identity % positive E value Organism coverage O07610.2 long-chain fatty acid CoA ligase 27 47 96% 2.00E-53 Bacillus subtilis Q9S725.2 4-coumarate CoA 29 47 98% 9.00E-51 Arabidopsis thaliana P69451.1 long-chain fatty acid CoA ligase 26 45 96% 1.00E-47 E. coli K12 AAC73148.2 crotonobetaine/carnitine-CoA ligase 28 46 94% 1.00E-43 E. coli K12 Q00594.1 medium-chain fatty acid CoA ligase 25 47 96% 6.00E-39 Pseudomonas oleovorans YP_559588.1 benzoate CoA ligase 27 45 93% 1.00E-35 Burkholderia xenovorans AAN32623.1 benzoate CoA ligase 25 41 93% 5.00E-28 Thauera aromatica AAA92151.1 benzoate CoA ligase 24 42 96% 5.00E-25 Rhodopseudomonas palustris A5JTM6.1 4-chlorobenzoate CoA ligase 27 41 70% 2.00E-22 Pseudomonas CBS-3 YP_931807.1 phenylacetyl CoA ligase 24 41 60% 1.00E-12 Azoarcus sp. CAA66100.1 phenylacetyl CoA ligase 25 40 57% 1.00E-11 E. coli K12 YP_002768992.1 phenylacetyl CoA ligase 25 40 54% 2.00E-09 Rhodococcus erythropolis ZP_05785221.1 phenylacetyl CoA ligase 23 40 55% 7.00E-08 13 Silicibacter lacuscaerulensis
14
162
Benzoate coenzyme A ligase Medium chain fatty acid coenzyme A ligase COOH OH COOH
Chlorobenzoatecoenzyme A ligase COOH
Cl Long-chain fatty acid coenzyme A ligase OH COOH
OH + N COOH
Carnitinecoenzyme A ligase
4-coumarate coenzyme A ligase COOH IpfF COOH
OH
Phenylacetate coenzyme A ligase COOH
1 2 Figure 4.5. Phylogenetic tree of the predicted aminoacid sequence of ipfF and characterized 3 coenzyme A ligases found in the Swiss-prot database and listed in Table 4.2. 4
5 Crude extracts from Sphingomonas Ibu-2 and E. coli epi300 pFOS3G7 were
6 shown to possess phenylacetyl-CoA ligase activity (Figure 4.1). Activity was further
7 shown to be completely dependent upon the presence of Mg 2+ and ATP (data not shown).
8 ipfF , which encodes the putative coenzyme A ligase, was further subcloned and
9 expressed in E. coli where it was shown to confer both phenylacetyl- and ibuprofen-CoA
10 ligase activity. This data establishes the role of IpfF in the first step of the ipf pathway as
11 postulated in chapter 3 (Figure 3.17).
163
1 The mode of activity of Sphingomonas Ibu-2 ibuprofen transport gene products
2 was studied via filter-based radio-labeled substrate uptake assays. Based on previous
3 findings regarding the mechanism by which Ibu-2 catabolizes ibuprofen, an uptake
4 system analogous to fatty acid uptake systems was hypothesized. It was previously
5 demonstrated that the ibuprofen catabolic machinery (encoded by ipfABDEF ) was
6 capable of acting upon phenylacetic acid and similar compounds in the same way that
7 ibuprofen was metabolized. Given its ready availability, 14 C phenylacetic acid was
8 therefore used as a surrogate for ibuprofen.
9 The roles played by different ipf gene products on the uptake of PAA was studied
10 with the aid of genetic constructs; E. coli epi300 pFOS3G7 is fully capable of
11 metabolizing ibuprofen to the catechol stage, while transposon mutant of ipfF is fully
12 defunct in this ability (Figure 3.6). The data presented in these figures is consistent with
13 a model of ibuprofen uptake via fatty acid-like vectorial acylation. The core result
14 supporting this model is the observation that E. coli epi300 pFOS3G7Tn:ipfF took up
15 less phenyacetate than. E. coli epi300 pFOS3G7 at all substrate concentrations tested.
16 This is consistent with previous findings that E. coli epi300 pFOS3G7Tn :ipfF was unable
17 to cause the disappearance of ibuprofen. Heterologous expression of ipfF was shown to
18 confer both ibuprofen and phenylacetate coenzyme A ligase activity on E. coli . Taken
19 altogether, it is clear from these findings that ipfF encodes the first step in ibuprofen
20 metabolism.
21 Phenylacetate uptake by E. coli epi300 pFOS3G7 was inhibited by the addition of
22 either potassium cyanide or 2,4-dinitrophenol. Cyanide directly interferes with electron
23 transport, thus slowing the pumping of protons outside of the inner bacterial membrane
164
1 (Gregus and Klaasen 2001). 2,4-DNP is a protonophore; it is believed to destroy the
2 electrochemical proton gradient by serving as a functional proton conduit across the inner
3 membrane. The impacts of both poisons relevant to this assay are the slowing of ATP
4 production, which may be used in active transport, and the loss of the electrochemical
5 proton gradient, which can be used to drive solute symport. This inhibition of uptake is
6 fully consistent with the model of IpfF-catalyzed coenzyme A ligase activity driving
7 uptake via vectorial acylation. Coenzyme A ligases are dependent upon energy provided
8 by ATP (Kunau, Dommes et al. 1995; Vellemur 1995; Trotter 2001). If ATP production
9 is inhibited by an uncoupler such as cyanide or a protonophore such as 2,4 dinitrophenol,
10 ATP will be in short supply and the substrate that diffuses into the cell will not be trapped
11 via acylation, rather, it will remain free to diffuse back out of the cell.
12 Alignment of the putative product of ipfF (IpfF) with three phenylacetyl
13 coenzyme A ligases and FadD showed that IpfF has the conserved P-loop, an ATP-
14 binding domain (Table 4.3). IpfF also has all three conserved residues associated with
15 the first adenylation step catalyzed by coenzyme A ligases (Table 4.4). IpfF also contains
16 five of the eleven residues that have been demonstrated to form the phenyacetate
17 substrate binding pocket in PaaK1 (Law and Boulanger 2011 ) (Figure 4.5).
18
19 20 Table 4.3 Alignment, positions, and identities of ATP-binding P-loop residues
P-Loop Residues PaaK2 B. cenocepacia 100 SSGTTGKPT 108 PaaK1 B. cenocepacia 93 SSGTTGKPT 101 PaaK E. coli 98 SSGTTGKPT 106 FadD E. coli 214 YTGTTGVAK 222 IpfF S. Ibu-2 165 TSGTTGVPK 173
165
1 Table 4.4 Positions and identities of conserved adenylating catalytic residues
Catalytic Residues PaaK2 B. cenocepacia Arg 333 Glu 248 Lys 432 PaaK1 B. cenocepacia Arg 327 Glu 242 Lys 424 PaaK E. coli Arg 331 Glu 246 Lys 427 FadD E. coli Arg 453 Glu 361 Asp 511 IpfF S. Ibu-2 Arg 410 Glu 309 Lys 510 2 3 4 5 Table 4.5 Positions and identities of residues that align with the binding pocket residues of 6 PaaK1 from B. cenocepacia . Residues that are identical to those found in PaaK are darkly- 7 shaded while similar residues (as judged by charge) are lightly-shaded.
Binding Pocket Residues
PaaK E. coli Tyr 141 Phe 146 Gly 148 Ala 152 Gly 218 Ala 219 Ala 241 Tyr 242 Gly 243 Gly 249 Pro 250 Pro 250 PaaK2 B. cenocepacia Phe 143 Phe 148 Gly 150 Ile 154 Gly 220 Ala 221 Ile 243 Tyr 244 Gly 245 Gly 251 Pro 252 Pro 252 PaaK1 B. cenocepacia Tyr 136 Phe 141 Gly 143 Ala 147 Gly 214 Ala 214 Ile 237 Tyr 238 Gly 239 Gly 245 Pro 246 Pro 246
FadD E. coli Pro 158 Phe 264 Leu 265 Cys 269 Gly 333 Ala 334 Gly 256 Tyr 257 Gly 258 Pro 364 Val 365 Val 365
IpfF S. Ibu-2 Pro 204 Trp 209 Val 211 Asn 215 Gly 280 Ala 281 Ala 304 Tyr 305 Ala 306 Gly 312 Ile 315 Ile 315 8 9 10 11 While PHDhtm predicted a single transmembrane motif for both FadD and the
12 putative product of ipfF , the lengths of the putative membrane-spanning helices (8-14
13 residues) are likely too small to span a lipid bilayer (Cuthbertson, Doyle et al. 2005).
14 True membrane-spanning helices tend to be 20-30 residues long (Cuthbertson, Doyle et
15 al. 2005). TMHMM made similar predictions (Figure 4.6), with putative but low
16 probability transmembrane-like motifs in the region of the amino acid chains where three
17 conserved binding pocket motifs are located approximately 30-40 amino acids
18 downstream of the ATP-binding P-loop domain. Neither program detected
19 transmembrane motifs in PaaK1. These kinds of short transmembrane-like motifs have
20 been termed “half-TMs” (Cuthbertson, Doyle et al. 2005).
166
bacteriorhodopsin PaaK1
IpfF FadD
1
2 Figure 4.6 Location and probabilities of putative transmembrane helix motifs in PaaK1, 3 IpfF, and FadD as predicted by the TMHMM algorithm (Krogh, Larsson et al. 2001). 4 Bacteriorhodopsin, an extensively membrane associated protein (Haupts, Tittor et al. 1999), 5 is presented for reference. Amino acid locations are shown on the x-axis while the 6 probability of membrane association is represented on the y-axis. 7
8 It has been demonstrated that FadD associates with the membrane; both
9 membrane and soluble enzyme fractions of induced E. coli K-12 contain FadD activity
10 (Weimar, DiRusso et al. 2002). It has been hypothesized that free substrate dissolved in
11 the membrane recruit FadD to become membrane-associated (Weimar, DiRusso et al.
12 2002), possibly involving a drastic conformational change common to this general class
13 of proteins (Law and Boulanger 2011 ). This membrane association might be due to the
14 short half-TMs present in each of these proteins, though there are other ways by which
15 proteins can transiently associate with membranes, such as amphipathic helices and
16 hydrophobic loops (Goni 2002). FadD has been shown to be sufficient to drive uptake of
167
1 fatty acids which diffuse into the inner bacterial membrane in E. coli and into the
2 cytoplasm where they are trapped via coenzyme A ligation (Weimar, DiRusso et al.
3 2002) (Figure 4.7).
4
COO-
1
Periplasm COOH
2
Cytoplasm HOOC
3 4
ATP + CoA FadD AMP + PPi
Fatty Acyl CoA 5 6 Figure 4.7 Model illustrating the role of FadD in the vectorial transport of exogenous fatty 7 acids (Weimar, DiRusso et al. 2002). 1, free fatty acids become protonated in the 8 periplasmic space following FadL-dependent transport across the outer membrane (not 9 illustrated here) and then partition into the inner membrane. 2, the protonated fatty acid 10 flips from the periplasmic face of the inner membrane to the cytoplasmic face. 3, free fatty 11 acids within the membrane signal FadD to partition into the inner membrane, presumably 12 in an ATP-bound form. 4, FadD functions to abstract fatty acids from the inner membrane 13 concomitant with the formation of fatty acyl-CoA for further metabolism. 14
168
1 The similarity of membrane-associating regions present in both FadA and the
2 putative product of ipfF are consistent with a similar vectorial acylation driven uptake
3 system for ibuprofen and phenylacetic acid by the ipf pathway in Sphingomonas Ibu-2.
4 The proximity of the putative half-TM (in the 200-220 residue range) to the ATP-binding
5 P-loop (residues 165-173) and three of the residues that align with conserved binding
6 pocket residues (Pro 204 , Trp 209 , Val 211 , and Asn 215 ) is consistent with an IpfF model
7 wherein a membrane-associating region binds with and adenylates membrane-dissolved
8 substrate.
9 It would be possible to test this model by separating membrane-associated and
10 cytoplasmic protein fractions and testing each for phenylacetate and ibuprofen coenzyme
11 A ligase activity using the methods employed by Weimar et al. in their description of
12 FadD membrane association (Weimar, DiRusso et al. 2002).
13 The vectorial acylation model is also consistent with the observation that the
14 background uptake of phenylacetate by E. coli epi300 pFOS3G7Tn :ipfF was not affected
15 by the addition of metabolic poisons.
16 It is important to keep in mind that this work was all performed using cloned
17 genes in an E. coli host. Uptake assays could not be performed using Sphingomonas Ibu-
18 2 because of the fact that the extensive exopolysaccharide clogged the filter membranes.
19 While vectorial acylation may be the prime driving force in E. coli harboring ipfF , it may
20 be the case that Ibu-2 requires more extensive machinery to take up ibuprofen due to
21 membrane structure or exopolychaccharide layer differences. Cloning ipfF into a
22 different Sphingomonad or another more closely related strain would begin to address the
23 question of whether such species specific factors are important to the transport system.
169
1 While benzoate and phenylacetate coenzyme A ligases have been described, in
2 each of these cases traditional symport or translocase driven uptake systems are required
3 for uptake (Olivera, Minambres et al. 1998; Clark, Momany et al. 2002; Ledger, Flores-
4 Aceituno et al. 2009; Wang, Xu et al. 2011). In contrast, the Ibu-2 subclone uptake data
5 presented herein provides preliminary evidence that the product of ipfF alone is sufficient
6 to drive PAA uptake via a membrane-associating coenzyme A ligase mediated vectorial
7 acylation mechanism akin to the uptake of fatty acid driven by FadD in E. coli (Weimar,
8 DiRusso et al. 2002). To this author’s knowledge, a vectorial acylation uptake system for
9 an aromatic acid would be novel.
10
11
12 Acknowledgements
13
14 R.W.M was supported in part by NIH-NIEHS Environmental and Molecular Toxicology
15 training grant ES 07052-27 and by the SUNY Graduate Diversity Fellowship Program.
16 Pseudomonas putida U was kindly provided by Jose M. Luengo.
17
170
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1 CHAPTER 5 2
3 CONCLUSIONS
4
5 Pharmaceuticals have been detected in bodies of water all over the world, in
6 treated wastewater used for agriculture (Kinney, Furlong et al. 2006; Siemens, Huschek
7 et al. 2008; Xu, Wu et al. 2009) and even in human drinking water (Fent, Weston et al.
8 2006; Benotti, Trenholm et al. 2008; Huerta-Fontela and Ventura 2008; Corcoran, Winter
9 et al. 2010; Kummerer 2010). The scientific community has seen a recent surge of
10 interest regarding the fate and effects of pharmaceutical compounds released into the
11 environment, prompted not only by the precautionary principle (Ferrari, Mons et al.
12 2004; Gardiner 2006) but due to widespread detection of such chemicals (summarized by
13 (Santos, Araújo et al. 2010)) and emerging reports of potential (see www.wikipharma.org
14 (Molander, Agerstrand et al. 2009) for a summary of available data) and observed
15 impacts (Oaks, Gilbert et al. 2004).
16 The prediction of environmental fate of such a diverse category of chemicals is
17 hampered in part, by limited understanding of the way microbes might degrade such
18 chemicals once they enter the environment. It is in response to such a challenge that this
19 dissertation was undertaken. As the most commonly used member of the diverse class of
20 phenylacetate-based non-steroidal anti-inflammatories, ibuprofen (2-(4-
21 isobutylphenyl)propionic acid) was selected so as to provide insight not only into the fate
22 of a common environmental contaminant but also to provide guidance to aid in the
23 prediction of the fate of similar pharmaceuticals. Indeed the results of the scientific
24 inquiries conducted suggest the utilization of a novel metabolic strategy for the
177
1 assimilative metabolism of such chemicals. While such work provides valuable insight,
2 the scope of the challenge posed by environmental release of pharmaceutical compounds
3 is great and will require a more attention from the scientific community.
4
5 Pharmaceuticals in the Environment: The scope of the challenge to the scientific
6 community
7
8 The lack of understanding regarding the issue of pharmaceuticals in the
9 environment can be categorized into several categories. The complex mixture of low
10 concentrations of biologically active compounds presents a quandary for ecotoxicology
11 and human toxicology (Borgert, Quill et al. 2004; Chou 2006; Baas, van Houte et al.
12 2007; Cedergreen, Christensen et al. 2008; Crofton 2008; Kortenkamp 2008). The
13 toxicological sciences are based upon a foundation of understanding acute toxic effects of
14 individual compounds and have only recently begun to investigate chronic exposure of
15 ecosystems to single chemicals. The subtle chronic effects of mixtures are largely
16 unexplored territory (Groten, Heijne et al. 2004). Ecotoxicology and ecology are tasked
17 with an even more complicated challenge; that of how to quantify the subtle toxicological
18 impact(s) of mixtures on not just single organisms, but on higher levels of biological
19 organization such as populations, communities, and ecosystems.
20 With respect to the latter problem, the toxicological sciences are faced with the
21 seemingly insurmountable challenge posed by the sheer number of different chemicals
22 present in the pharmaceutical waste stream. Not only is that number already vast, but it
178
1 grows ever larger as new products are continually brought into use. The cost of bringing
2 a single pharmaceutical to market has been estimated to be an average of $897 million
3 dollars (Kaitin 2003), of which $466 million is spent on human testing phases and $2-9
4 million on testing on model organisms (DiMasi, Grabowski et al. 2004). These
5 procedures take years. It is highly doubtful that it will ever be feasible using existing
6 methods to test every compound for subtle and mixture effects. It is in response to such
7 challenges that computational and predictive methods are being increasingly used
8 (Kulkarni, Zhu et al. 2005; Collins, Gray et al. 2008 ), along with the development of
9 cell-based high through put studies (Inglese, Auld et al. 2006 ). Large-scale projects by
10 the U.S. EPA, National Institutes of Environmental Health Sciences and National
11 Institute of Health are under way to create comprehensive toxicology databases and focus
12 high-throughput screening efforts, although almost none of these efforts take into account
13 microbial metabolism of these compounds (NIH 2011; Tox21 2011; ToxCast 2011).
14 If one is presented with several novel chemicals yet have only the capacity to
15 investigate the toxicology of a single compound, one must employ some rational method
16 of deciding which chemical to analyze. While computational tools based on structure
17 activity relationships exist for the prediction of ecotoxicological effects of a given
18 chemical which can help guide such decisions (reviewed in (Netzeva, Pavan et al. 2007;
19 Pavan and Worth 2008; Piparo and Worth 2010; Serafimova, Gatnik et al. 2010) these
20 models are dependent upon thorough scientific data (Scior, Medina-Franco et al. 2009)
21 describing known endpoints or mechanisms of action which calls in to doubt their ability
22 to predict toxicity in the case of novel mechanisms of intoxication or when appropriate
23 data sets are lacking (Greene 2002). An added level of complexity comes from the fact
179
1 that the overall impact of a given chemical is not based only upon the chemical itself, but
2 also upon the breakdown products of that chemical. The type and importance of
3 metabolites can vary due to inter-individual differences in human metabolism (Baillie
4 2007; Uetrecht 2007; Ulrich 2007; Li 2009), human gut metabolism (Doherty and
5 Charman 2002; Li, Wang et al. 2008 ; Thelen and Dressman 2009; van Herwaarden, van
6 Waterschoot et al. 2009) metabolism by environmental microbes (Neuwoehner,
7 Zilberman et al. 2010), and abiotic breakdown products. Science is beginning to develop
8 tools for predicting the breakdown products of a particular pharmaceutical compounds
9 (Kulkarni, Zhu et al. 2005) and make predictions of likely metabolites, but once again,
10 prediction of metabolism relies upon known pathways for similar chemicals. Reliable
11 predictions of a novel compound’s metabolism and toxicity are most likely to be accurate
12 only when they are based on experimental data from closely related compounds. Thus
13 we are still left with a daunting task when it comes to hazard identification and risk
14 characterization which underscores the value of the precautionary principle for protecting
15 human and environmental health.
16 The work presented in this dissertation represents the first investigation into the
17 assimilative bacterial metabolism of an alpha-branched phenylacetic acid with the goal of
18 providing insight that may eventually help inform model development and pathway
19 prediction accuracy
20
21
22
23
180
1 Metabolism of Ibuprofen by Sphingomonas Ibu-2
2
3 Sphingomonas Ibu-2, which was isolated by enrichment culture from sewage
4 sludge, has the ability to grow using ibuprofen as sole carbon and energy source. Ibu-2
5 exhibits a slight tendency to degrade the R-enantiomer more quickly than the S, but is
6 capable of growth on either (Figure 2.1). Ibu-2, in an unprecedented fashion,
7 accumulates de-acylated catechols when incubated with a ring-cleavage inhibitor
8 (Murdoch and Hay 2005). The expression of seven open reading frames, ipfABDEFHI,
9 proved sufficient to transfer this metabolic activity upon an E. coli host. Uptake assays
10 performed using an Ibu-2 chromosomal library clone capable of performing this
11 deacylating reaction suggests that Ibu-2 accumulates ibuprofen intracellularly through the
12 action of a coenzyme A ligase enzyme encoded by ipfF and powered by ATP. Coenzyme
13 A ligation encourages uptake on two general principles. Firstly, the coenzyme group is
14 large and hydrophilic, thus trapping ibuprofen inside the cell. In the absence of ligation,
15 ibuprofen is able to cross cellular membranes in both directions, resulting in no net
16 intracellular accumulation. However, by chemically altering it, ibuprofen is effectively
17 taken out of the thermodynamic equation therefore encouraging continued influx. The
18 combination of intracellular trapping and continued influx down a concentration gradient
19 through a ligative process is termed vectorial acylation and has also been observed in the
20 metabolism of fatty acids by bacteria and eukaryotes (Weimar, DiRusso et al. 2002;
21 Black and DiRusso 2003; Zou, F. et al. 2003).
22 Ibuprofen coenzyme A (ipfCoA) is the likely target of further metabolism by a
23 dioxygenase system in Ibu-2. This conclusion is based on the requirement of functional
181
1 copies of ipfA and ipfB (genes share homology with the two primary components of
2 aromatic dioxygenase systems) in pFOS3G7 for isobutylcatehcol production and upon
3 the observation that subcloned ipfABDEF is not capable of efficient metabolic activity
4 without the co-expression of ipfHI, a ferredoxin reductase and ferredoxin respectively
5 (Figures 3.9 - 3.11). The presumed 1,2 dearomatized diol that results from IpfAB-
6 mediated transformation of ibuprofen CoA is predicted to be the substrate that leads to
7 isobutylcatechol production via IpfDE.
8 The predicted amino acid sequence of ipfD shares homology with genes encoding
9 acyl-transferases that are members of the sterol carrier protein X (SCPx) family and
10 which are known to catalyze the removal of an acyl-CoA group. Despite this family
11 being numerous and diverse, it is poorly characterized. In fact, the limited experimental
12 exidence presented in chapter 3, which shows that expression of ipfDE is essential for
13 isobutyl catechol production from ibuprofen, makes it only the third bacterial putative
14 SCPx protein that has been characterized at the reaction level. Via an unknown
15 mechanism, ibuprofen CoA diol is rearomatized to isobutylcatechol during or after the
16 deacylation reaction. The role played by the domain of unknown function 35 (DUF35)
17 protein IpfE in this process is unknown, but it likely associates with the acid side chain
18 and is necessary for the activity of IpfD given the fact that many DUF35 genes are found
19 next to a SCPx gene in sequenced genomes. The likely involvement of an SCPx thiolase
20 and DUF35 protein in the direct conversion of a phenylacetic acid derivative directly to a
21 de-acylated catechol was not forseen and is without precedent. This novel reaction
22 mechanism may prove relevant for the prediction of possible environmental breakdown
23 products of other alpha-branched phenyalcetic acids such as ketoprofen and naproxen
182
1 (figure 1.1). While the recently characterized paa bacterial pathway might prove more
2 useful for the breakdown prediction for non-alpha-branched NSAIDs with a phenylacetic
3 acid core such as diclofenac, etodolac, indomethacin, sulindac, and tolmetin, it is at this
4 point unknown how wide spread ipf-like pathways might be and what their specifity
5 might be.
6
7 Points of Uncertainty in the Ibu-2 Ibuprofen Metabolism Model
8
9 The vectoral acylation mechanism conclusion was based partially upon the
10 observation that adding the metabolic poisons potassium cyanide or dimethylphenol to E.
11 coli epi300 pFOS3G7 inhibited uptake, indicating the presence of an active transport
12 system. Using the same poisons on E. coli epi300 pFOS3G7 with a knocked-out ipfF did
13 not impact uptake to any degree; this is a critical piece of data indicating that IpfF is the
14 only source of active transport amongst the DNA expressed on pFOS3G7. It may be
15 pointed out that cyanide and 2,4-DNP poison both affect the chemiosmotic gradient and
16 as a consequence ATP production and therefore the loss of uptake upon poisoning could
17 result in the disruption of gradient-driven transport by a major facilitator porin transport
18 system. A thorough approach would have utilized an ATP synthase inhibitor such as
19 venturicidin (Hong and Pedersen 2008) while leaving the chemiosmotic gradient
20 undisrupted. However, the E. coli epi300 pFOS3G7Tn: ipfF data obviates the need for
21 this by demonstrating that no other active transport system is active in E. coli epi300
22 pFOS3G7.
183
1 While IpfF is sufficient for uptake in E. coli , it may not be so in Sphingomonas . It
2 is possible that transport in Ibu-2 also involves a FadL-like translocase such as has been
3 proven to enhance the uptake of fatty acids (Ferguson and Deisenhofer 2002; van den
4 Berg, Black et al. 2004) and of toluene (Wang, Rawlings et al. 1995; Kahng, Byrne et al.
5 2000).
6 While ipfABDEFHI were shown to be sufficient for metabolic activity,
7 conclusions regarding the activity of the putative enzymes IpfAB and IpfDE are based on
8 indirect experimental data . Each of the associated genes has been demonstrated to be
9 necessary for ibuprofen metabolism. The hypothesis that IpfAB acts upon the coenzyme
10 A CoA-ligate that is the product of IpfF is supported by the statistically significant
11 greater loss of phenylacetyl-CoA in E. coli pGEM: ipfAB cell-free extracts. However, the
12 difference between the clone and the control was modest (though statistically significant)
13 and no reaction products could be identified via chromatographic methods. Additionally,
14 the requirement of subcloned ipfABDEF on the co-expressed electron transport chain
15 ipfHI (Figure 3.10 – 3.11) offers strong but indirect evidence for the involvement of
16 ipfAB.
17 The activity of IpfDE is largely speculative and based primarily on the inference
18 that IpfDE is likely responsible for metabolizing dioxygenated ibuprofen-CoA to
19 isobutylcatechol. This inference is supported by catalytic activity described for orthologs
20 of IpfD; SCPx acyl-transferase proteins which deacylate alpha-branched 3-oxo CoA-
21 ligated fatty acids in both eukaryotes (Wanders, Denis et al. 1997; Verhoeven and Jakobs
22 2001; Westin, Hunt et al. 2007 ) and bacteria (Leuthner and Heider 2000; Kube, Heider et
23 al. 2004; Kuhner, Wohlbrand et al. 2005). The inferred involvement of IpfE in the
184
1 process is based upon the fact that ibuprofen metabolism is dependent upon its presence
2 and that an IpfE-like DUF35 protein and an SCPx thiolase have been shown to be
3 intimately associated in the case of benzylsuccinate metabolic proteins BbsB and BbsC
4 (Leuthner and Heider 2000; Kube, Heider et al. 2004; Kuhner, Wohlbrand et al. 2005).
5
6 Environmental Relevance of the Ibu-2 Ibuprofen Pathway
7
8 A number of questions can be raised regarding the relevance of this pathway.
9 Firstly, how common is this pathway? Is it a common paradigm for dealing with
10 ibuprofen and similar chemicals or is it an anomaly? Additionally, it is not at all clear
11 that this is the mechanism whereby ibuprofen would be metabolized in the environment
12 where it is found at much lower concentrations than were used in this work.
13 To approach the question of whether or not ibuprofen is degraded via the Ibu-2
14 type pathway by organisms that have only been exposed to low concentrations, active
15 sewage sludge was spiked with ibuprofen and ring-cleavage inhibitor and metabolites
16 were analyzed within only a few hours, presumably before any genetic acclimatization
17 could take place (Appendix 4). While many structural variations of hydroxyibuprofen
18 and carboxyibuprofen were detected, isobutylcatechol was not detected, suggesting that
19 the resident microbial community was not expressing an Ibu-2 type pathway. This
20 preliminary piece of data suggests that the isobutylcatechol pathway may not be relevant
21 in the environment in a strict sense although catechols could have sorbed or reacted with
22 the abundant organic matter present in the sewage sludge and therefore went undetected.
23 This issue was addressed directly in a pilot project that applied a bacterial fractionation of
185
1 environmental samples in order to remove the presumptive solid phase catechol sink
2 (outlined in Appendix 4). The bacterial fractions did indeed produce traces of acid labile
3 yellow color consistent with meta cleavage product when spiked with the ibuprofen
4 analog m-tolylacetica acid, suggesting that such pathways might indeed be present at
5 appreciable levels in the environment.
6 The apparent discrepancy regarding the fate of ibuprofen at high versus low
7 concentrations is one of far-reaching relevance. It is not entirely clear what happens to
8 low-concentration organic chemicals in the environment. Martin Alexander’s laboratory
9 (Rubin, Subba-Rao et al. 1982; Schmidt, Alexander et al. 1985) approached this topic and
10 presented evidence that an entirely different set of organisms acts upon organics at low
11 versus high concentrations. Low concentrations of organics were mineralized via
12 unknown and apparently non-assimilative mechanisms, which is reminiscent of the
13 seemingly non-specific oxidation of ibuprofen by the biological activity present in
14 sewage sludge. The mineralization reported by Alexander and coworkers obviously
15 excludes the assimilative types of metabolism that are present in microorganisms isolated
16 by the enrichment techniques used in this study. The possible mechanisms of low
17 concentration metabolism could be but are not limited to:
18
19 1. Fungal extracelluar lignin-degrading enzymes such as fungal laccases;
20 Laccases, which are present in prokaryotes, fungi, and plants, have a wide variety of
21 functions (Mayer and Staples 2002; Claus 2004). Fungi are known to secrete laccases for
22 the putative purpose of catalyzing the oxidative degradation of the lignin polymers into
23 smaller molecules that can be been absorbed (Claus 2003). Because lignin is an
186
1 extremely structurally complex compound, a wide variety of laccases with varying
2 specificity are secreted. While it has never been directly investigated, it seems likely that
3 this constant presence of laccases in biological slurries is likely to have the capacity to
4 non-specifically oxidize many organic xenobiotics. Indeed, it has been demonstrated that
5 laccases can oxidize a wide variety of phenolics and aromatic anilines (Claus 2003) and
6 that they oxidize the carbons of a xenobiotics in a fairly random manner (Ullrich and
7 Hofrichter 2007) such as was observed during incubations of ibuprofen in sewage sludge.
8 2. Cometabolism, or the biotransformation of xenobiotics by metabolic
9 processes designed for another purpose but with loose specifity; cometabolic processes
10 have been found to be significant in the fate of MTBE (Fiorenza and Rifai 2003),
11 chlorinated ethylenes (Arp, Yeager et al. 2001), and polycyclic aromatic hydrocarbons
12 (Prak and Pritchard 2001). Such processes may be at work in metabolizing very low
13 concentrations of xenobiotics when the enzymes in question have been induced by other
14 chemicals for which they are specific.
15 3. Organisms that are poorly represented in enrichment and culturing
16 processeses; it is widely acknowledged that the majority of prokaryotes present in the
17 environment cannot be recovered using traditional culturing techniques (Amann, Ludwig
18 et al. 1995). This means that any given bacterium performing a relevant metabolic
19 process in the environment is likely to be uncultivable.
20
21
22
23
187
1 Future Work
2
3 The relevance of this work straddles two realms, environmental toxicology and
4 microbial metabolism. From the microbiological perspective, this work’s contribution
5 lies in providing preliminary evidence for new types of enzymes and metabolic pathways
6 that may provide a deeper understanding of bacterial metabolic potential in general.
7 From an environmental perspective, this work was performed with the hope that
8 information would be gained that would aid in the prediction of the environmental fate of
9 similar xenobiotics. For example, the metabolic pathways responsible for the elimination
10 of the phenylacetic acid based pharmaceuticals diclofenac, naproxen, ketoprofen, and
11 benzafibrate are all currently poorly understood (Quintana, Weiss et al. 2005; Kosjek,
12 Heath et al. 2009). Degradation of each of these chemicals in environmental systems has
13 been observed, but the intermediates have been scarcely described. The 1,2 dioxygenase,
14 SCPx, DUF35 deacylating pathway offers novel hypothetical metabolites to search for in
15 each case.
16
17 Future Work: Environmental
18 First of all it would be informative to know whether or not ipf-type genes are
19 found with any frequency in the environment. While it would be informative to examine
20 environmental samples using degenerate primers or probes for bacterial SCPx genes, this
21 family of genes is very divergent; sequence alignment does not reveal any suitably
22 conserved targets for analysis. In order begin to address the general question of whether
23 such a pathway is present at appreciable levels in the environment, a pilot experiment
188
1 was conducted in which ibuprofen and m-tolylacetic acid were spiked into the bacterial
2 fraction of Cayuga lake water (described in Appendix 4). A yellow coloration consistent
3 with the presence of meta-cleavage product of methylcatechol was present though the
4 absorbance peak was somewhat broad and inconclusive. A GC/MS analysis of such
5 samples might reveal the presence of the meta-cleavage semi-muconic acid metabolite.
6 The presence and unique qualities of the ipf pathway are puzzling for a variety of
7 reasons. For instance, why is this pathway fundamentally different from the widely
8 distributed paa pathway which degrades phenylacetatic acid and phenylalanine via an
9 epoxide intermediate? It is perhaps informative that Ibu-2 grows using the same ipf
10 pathway on tolylacetic acids but is unable to grow on phenylacetic acid. This suggests
11 that the ipf pathway is suited to the metabolism of methylated phenylacetic acids as
12 opposed to unsubstituted phenylacetic acid. It would be informative to investigate
13 whether the paa pathway is capable of activity towards tolylacetic acids. An inability of
14 the epoxide-mediated paa metabolic strategy to act upon tolylacetic acids would take a
15 step towards explaining the presence of the unique ipf pathway. However, it remains
16 puzzling that while the ipf pathway strategy is apparently very well-suited towards the
17 metabolism of phenylacetic acid to catechol and subsequent ring cleavage products, such
18 a strategy has not been reported in the literature for the metabolism of such a ubiquitous
19 and well-studied chemical.
20 In this and other studies, several varieties of hydroxylated ibuprofen have been
21 identified in spiked sewage sludge or environmental samples. In order to approach the
22 role of fungal laccases in the production of the hydroxylated metabolites that have been
23 identified, the following research strategies could be utilized. Unfortunately, laccase
189
1 inhibition is out of the question due to the fact that the only true laccase inhibitor is azide,
2 which also has broad biocidal properties (Johannes and Majcherczyk 2000).
3 Alternatively, it would be very informative to use pure fungal strains or to clone laccases
4 and specifically examine their capacity to oxidize ibuprofen and other xenobiotics.
5 Radiolabelled ibuprofen could be used to trace the fate of ibuprofen in
6 environmental samples. Unfortunately only ibuprofen that is labeled on the carboxy
7 carbon is currently available commercially. This is a very limiting factor due to the
8 observation that Ibu-2 removes the carboxy atom early in the metabolic process. If ring
9 labeled ibuprofen could be synthesized, much more could be learned. For example, trace
10 amounts of labeled ibuprofen could be added to sewage sludge, the carbon dioxide
11 released during mineralization could then trapped and quantified or HPLC and GC/MS
12 could be used to detect and identify radiolabelled metabolites. Further analysis could be
13 performed on the biological fraction to detect any assimilated radioactivity. For example,
14 a radioactive variation of Stable Isotope Probing (SIP) (Madsen 2006) which uses cesium
15 chloride gradients to separate DNA based on the mass of the carbon isotope it contains
16 could be used to obtain a phylogenetic snap-shot of the organisms that are assimilating
17 labeled ibuprofen.
18
19 Future Work: Microbiological
20 From a microbiological perspective, the most intriguing aspect of the system by
21 far is the apparently novel removal of the acyl-CoA group and associated re-
22 aromatization of the ring. No such chemical reaction has been reported in the literature.
190
1 The pathway was successfully reconstructed using subcloned ipfABDEFHI. Presently
2 there is only indirect evidence that IpfABHI dioxygenates CoA ligated substrate.
3 Directly identifying the oxygenated metabolite of ipfABFHI would both remove
4 uncertainty on this point and also begin to offer insight into the mechanism of ipfDE.
5 The SCP-x thiolase and DUF35 ipfDE are the most exciting aspect of this system given
6 the ubiquity of these types of genes and the near complete lack of insight into the
7 functions of their products. The pFOS3G7∆D mutant should be adequate for production
8 of the dioxygenase metabolite. The fosmid, when upregulated, is at a high copy number.
9 However, the activity of the pFOS3G7 system is not remarkably high. It is possible that
10 cloning ipfABFHI into a high copy number vector with a strong E. coli promoter will
11 prove more fruitful. An ipfABF construct in pGEMt-easy, which is a high copy number
12 vector with a strong lac promoter, has already been constructed. pBBR1mcs could be
13 used to express ipfHI. Alternatively, the ipfABF cassette could be combined with ipfHI
14 on a single vector using strand overlap extension.
15 In order to definitively identify the dioxygenated metabolite, NMR should be
16 employed. GC/MS identification of a diol is possible but challenging, requiring the use
17 of a butylboronic acid derivitization. Even then, GC/MS does not provide definite
18 identification of a chemical in the absence of a standard. In order to perform NMR,
19 substrate completely labeled with carbon-13 must be obtained. [U-13C]phenylacetate is
20 not commercially available, but a farily simple method for it’s production is in
21 publication (Ismail, Mohamed et al. 2003) by which phenylalanine is deaminated using
22 commercially available L-amino acid oxidase. The reaction is then acidified and
23 extracted with ethyl acetate in order to purify the reaction product.
191
1 Teufel et al. (Teufel, Mascaraque et al. 2010) produced 13C-labelled
2 intermediates in bacterial phenylacetate production by first developing methods by which
3 to purify the proteins. Maltose binding protein tagging was used instead of traditional
4 histidine-tags in most cases for unspecified reasons. Developing methods by which to
5 purify IpfABFHI will be required as a first step. Teufel et al. column-purified
6 overexpressed proteins and then exposed them to substrate with all necessary cofactors,
7 which in the case of IpfABFHI will likely require a pool of ATP, CoA, and NADH.
8 Alternatively, the reaction could be split into the two substituent steps, i.e. separate
9 reactions and product purifications for first the IpfF coenzyme A ligation and then the
10 IpfABHI dioxygenation. The reaction products can be purified by HPLC. Teufel et al.
11 also included radioactive 14C-labelled substrate as a tracer to aid in this step. The 14C
12 substrate would also have to be prepared from 14C phenylalanine using the amino acid
13 oxidase method described above. Regarding product stability, Teufel et al. found that
14 most intermediates in the paa pathway had half-lives in methanol ranging from 2.5 to 6
15 hours. Half-lives in water were very low. Following product purification, the samples
16 should be dried, resuspended in deuterated water and subjected to NMR analysis.
17 Further characterization of ipfD and ipfE will prove challenging given that these
18 types of gene products have never been characterized at the reaction level. I have
19 constructed histidine-tagged ipfD and ipfE on protein overexpression vectors and they
20 might be prove useful for further characterizations by allowing for the purification of the
21 protein products. Unfortunately, the likely instability of the hypothetical diol metabolite
22 will make generation of a stable chemical stock by an ipfABFHI construct challenging if
23 not impossible. If the reaction product of IpfABFHI can be identified by NMR as
192
1 described above, the reaction product can be exposed to purified IpfD and/or IpfE in
2 order to verify that it is indeed the substrate for the subsequent and final reaction.
3 Chapter 4 described the investigations into the transport mechanisms employed by
4 the ipf system. The results suggested that the predicted protein product of ipfF was
5 responsible for uptake of the substrate via a CoA-mediated vectorial ligation mechanism.
6 However, these experiments were performed exclusively in an E. coli host using
7 radiolabelled phenylacetic acid. Ibu-2 proved difficult to work with due to the fact that it
8 clogged the filter membranes and phenylacetic acid was used due to the inexpensiveness
9 of radiolabelled phenylacetic acid versus ibuprofen. While the results were consistent
10 with the known mechanisms for fatty acid uptake systems, they were at odds with other
11 described systems for aromatic acid uptake, including those for phenylacetic acid. The
12 bulk of phenylacetic acid uptake systems involve specific porin-type active-transport
13 systems. While IpfF may have been sufficient to mediate uptake in the E. coli host, it is
14 possible that other transport proteins are involved or perhaps even required in
15 Sphingomonas Ibu-2. In order to study uptake in Ibu-2, a different approach would have
16 to be taken. Either an efficient way to remove the extracellular matrix from Ibu-2 would
17 have to be developed or an uptake assay that does not involve filter membranes would
18 have to be utilized. If an alternative uptake protocol could be developed, the question of
19 the sufficiency of IpfF to drive uptake could be addressed by a targeted knockout of ipfF
20 in Ibu-2 and measuring uptake. Initial attempts to generate genetic knockouts in Ibu-2
21 using targeted double recombination or random transposon based approaches proved
22 untenable (Appendix 4). Alternatively, ipfF could be cloned into a more closely related
193
1 species, one that is more amenable to filter-based uptake assays, and the uptake studied
2 therein.
3
194
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202
1 APPENDIX 1
2 A NOVEL MECHANISM FOR THE BIODEGRADATION OF
3 IBUPROFEN
4
5 Preface
6
7 The following manuscript was prepared in 2002 and submitted to Environmental
8 Science and Technology. It was accepted on the condition that certain experiments
9 be elaborated upon. Attempts to revive the organism that was supposedly isolated
10 and identified as a Variovorax sp. Ibu-1 were unsuccessful. It seemed the case that
11 Ibu-1 was not a pure culture and in fact had been, at some point in the course of
12 regular maintenance of the organism, heavily contaminated. Whether the dihyroxy-
13 and trihydroxyibuprofen detected in culture media were actually utilized in a
14 catabolic pathway remains uncertain. Subsequent attempts to isolate an organism
15 utilizing a similar pathway, distinct from the isobutylcatechol pathway utilized by
16 Ibu-2, proved fruitless.
17
18 Abstract
19
20 A bacterium was isolated from sewage sludge that has the ability to use
21 ibuprofen as its sole carbon and energy source. 16S rRNA gene sequence
22 analysis revealed 98% homology to species of the Variovorax genus.
203
1 When grown on ibuprofen it accumulated a transient yellow intermediate
2 that disappeared upon acidification, a trait consistent with meta ring-
3 fission metabolites. GC/MS analysis of the derivatized culture supernatant
4 yielded two spectra consistent with trihydroxyibuprofen, bearing all three
5 hydroxyl groups on the aromatic ring. However, these metabolites were
6 only detected when 3-fluorocatechol, a meta ring-fission inhibitor, was
7 added to Ibu-1 cultures and the supernatant was then derivatized with
8 aqueous acetic anhydride and diazomethane. These findings suggest the
9 possibility of ibuprofen metabolism proceeding via a meta ring-fission
10 pathway. Identical spectra, consistent with these putative ring-
11 hydroxylated trihydroxyibuprofen metabolites, were also obtained from
12 ibuprofen-spiked sewage sludge, but only when it was poisoned with 3-
13 fluorocatechol and derivatized. This demonstrates the biological potential
14 for ibuprofen degradation via previously unidentified intermediates. The
15 presence of the same metabolites in both spiked sewage sludge and culture
16 supernatants suggests that a ring-cleavage pathway for the degradation of
17 ibuprofen may have environmental relevance.
18
19 Introduction
20
21 Ibuprofen (2-(4-isobutylphenyl-propionic acid)) is a non-steroidal anti-
22 inflammatory drug (NSAID) used for its analgesic, antipyretic, and anti-inflammatory
23 properties. Two decades ago, it was labeled as being “inherently biodegradable”
204
1 (Richardson and Bowron 1985). In the decades since, ibuprofen has become the third
2 most highly consumed drug in the world (Buser, Poiger et al. 1999). Given that 8.9% -
3 14% of the administered dose is excreted unmodified or as the easily hydrolysed
4 glucuronide conjugate (Lee, Williams et al. 1985; Rudy, Knight et al. 1991; D'Ascenzo
5 2003), a large amount of ibuprofen has the potential to enter the environment.
6 Environmental concentrations of ibuprofen have been found to range from low ppt
7 (Buser, Poiger et al. 1999; Stumpf, Ternes et al. 1999; Farre, Ferrer et al. 2001; Winkler,
8 Lawrence et al. 2001; Kolpin, Furlong et al. 2002) to low ppb levels (Buser, Poiger et al.
9 1999; Farre, Ferrer et al. 2001). However, these studies analyzed only the ibuprofen in
10 aqueous solution. With a log K ow of 3.5 (Stuer-Lauridsen, Birkved et al. 2000), ibuprofen
11 would be predicted to partition predominantly to sediment.
12 Despite the large environmental load of ibuprofen, little is known regarding how
13 ibuprofen is degraded by bacteria in the environment. No organisms have been reported
14 to grow on ibuprofen in pure culture, although Chen and Rosazza (Chen and Rosazza
15 1994) reported that a Nocardia species could reduce the carboxylic acid moiety to an
16 alcohol and then acetylate it. In laboratory reactor experiments, Winkler et al. (Winkler,
17 Lawrence et al. 2001) reported an increase in the concentration of ibuprofen with a
18 hydroxyl- or carboxyl-group on a side-chain concomitant with the decrease in
19 concentration of ibuprofen. In addition to these metabolites, Zwiener et.al . (Zwiener,
20 Seeger et al. 2002) also reported 4-carboxyhydratropic acid as a degradation product of
21 ibuprofen. However, these metabolites did not account for all ibuprofen degradation,
22 representing only 10% of total ibuprofen disappearance in one case (Zwiener, Seeger et
23 al. 2002).
205
1 Like ibuprofen, many other NSAIDs, including naproxen, ketoprofen, and
2 diclofenac, are aliphatic-substituted phenylacetic acids. There is extensive literature
3 describing the bacterial metabolism of hydroxyphenylacetic acids (Sparnins and
4 Chapman 1976; Blakley 1977; van den Tweel, Smits et al. 1988) and some recent work
5 regarding phenylacetic acid, which proceeds via a phenylacetyl CoA intermediate
6 (Ferrandez, Minambres et al. 1998; Olivera, Minambres et al. 1998). However, there has
7 been almost no work describing the biodegradation of aliphatic-substituted phenylacetic
8 acids, including ibuprofen. As perhaps the most ubiquitous member of these two
9 overlapping classes of compounds, ibuprofen serves as a useful model for how these
10 compounds may be metabolized.
11 With the exception of unsubstituted phenylacetic acids, the literature regarding
12 the aerobic metabolism of aromatic compounds by bacteria reveals a near-constant
13 paradigm. The aromatic ring is dioxygenated on vicinal carbons and dehydrogenated to
14 form a catechol, followed then by the insertion of molecular oxygen to accomplish ring
15 opening (Butler and Mason 1997). para -dioxygenation is more rare, but is also a
16 sufficient prerequisite for ring-opening (van den Tweel, Smits et al. 1988). If bacteria are
17 to completely metabolize ibuprofen under aerobic conditions, they might do it by
18 dioxygenating the ring.
19 While catechols seem to be a common intermediate in the aerobic metabolism of
20 aromatic compounds, they are ephemeral, being transformed rapidly by both enzymatic
21 and non-enzymatic mechanisms (Schweigert 2001). For these reasons, they are rarely
22 detected in the environment. As catechols are known to be toxic (Schweigert 2001), most
23 organisms capable of degrading aromatic compounds do not normally accumulate
206
1 catechols without either prior genetic manipulation or the use of enzyme inhibitors.
2 Given the uncertainty regarding the fate of most of the ibuprofen entering the
3 environment, we hypothesized that ibuprofen may be metabolized to intermediates that
4 are not readily detected using standard sample preparation techniques, and have therefore
5 previously gone undetected. Specifically we hypothesized that ibuprofen might be
6 degraded via initial ring dioxygenation followed by meta -cleavage. In order to test this
7 hypothesis we characterized ibuprofen metabolites from an environmental bacterium
8 named Ibu-1, which is capable of using ibuprofen as a sole carbon and energy source.
9 We then examined ibuprofen-supplemented sewage sludge for the presence of the same
10 metabolites.
11
12 Materials and Methods 13
14 Materials. All chemicals were purchased from Acros (Morris Plains, NJ).
15 Sewage sludge was collected from the City of Ithaca, NY sewage treatment plant.
16 Enrichment of Sewage Sludge. Sewage sludge was enriched with S-ibuprofen
17 according to standard protocols (Krieg 1981). After five serial transfers, the final
18 enrichment was streaked onto a solid mineral salts medium (MSM) containing 500 mg/L
19 S-ibuprofen. MSM was composed of 1 mM MgSO 4, 10 mM K 2HPO 4, 3 mM NaH 2PO 4,
20 10 mM (NH 4)2SO 4, 10 µM Fe(NO 3)3, and 100 µM Ca(NO 3)2 (McCullar 1994). A single
21 colony was isolated and subjected to further analysis.
22 Species Identification. A portion of the 16S ribosomal rRNA gene of Ibu-1 was
23 PCR amplified using primers 1055F (ATGGCTGTCGTCAGCT) (Ferris 1996) and
24 1492R (TACGGYTACCTTGTTACGACTT) (Lane 1991). The resulting product was
207
1 then ligated into pGEMt-easy (Promega, Madison, WI) and electroporated into
2 Esherichia coli DH5 α. The insert contained within a single colony was amplified using
3 T7/M13 primers and sequenced. The sequence was then analyzed via BLAST (Altschul
4 1990), an online database and search engine which identifies analagous DNA sequences.
5 Growth Conditions of Ibu-1. The isolate was grown in liquid MSM and
6 containing 500 mg/L S-ibuprofen and supplemented with 0.2% Luria-Bertani broth (LB)
7 to speed growth. Growth was monitored by measuring absorbance at 600 nm using a 96
8 well µQuant spectrophotometer from BioTek instruments (Winooski, VT).
9 Monitoring Ibuprofen Concentrations via HPLC. 1 ml of Ibu-1 culture was
10 centrifuged at 15,800 x g for 2 minutes. The supernatant was then passed through a 0.2
11 µm filter. Results from the HPLC analysis of samples and a standard curve were used to
12 determine the ibuprofen concentration in the supernatant. The eluent was 70% methanol
13 and 30% 40 mM acetic acid. The sample was pumped at a rate of 1 ml/min using a
14 Waters Model 590 pump. The column was a Varian Microsorb-MV C18 column, 250
15 mm by 4.6 mm. Under these conditions, ibuprofen elutes at approximately 15 minutes.
16 Samples were injected by a Shimadzu SIL-10AD AP autoinjector. Detection was
17 accomplished with a Shimadzu SPD-10A VP UV-Vis detector measuring absorbance at
18 220 nm.
19 GC/MS Analysis of Variovorax Ibu-1 Culture Supernatant. Two 100 ml ibu-1
20 cultures were grown on 500 mg/L S-ibuprofen until late exponential phase. To one
21 culture, 3-fluorocatechol (3FC) was added to a final concentration of 50 mg/L. After
22 thirty minutes at room temperature, the cultures were centrifuged and the supernatants
23 pulled through a 0.2 µm filter. Aqueous acetylation was performed by adding potassium
208
1 carbonate and acetic anhydride to the supernatant to final concentrations of 1.5% and
2 0.5% respectively (Mars 1997). After thirty minutes, the samples were acidified to pH 3,
3 extracted with ethylacetate, and dried over a sodium sulfate column. The extracts were
4 then methylated with diazomethane. Diazomethane was generated in a 40 ml EPA vial
5 by combining 5 ml ethyl ether with 3–5 g of N-nitroso-N-methylurea, a single pellet of
6 sodium hydroxide, and 0.1–0.2 ml of water. The ethyl ether containing diazomethane
7 was dried over a sodium sulfate column, and then added to the ethylacetate extracts.
8 After thirty minutes at room temperature, the samples were evaporated to a minimal
9 volume under a nitrogen stream and separated using an HP 6890 GC equipped with an
10 HP-5MS column (5% phenyl methyl siloxane 30 m x 0.25 mm, 0.25 µm film thickness)
11 with helium as the carrier gas at a flow rate of 1 mL/min. The injector temperature was
12 250°C. The initial oven temperature of 40°C was held for 1 min, then ramped at a rate of
13 10°C/min. to 250°C. The temperature was held at 250°C for 7 min and then ramped up at
14 30°C/min until 300°C. The detector was an HP 5973 MSD with quadrapole and source
15 settings of 150 ºC and 230 ºC respectively.
16 GC/MS Analysis of Ibuprofen Supplemented Sewage Sludge. 500 mg/L R/S
17 ibuprofen was added to 1 L of sewage sludge. Ibuprofen concentration was monitored
18 via HPLC as described above. After one week the ibuprofen concentration had dropped
19 to approximately 50% of original level and the sample was split in two. To one half, 3-
20 fluorocatechol was added to a final concentration of 1000 mg/L. After thirty minutes, the
21 sludge samples were centrifuged. The supernatant was then filtered sequentially over a 4
22 µm filter followed by a 0.4 µm filter. The samples were then extracted, derivatized, and
23 analyzed via GC/MS as described above.
209
1 Results and Discussion
2
3 Isolation of a Bacterium capable of growth on Ibuprofen. Ibu-1 was isolated
4 from an ibuprofen enrichment culture that had the ability to use S-ibuprofen as a sole
5 carbon and energy source. Growth of Ibu-1 correlated directly with the disappearance of
6 ibuprofen (Figure A1.1).
7
600 0.2
0.15 400 0.1 200 0.05 ppm ibuprofen ppm
0 0 absorbance600nm @ 0 50 100 150 200 time from inoculation (hrs.) 8
9 Fig. A1.1. Growth of Ibu-1 on ibuprofen as measured by increase in Ibu-1 culture density 10 via spectrophotometry at 600nm ( uuu ). Concomittant loss of ibuprofen during growth as
11 measured via HPLC ( ppp ).
12
13 Ibu-1 entered exponential growth phase 10 to 20 hours after inoculation into 500 mg/L
14 ibuprofen in MSM (fig. A1.1). Stationary phase was reached after approximately 75
15 hours. By this time, approximately 40% of the initial ibuprofen had been metabolized.
16 When Ibu-1 was grown in ibuprofen/MSM liquid, a yellow color appeared in the
17 supernatant. This yellow color disappeared upon acidification, and reappeared upon
210
1 neutralization. This pH-dependent yellow color is indicative of typical meta -cleavage
2 products (McCullar 1994).
3 BLAST (Altschul 1990) analysis of the 16S ribosomal rRNA gene sequence
4 revealed Ibu-1 to be a β-proteobacterium, having 98% homology with species of the
5 Variovorax genus. This genus is represented by a large number of environmental isolates
6 that have been best characterized with respect to their ability to metabolize 2,4-
7 dichlorophenoxyacetic acid (2,4-D) (Giovanni, Neilson et al. 1996; Vallaeys, Albino et
8 al. 1998; Smejkal, Vallaeys et al. 2001). In addition to utilizing 2,4-D, some members of
9 this genus such as V. paradoxus have also been reported to be capable of growth on
10 benzoate (Maskow and Babel 2001). However, degradation of both of these compounds
11 by Variovorax has been reported to take place via ortho -cleavage.
12 GC/MS of Variovorax Ibu-1 Culture Supernatant. Ibu-1 cultures were
13 exposed to 3FC, which inhibits meta -cleavage enzymes (Bartels 1984), and resulted in
14 the accumulation of ibuprofen catechols (Mars 1997). Underivatized catechols are not
15 usually amenable to analysis via gas chromatography. To overcome this problem the
16 supernatant was derivatized with aqueous acetic anhydride, a procedure that adds acetyl
17 groups specifically to aromatic hydroxyl-groups (Fujimoto 1997). Thus, derviatization
18 not only permitted good analyte separation but, when coupled with mass selective
19 detection, gave diagnostic mass spectra that allowed for the identification of ibuprofen
20 metabolites substituted with aromatic hydroxyl groups. Two mass spectra consistent with
21 ring-hydroxylated ibuprofen were detected in 3-fluorocatechol-treated Ibu-1 supernatant
22 that had been derivatized with acetic anhydride (Fig. A1.2).
23 24
211
COOMe a O-Ac b Ac-O 1.5 O-Ac 1.2 1 268 1 268 0.8 0.6 0.5 0.4 310 352 235 310 352 0.2 235 207 394 207 394 0
0 relativeabundance relative abundance relative 50 100 150 200 250 300 350 50 100 150 200 250 300 350 400 m/z m /z c d
1.2 1.2 268 1 268 1 0.8 0.8 0.6 0.6 0.4
0.4 149 310 352
310 0.2 352 253 196 225 209 0.2 394 253 394 225 196 209 relativeabundance 0 relative relative abundance 0 50 100 150 200 250 300 350 400 50 100 150 200 250 300 350 m /z m/z 1
2 Fig. A1.2. Spectra of putative metabolites identified via GC-MS analyses. Figures (a) and 3 (b) (Rt 22.7 min) from Ibu-1 culture supernatant and sludge respectively. Figures (c) and 4 (d) (Rt 27.4 min) from Ibu-1 culture supernatant and sludge respectively. These spectra 5 were only detected in extracts after 3-fluorocatechol poisoning followed by aqueous 6 acetylation. “Ac” is an acetyl group, “Me” is a methyl group. 7
8 These spectra were not detected in the absence of either 3-fluorocatechol or
9 derivatization. Both spectra contained a parent ion at 394 and were consistent with
10 methylated triacetylated trihydroxyibuprofen. Three separate losses of 42 resulted in
11 peaks at 352, 310, and 268. A loss of 42 is diagnostic of the fragmentation of an acetyl
12 ester. The aqueous acetylation step employed during derivatization has been shown to
13 acetylate only aromatic hydroxyls (Fujimoto 1997). Two such spectra appeared, one at
14 22.7 minutes and the other at 27.4 minutes, presumably representing the two possible
15 isomers of ring hydroxylated trihydroxyibuprofen.
16 Dihydroxyibuprofen was the intermediate that was expected based upon analogy
17 to metabolic pathways employed to degrade similar compounds, such as ρ-cumate
18 (Defrank and Ribbons 1976; Defrank and Ribbons 1977; Eaton 1996; Eaton 1997). ρ-
212
1 Cumate (4-isopropylbenzoate) is similar to ibuprofen in that it too is an aromatic acid
2 with a branched aliphatic group in the para position. It is the only such compound whose
3 degradation pathway has been described. ρ-Cumate is dioxygenated at the 2,3 position
4 by Pseudomonas putida F1 carrying the cmt operon. It is then subsequently
5 dehydrogenated to yield 2,3-dihydroxy-4-isopropylbenzoate. This compound is then
6 cleaved via dioxygenation across the 3,4-bond to yield a diagnostic pH-dependent yellow
7 meta -cleavage product.
8 Although not a canonical intermediate of aromatic metabolism, the presence of
9 trihydroxyibuprofen could be explained two ways. First, it may be a dead-end metabolite
10 produced only in the presence of 3-fluorocatechol. Under normal growth conditions, i.e.
11 when the ring cleavage inhibitor was not present, these poly-hydroxy metabolites were
12 not observed. When ring-cleavage is inhibited, the putative ibuprofen hydroxylating
13 enzyme(s) may fortuitously oxygenate dihydroxyibuprofen, producing
14 trihydroxyibuprofen . We have observed similar nonspecific oxygenase activity when the
15 same techniques were employed to inhibit ring cleavage of phenol metabolites by
16 Pseudomonas sp. CF600 (data not shown). Alternatively, trihydroxyibuprofen may be
17 the true ring-cleavage substrate. Although there is precedent for the degradation of
18 trihydroxybenzenes via ring-cleavage (Haigler 1999), established theory in the field of
19 aromatic metabolism holds that dihydroxylation sufficiently activates the phenyl ring so
20 as to permit ring-cleavage (Harayama and Timmis 1989). As oxygenation is an
21 energetically expensive reaction, it seems unlikely that an organism would waste energy
22 by performing an unnecessary oxygenation unless this somehow facilitated the further
213
1 degradation of the ring cleavage product. Additional analysis is needed before the nature
2 of the actual substrate for ring cleavage can be confirmed.
3 As the vast majority of ring-cleavage pathways involve catecholic intermediates,
4 such a compound is most likely to be a substrate for ring-cleavage. This is supported by
5 the observation that catecholic metabolites were not detected unless the system was
6 poisoned with 3FC, a known catechol dioxygenase inhibitior. Although a yellow
7 metabolite accumulated in culture supernatant which had the characteristics of a ring-
8 cleavage product, GC-MS analysis of methylated extracts failed to yield spectra
9 consistent with a ring fission product of an ibuprofen catechol. This is not surprising
10 however, as meta-ring cleavage products have been reported to be recalcitrant to isolation
11 and analysis (Schweigert 2001).
12 GC/MS of Activated Sludge Supplemented with Ibuprofen. After an initial
13 lag phase of approximately 5 days, the ibuprofen concentration began to drop. Seven
14 days after the ibuprofen addition, approximately 50% of the original 500 mg/L had been
15 degraded. At this point, the liter of sludge was split in half, one half being poisoned with
16 1000 mg/L 3-fluorocatechol. Analysis of derivatized products from the sludge
17 supernatant via GC/MS revealed spectra identical in all major aspects to those putatively
18 identified as trihydroxyibuprofen from extracts of Ibu-1 supernatant, including retention
19 time (Fig. A1.2). The fragmentation patterns clearly demonstrated these to be acetylated
20 metabolites, indicating that all of the hydroxyl groups were of an aromatic nature. As
21 with the pure culture study, the ring-hydroxylated metabolites were detected only in the
22 3-fluorocatechol treatment, again suggesting a meta -cleavage pathway.
214
1 In addition to the ring hydroxylated intermediates, two mass spectra consistent
2 with 2-(4-(2-methylhydroxypropyl)phenyl)propionate (15.28 and 16.31 min.)
3 (hydroxyibuprofen) and one consistent with 2-(4-(2-methylpropionyl)phenyl)propionate
4 (carboxyibuprofen) (16.52 min.) were detected in both treatments (Table A1.1). Similar
5 mass spectra identified as these ibuprofen metabolites have been identified by other
6 researchers (Buser, Poiger et al. 1999; Winkler, Lawrence et al. 2001; Zwiener, Seeger et
7 al. 2002). Two spectra consistent with hydroxyibuprofen were identified in this study,
8 presumably representing isomers.
9 Table A1.1. Ion abundance data from mass spectra obtained for previously identified 10 ibuprofen metabolites isolated from sewage sludge Name retention time (min.) mass (relative abundance) Hydroxyibuprofen 15.24 178(71) 119(100) 118(95) 117(73) 91(72) 59(42) Hydroxyibuprofen 16.31 178(63) 161(25) 119(21) 118(30) 117(32) 91(16) 84(100) 59(2) Carboxyibuprofen 16.52 264(28) 233(9) 205(100) 11
12 Although ibuprofen metabolites with side chain modifications such as these have
13 been previously reported in environmental samples, we also detected unique, ring-
14 hydroxylated ibuprofen metabolites identical to those detected in the supernatant of
15 Variovorax Ibu-1 pure cultures. It is possible that these ring-hydroxylated metabolites
16 were not detected previously because they are an artifact of the high concentrations of
17 ibuprofen employed in this study. However, the techniques employed in earlier studies
18 were not capable of detecting such catechols. In this report, 3-fluorocatechol was used to
19 inhibit meta -cleavage, resulting in an accumulation of catecholic metabolites, while
20 aqueous acetylation allowed for their detection and characterization via GC/MS. It is
215
1 important to note that the combination of these two techniques was necessary, as neither
2 technique alone was be able to reveal the presence of the catecholic metabolites.
3 To date, no reports have been published detailing bacterial growth on ibuprofen or
4 suggesting that environmental microbes may metabolize ibuprofen via ring-
5 dioxygenation and subsequent ring-cleavage. While the concentrations of ibuprofen used
6 in this study are far in excess of those detected in the environment, this study
7 demonstrates the biological potential for ibuprofen degradation via direct oxidation of the
8 ring followed by further metabolism using a putative meta-cleavage pathway. Such a
9 degradation pathway is one mechanism that may account for a portion of the biological-
10 removal of ibuprofen from wastewater treatment plants (Buser, Poiger et al. 1999) and
11 laboratory reactors (Winkler, Lawrence et al. 2001; Zwiener, Seeger et al. 2002) that has
12 previously gone uncharacterized.
13
216
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1 Stuer-Lauridsen, F., M. Birkved, et al. (2000). "Environmental risk assessment of human 2 pharmaceuticals in Denmark after normal therapeutic use." Chemosphere 40 : 783- 3 793. 4 Stumpf, M., T. A. Ternes, et al. (1999). "Polar drug residues in sewage and natural waters 5 in the state of Rio de Janeiro, Brazil." The Science of the Total Environment 225 : 6 135-141. 7 Vallaeys, T., L. Albino, et al. (1998). "Isolation and characterization of a stable 2,4- 8 dichlorophenoxyacetic acid degrading bacterium, Variovorax paradoxus , using 9 chemostat culture." Biotechnology Letters 20 (11): 1073-1076. 10 van den Tweel, W. J. J., J. P. Smits, et al. (1988). "Catabolism of DL-alpha 11 phenylhydracrylic, phenylacetic, and 3 and 4-hydroxyphenylacetic acid via 12 homogentisic acid in a Flavobacterium Sp." Archives of Microbiology 149 (3): 13 207-213. 14 Winkler, M., J. R. Lawrence, et al. (2001). "Selective degradation of ibuprofen and 15 clofibric acid in two model river biofilm systems." Water Research 35 (13): 3197- 16 3205. 17 Zwiener, C., S. Seeger, et al. (2002). "Metabolites from the biodegradation of 18 pharmaceutical residues of ibuprofen in biofilm reactors and batch experiments." 19 Analytical and Bioanalytical Chemistry 372 (4): 569-575. 20
220
1 APPENDIX 2
2
3 E. COLI EPI00 PFOS3G7 METHODOLOGICAL CONSIDERATIONS
4
5 The metabolic performance of E. coli epi300 pFOS3G7 proved to be very
6 inconsistent throughout the project. The ibuprofen and phenylacetate metabolic pathway
7 encoded by the ipf genes present on the fosmid showed widely different activity rates
8 from week to week and month to month, often forcing a complete shutdown of particular
9 routes of inquiry. Throughout the project, several attempts were made to identify the
10 causes of the erratic behavior. The logic, methods, and results of each line of inquiry are
11 outlined briefly below.
12
13 The effect of pH on fosmid activity
14
15 Even at it’s most optimal, E. coli epi300 pFOS3G7 metabolized ipf and paa at
16 remarkably slow rates, often taking four days to achieve the metabolism of a few
17 milimolar of substrate. Mature stationary-state cultures might begin to show drastic
18 deviations from optimal physiological pH; considering that uptake of ibuprofen was
19 shown to be possibly dependent upon passive diffusion followed by coenzyme A ligation
20 (Chapter 4), a rising pH may negatively impact metabolic rate. In order to address this
21 possibility, attempts to stabilize and lower the pH were made.
22
23
221
1 Methods
2
3 Culture media was inoculated from overnight LB cultures into LB with 25ppm
4 chloramphenicol, 2mM arabinose, and 100 ppm ibuprofen with the following
5 modifications.
6
7 1. unbuffered (control) (pH 7)
8 2. 25mM HEPES, pH unmodified (7)
9 3. 25mM HEPES, reduced to pH 6 with 4ul / 5ml ¼ concentrated sulfuric acid
10
11 Ibuprofen concentration was measured at 24 hours via standard HPLC protocols.
12
13 Results
14
15 At 12 hours, the pH was;
16 1. pH 8
17 2. pH 7-7.5
18 3. pH 7-7.5 (indistinguishable from the non-acidified samples)
19
20 18 hours;
21 1. pH 8.5 (note the increase)
22 2. pH 7-7.5
23 3. pH 7-7.5 (slight difference is noticeable)
222
58% 56% 54% 52%
ipf remaining 50% 48% unbuffered HEPES HEPES acid 1
2 Figure A2.1. Percent ibuprofen remaining after growth of E. coli pFOS3G7 in LB media 3 with or without 25mM HEPES buffer and with or without additional acidification of the 4 media to pH6. Y-axis amplified to demonstrate the slight differences. 5
6 Conclusions
7
8 There is a clear but small effect of pH control on ibuprofen levels. OD600s were
9 measured at 24 hours; no differences were found, though it is possible that a 5%
10 difference (such as the difference in remaining ibuprofen between the cultures) would not
11 have been detected. PIPES, because it buffers in the pH 6.5 range, might be more
12 appropriate. The small size of the effect of buffering on metabolism makes this approach
13 non-ideal. Further acidification of the media and use of a more ideal buffer could be a
14 useful approach for optimizing metabolic rate.
15
16 Fosmid aeration
17
18 At the onset of the project, a vertical drum shaker was used for aerating fosmid
19 cultures in snap cap falcon tubes. At some point, the drum shaker was no longer in the
20 lab and the cultures were thenceforth incubated in snapcaps at a 45 degree angle with
223
1 vigorous shaking. A careful reading of the protocol for the fosmid kit revealed that
2 Epicentre mentions that oxygen levels have a strong effect on fosmid copy number up-
3 regulation by arabinose (p.16, (Epicentre)), though no explanation is given. In order to
4 investigate the impact of aeration on metabolism by fosmid-cloned genes, a more
5 thorough aeration protocol was tested against the traditional protocol.
6
7 Methods
8 Culture media was LB with 25ppm chloramphenicol, 10mM arabinose, and 500
9 ppm ibuprofen. Two different aeration methods were compared; 15ml snapcap vials
10 versus 50ml culture tubes, both containing 5ml of culture shaken at 45 degree angle at 37
11 degrees centigrade. For rapid visualization of catechol product, 1.5mM ferric chloride
12 was added to the cultures. Ibuprofen concentration was measured at 24 hours via HPLC.
13
14 Results
15 At 18 hours there was no visible difference in the cultures. At 24 hours, the
16 conical cultures were remarkably dark.
17
18
19 Figure A2.2. Growth of E. coli pFOS3G7 in LB media with addition of 10mM arabinose, 20 1.5mM ferric chloride, and 500ppm ibuprofen in two different types of vessel; falcon tube 21 on the left and 40mL conical on the right.
224
1 transferring the snapcap cultures to conicals for visualization purposes clearly showed the
2 difference in product levels (Figure A2.2).
3
4
5 Figure A2.3. Same cultures as shown in Figure A2.2 but with the falcon tube grown culture 6 transferred to conicals for the sake of visual comparison. 7
8 Centrifuging a single sample of each for the sake of concentrating the dark iron-
9 catecholic pigment made the visual results more dramatic (Figure A2.4)
10
11
12 Figure A2.4. One of each culture set shown in figures A2.2 and A2.3 centrifuged in order to 13 concentrate the dark catecholic polymer. 14
225
100% 80% 60%
hours 40% 20% 0% Ibuprofen remaining at 24remaining Ibuprofen 1 2 culture condition 1
2 Figure A2.5. Percent ibuprofen remaining in E. coli pFOS3G7 cultures grown in falcon 3 tubes (1) or 40mL conicial vials (2). 4
5 Conclusions
6 The snapcap cultures had barely begun to metabolize ibuprofen while the conical
7 cultures had metabolized somewhere in the range of 100ppm of the ibuprofen initially
8 present. Oxygen level clearly had a strong effect on overall metabolic activity,
9 presumably through fosmid copy-number up-regulation, though dependence on oxygen
10 of the dioxygenase step could have also been responsible. It is likely that the higher
11 levels of activity observed during the initial stages of the project were due to much more
12 vigorous aeration of the cultures by the vertical drum shaker.
13
14 Addressing possible instability of the fosmid
15
16 While the two assays above addressed optimization of the system and did reveal a
17 possible reason that the system performed more robustly during the initial stages of the
18 project when a drum shaker was used, the erratic behavior of the system on a shorter term
19 basis is still unclear. Other researchers using fosmid-based systems have reported similar
226
1 erratic behavior (Abbie Wise and Anthony Hay, personal communication) leading to the
2 suspicion that fosmids are particularly prone to mutagenesis or recombination.
3 Additionally, sequencing of pFOS3G7 revealed it to be peppered with transposases (two
4 of which are shown flanking the core ipf genes in figure 3.20, the locations of others are
5 outlined in Appendix 5), suggesting that the nature of the insert DNA itself might make it
6 prone to reorganization and possible reduction of activity. Such a
7 mutagenic/recombinative loss-of-function might actually be selected for given the
8 apparently toxic nature of certain elements of the ipf pathway (See Appendix 4). In order
9 to address this hypothesis, three individual E. coli epi300 pFOS3G7 clones were cultured
10 separately and their activity levels compared to one another.
11
12 Methods
13
14 E. coli epi300 pFOS3G7 was streaked from the culture collection. Three
15 individual colonies were inoculated into LB, grown overnight, and 100 ppm ibuprofen
16 with 2mM arabinose. Ibuprofen remaining at four days was measured via HPLC.
4000
3000
2000
1000 ipf area peak
0 culture 1 culture 2 culture 3 17
18 Figure A2.6. Comparison of remaining ibuprofen in E. coli pFOS3G7 cultures descended 19 from different colonies.
227
1 Results
2
3 Culture 2 degraded significantly more ibuprofen than the other two as determined by
4 unpaired students t-test (Figure A2.6).
5
6 Conclusions
7 The results demonstrate the erratic behavior of the pFOS3G7 system. In this case,
8 it appears that two of three cultures develop lower activity levels than would be expected
9 in accordance with the hypothesis.
10
11 Complete system shutdown
12
13 In November of 2010, E. coli epi300 pFOS3G7 and all derivatives nearly
14 completely ceased to function in any capacity. Virtually every aspect of the system was
15 varied in hopes of identifying the culprit for the across-the-board loss-of-function
16 including;
17 • Ultra-pure mili-Q water versus distilled water
18 • Stocks of every media component including sodium chloride, tryptone, arabinose,
19 chloramphenicol, yeast extract
20 • Culture vessel material; glass versus plastic
21 • Culture temperature was tested and verified to be 37 degrees centigrade
22 • Assuring that cultures were as freshly out of freezer stocks as possible
228
1 Despite aggressive testing, the cause of the shut-down was not identified, bringing the
2 project to a temporary halt.
3
229
1 REFERENCES 2 3 Epicentre CopyControl™ Fosmid Library Production Kit with pCC1FOS™ Vector.
4
5
230
1 APPENDIX 3
2
3 IDENTITY AND INITIAL CHARACTERIZATIONS OF OTHER GENES OF
4 INTEREST IN SPHINGOMONAS IBU-2 FOSMID LIBRARY CLONES
5
6 Abstract
7
8 Additional sequencing of pFOS3G7 and pFOS4F6, a clone that is capable of
9 ibuprofen metabolism but does not accumulate catechols, revealed several genes
10 whose sequence identities suggested possible involvement in the ipf pathway in
11 some capacity. Herein, in silico analyses of the the putative gene products are
12 presented along with initial inquiries into what role they may play in ibuprofen
13 metabolism. The gene names should be regarded as tentative since in no case was
14 the role of the gene or its requirement for metabolism or transport substantiated.
15 Briefly, elements of an aromatic dioxygenase complex, both the reductase (IpfH)
16 and the ferredoxin (IpfI) were identified. Knockout of ipfH slowed substrate
17 disappearance. The two genes were cloned into E. coli epi300 pJ25( ipfABDEF )
18 but failed to complete the pathway. An aromatic 1,2-dioldehydrogense gene ipfL
19 was knocked out with no consequence on pFOS3G7. A putative regulatory
20 element ipfR was identified and initial HPLC data suggests that it plays an
21 inhibitory role. Two other elements of interest, an enoyl-CoA hydratase ( ipfY )
22 and an acyl-CoA racemase ( ipfT ) were identified via sequence analysis, though
23 initial data suggests that they do not play an essential role in the ipf pathway.
231
1 Interestingly, ipfT shares remarkable sequence similarity with human alpha-
2 methylacyl-CoA racemase, an enzyme that performs a fortuitous chiral inversion
3 of ibuprofen.
4
5 Ferredoxin Reductase ipfH
6 Please see Chapter 3 for a description of this ORF.
7
A B C ipfI ipfH ipfL
8
9 Figure A3.1. ORFs found on the SeqMan assembly contig containing ferredoxin reductase 10 ipfH, ferrdoxin ipfI, and diol-dehydrodgenase ipfL. Black arrows represent sequences with 11 high similarity to conserved transposases. Regions A, B, and C have similarities to 4- 12 hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase, and 4-oxalocrotonate 13 isomerase. 14
15
16 Plant-like ferredoxin ipfI
17
18 Near ipfH, a small ORF was located via random sequencing (Figure A3.1). No
19 transposon mutant with an insertion in this small 333bp ORF was found. Conserved
20 domain analysis of the translated ORF located a 2Fe-2S ferredoxin conserved domain;
21 alignment with stereotypical members of the group revealed that each of the conserved
22 iron-coordinating cysteines is present (Figure A3.2).
232
1
2 Figure A3.2. Alignment between ferredoxin reductase IpfH (“query”) and several other 3 ferredoxin reductases found in the NCBI database showing the four conserved iron- 4 coordinating cysteine residues indicated by “#”. 5
6 Addition of the electron transport genes to sublconed ipfABDEF (pJ25)
7
8 **Important Note** The remainder of this section on ipfHI was created before the
9 modification of the methodology that allowed for strong conversion of substrates to
10 corresponding catechols by pJ25 pGEM: ipfHI .
11
12 Consideration of the ipfH knockout data, the proximity of ipfI , and the sequence
13 characteristics of both led to the hypothesis that the two gene products are the electron
14 transport chain of the dioxygenase IpfAB. The lack of the presence of these two genes
15 on pJ25 might explain the very poor catechol generation displayed by pJ25-expressing
16 clones. To address this hypothesis, ipfH and ipfI were targeted for cloning. In order to
17 place both genes on the same plasmid despite their distance from one another (it was not
18 known at the time that they were so closely located to one another), a PCR strand overlap
233
1 extension (SOE) strategy was employed. SOE employs PCR primers with additional 5’
2 homology overlaps between the two PCR products that allows for artifical joining of the
3 two PCR products. An initial round of PCR using a 50C anneal and 1.5 minute extension
4 with a pFOS3G7 template and the SOE primers (Table A3.1) was successful (Figure
5 A3.3)
6 Table A3.1. Primers designed for use in the creation of novel Ibu-2 fosmid library 7 constructs. Italicized nucleotides indicate the manufactured overlap sequence for use in the 8 strand overlap extension combination of ipfI and ipfH , while the lower-case nucleotides 9 indicate sequence that is specific for the pKD4-borne kanamycin resistance insertion 10 cassette used in the creation of gene-replacement mutants via the lambda red system. primer name primer sequence ipfFeDox F sew TAC CGC CGA GCA GGA ATA TTA CAG CCG CGA CA ipfFeDox R sew 2 GAA GCG GGT ACA TTC CTC CAG ACG GTC CTC ipfFeDoxRed F sew GGA GGA ATG TAC CCG CTT C AC GCA CAC AAT CTA ipfFeDoxRed R sew TTT CAC CGC AGG CCT ATG CCG C ipfLlambdaF ACGGCGCGCCTGAGAGGCCGAGATCCAGCGCTCCTT gtgtaggctggagctgcttc ipfLlambdaR ACGGAAGAGGGTGGGCGCCTTGGACGCCCCCTCAGA 11 atgggaattagccatggtcc
12
234
1 2 Figure A3.3. The FedoxSOE project. The sizes of the ipfI and ipfH amplicons are shown 3 above, while the successful strand overlap extension product is shown on the lower right. 4 5 The products, which were of the predicted size, were gel purified and used as template
6 for three cycles of primer-less PCR under the same conditions. IpfFeDox F sew and
7 ipfFeDoxRed R sew were then added and PCR performed under the same conditions,
8 yielding a combined product of the predicted size (Figure A3.3). The SOE product was t-
9 cloned into pGEMt-easy, sequence verified, and transformed into E. coli epi300 pJ25.
10 The sequence was searched in silico for the presence of any E. coli –recognizable
11 transcriptional terminators, of which none were present.
12 While E. coli epi300 pJ25 pGEM: ipfHI did turn a slightly darker color when
13 induced with IPTG (Figure A3.4), no detectable catechols were generated as determined
14 by ferric chloride assay and HPLC analysis (method described in Chapter 2); the dark
15 color was likely due to high expression of the ferredoxin or to interaction of the electron
16 transport proteins with the ferric chloride present in the diagnostic media.
235
1 ipfHI ipfHI ipfHI ipfHI ipfHI ipfHI pJ25 pJ25 pJ25 pGEM: pJ25 pGEM: pJ25 pGEM: pJ25 pGEM: pJ25 pGEM: pJ25 pGEM:
2
3 Figure A3.4. E. coli epi300 pJ25, E. coli epi300 pJ25 pGEM: ipfHI , and E. coli JM109 4 pGEM: ipfHI , all with IPTG induction, grown in LB with 500 ppm ibuprofen and 1.5mM 5 ferric chloride. The slightly dark color that appeared in the pJ25 pGEM: HI culture (poorly 6 visible in this photograph) did not pellet in the same manner as the catecholic polymers 7 produced by E. coli epi300 pFOS3G7 (Figure 3.18). pGEM: ipfHI expression in JM109 led 8 to a dark coloration in the culture media and in deceased cell material (indicated by arrow). 9
10 HPLC analysis for accumulation of 2-hydroxyphenylacetic acid, presumably a
11 byproduct of the abiotic degradation of the 1,2-diol product of dioxygenase activity,
12 showed that E. coli pJ25 pGEM: ipfHI accumulated the 2-hydroxy to a higher extent when
13 cultured with 1mM IPTG (Figure A3.5) and that the phenylacetate substrate concurrently
14 disappeared at a faster rate. However, given that the pBBR1mcs plasmid that is the
15 backbone of pJ25 and the pGEMt-easy plasmid that is the backbone of the pGEM: ipfHI
16 plasmid both have a lacZ promoter, it is not clear which aspect of the pathway the IPTG
17 was inducing. However, this data strongly suggests that the dioxygenase system is
18 functioning in E. coli pJ25 pGEM: ipfHI , especially given that induction of the cloned
19 elements speeds the production of this hydroxylated byproduct. However, this data was
236
1 poorly reproducible; in subsequent assays the pJ25 pGEM: ipfHI construct behaved
2 differently until the methodological adjustments described in Chapter 3, namely using a
3 higher concentration of chloramphenicol and delaying addition of substrate until 24 hours
4 of growth.
5
2.5
2
1.5
PAA lost
mM 2OHPAA gained
1
0.5
0 pGEM:ipfHI J25HI J25HI IPTG 6
7 Figure A3.5. 2-hydroxyphenylacetete accumulation and phenylacetate disappearance by 8 two E. coli JM109 pGEM: ipfHI , E. coli epi300 pJ25, and E. coli epi300 pJ25 pGEM :ipfHI . 9 Effect of lacZ induction on the metabolic behavior of J25F was examined by addition of 10 1mM IPTG. All data points are significantly different from one another as determined by 11 t-test (p<0.05) 12
13 In later assays performed a year later using the same strains and techniques
14 described above but with the addition of a vectorless E. coli epi300 negative control
15 yielded different results. E. coli epi300 pJ25 pGEM: ipfHI cultures degraded 1.4mM of
237
1 phenylacetate and accumulated 0.7mM (+/- 0.05mM) catechol as detected by HPLC
2 analysis of two day old cultures (Figure A3.6) and dark color generation by two weeks
3 (Figure A3.7) while the other strains did not.
4
catechol phenylacetate
5
6 Figure A3.6. HPLC of E. coli epi300 pJ25 pGEM: ipfHI grown in LB with 5mM 7 phenylacetate for two days. Strains with single or no vector accumulated no detectable 8 catechol. This culture went on to turn dark brown as seen in Figure A3.7.
238 ipfHI ipfHI pJ25 Negative control pJ25 pJ25 pGEM: pGEM:
1
2 Figure A3.7. E. coli pJ25 pGEM: ipfHI after two weeks incubation in LB media with 5mM 3 phenylacetate compared to E. coli with only one or neither of the plasmids. 4
5 Ultimately, this strong catechol generation was reproduced by using four times as
6 much chloramphenicol and by spiking stationary phase cultures with substrate instead of
7 growing cultures in the presence of substrate from inoculation. In addition to the
8 phenylacetate and ibuprofen metabolism presented in Chapter 3, E. coli epi300 also
9 metabolized m-tolylacetate and 2-phenylacetate to 3-methylcatechol and catechol
10 respectively. For direct visualization of catechols, ferric choride was added to 150ul of
11 culture to a final concentration of 1.5mM in 96-well plate format. HPLC was used to
12 directly quantify substrate and catechols. 40:60 methanol:40mM acetic acid running
13 buffer was used for separation of phenylacetate (10.6 minutes), catechol (4.3 minutes),
14 and 3-methylcatechol (7.8 minutes). 50:50 methanol:40mM acetic acid was used to
15 separate m-tolylacetate (11.8 minutes) and 2-phenylpropionate (11 minutes). A detection
16 wavelength of 220nm was used for the aromatic acids while a detection wavelength of
17 280nm was used for the catechols.
239
1 The activity of E. coli epi300 harboring both pJ25 and pGEM: ipfHI towards m-
2 tolylacetate and 2-phenylpropionate was consistent with the results presented in Chapter
3 2 (Figure A3.8). The dual vector system degraded approximately 0.55 mM of the m-
4 tolylacetate added and accumulated 0.18 mM of 3-methylcatechol (Figure A3.9). The
5 dual vector system also degraded 0.62 mM 2-phenylpropionate and accumulate 0.19 mM
6 catechol (Figure A3.10).
2PPA mTAA pJ25 + pGEM:HI
pJ25
pGEM:HI
Vectorless control
1 75 .5 .2 .1 0 0. 0 0 0 Catechol standard
7 curve (mM)
8 Figure A3.8. 150ul of E. coli epi300 harboring the indicated vector exposed to either 2- 9 phenylpropionate (2PPA) or m-tolylacetate (mTAA). 1.5 mM ferric chloride has been 10 added as a catechol indicator. A standard curve of catechol with 1.5 mM ferric chloride is 11 presented for reference. 12
240
1 0.9 0.8 0.7 0.6 mtaa 0.5 mM 3mcat 0.4 0.3 0.2 0.1 0 pJ25 + pHI pJ25 pHI negative 1
2 Figure A3.9. m-tolylacetate and 3-methylcatechol concentration in E coli epi300 cultures 3 harboring pJ25 and/or pGEM: ipfHI (pHI) following 18 hours of incubation 1 mM 4 ibuprofen. 5 6
1.2
1
0.8
2ppa 0.6 mM cat
0.4
0.2
0 pJ25 + pHI pJ25 pHI negative 7
8 Figure A3.10. 2-phenypropionate and catechol concentration in E coli epi300 cultures 9 harboring pJ25 and/or pGEM: ipfHI (pHI) following 18 hours of incubation 1 mM 10 ibuprofen. 11 12 13
241
1 Additional Genes of Interest
2
3 The early unreliability of the E. coli pJ25 pGEM: ipfHI system suggested that a
4 further gene product might be required for efficient function of the ipf pathway. In order
5 to address this now defunct hypothesis, available pFOS3G7 and pFOS4F6 sequence
6 information was scoured for possible genes of interest, of which four, detailed below,
7 were identified. The characteristics of these putative genes and attempts to create and/or
8 characterize knockouts of each are detailed below.
9
10 Diol-dehydrogenase ipfL
11
12 Aromatic dioxygenase pathways often employ a diol-dehydrogenase step
13 following the initial dioxygenation. While the putative ipf pathway does not allow for the
14 involvement of a classical diol-pdehydrogenase, the possibility remains that a modified
15 protein serves a similar function in Ibu-2. Indeed, adjacent to ipfH, an ORF was
16 identified with high similiarity to x ylL, a diol-dehydrogenase that acts on the 1,2-diol of
17 benzoate (Figure A3.11). The existing data regarding the activity of E. coli pJ25
18 pGEM: ipfHI suggested that there might be an additional required protein at the later
19 stages of the pathway.
20
242
HOOC OH OH H
+ NAD XylL + NADH + H + CO 2
OH OH
1
2 Figure A3.11. General reaction performed by the diol-dehydrogenase XylL. 3
4 As no existing sequenced transposon mutants had insertions in this small 432bp
5 ORF, the lambda red system (Chapter 3) was used to create an ipfL knockout in
6 pFOS3G7. pKD4 ipfL primers and a primer flaking the ipfL ORF (for verifying
7 successful insertion) were designed (Table 3.7) and incorporation into pFOS3G7 was
8 successful (Figure A3.6), the ipfLlambdaF and ipfLR primer set yielding a 2.2 kb product
9 indicative of successful incorporation of the pKD4 kanamycin cassette. Screening with
10 two flanking ipfL gene primers was consistent with gene replacement. However,
11 screening the insertion clones on diagnostic plates (LB with 100 ppm p-toluidine,
12 100ppm ibuprofen, and 1.5mM ferric chloride, Figure A3.12) clearly showed that the
13 pathway was still intact. Liquid culture assays with proper control strains clearly
14 confirmed this result (data not shown).
243
1
2 Figure A3.12. Dark catecholic accumulation by E. coli epi300 pFOS3G7 with ipfL replaced 3 by the pKD4 insertion cassette (confirmed by PCR in Figure A3.13). The media consists of 4 LB with 100 ppm ibuprofen and 1.5mM ferric chloride. 5
6
7 Figure A3.13. PCR screening of several E. coli epi300 pFOS3G7 ipfL pKD4 insertion 8 cassette mutants using primers that flank the ipfL gene showing successful gene 9 replacement; ipfL is 432 bp while the insertion cassette is approximately 1.6kb. 10
11 The presence of a xylL analog directly adjacent to a gene strongly suspected to be
12 part of the ipf pathway in addition to the data leading to the conclusion that an additional
13 late-upper-pathway gene is required for efficient metabolism proved too good to be true.
14 Knocking out ipfL did not lead to loss of function to any degree. However, the possibility
15 remains that there is another copy of ipfL present on pFOS3G7 in a different genetic
16 context that the flanking primers might not be able to detect nor the pKD4 ipfL primers
17 able to knock out (given that they target flanking DNA for recombination). The still
244
1 leaves open the possibility that such a gene is required for metabolism. This hypothesis
2 can be tested by cloning ipfL into E. coli pJ25 pGEM: ipfHI in order to see if the pathway
3 is completed; given that in E. coli pJ25 pGEM: ipfHI , both pBBR1 and pGEM plasmid
4 systems are present, the pOFX low copy number expression system may prove useful.
5
6 AAA+ ATPase ipfR
7
8 Approximatly 3.5kb downstream of ipfF a AAA+ ATPase domain was located.
9 The sequence data in this region was of fairly low quality, prohibiting identification of
10 the full ORF. The identified ORF at present is represented by approximately 1kb. The
11 identity with the conserved AAA+ ATPase domain via BLAST conserved domain search
12 was quite confident (E-value = 3x10 -31 ). The AAA+ ATPase domain is a molecular
13 motor that couples ATP hydrolysis to mechanical motion. This domain is found in all
14 domains of life in a very wide variety of proteins, often those with proteolytic or DNA
15 regulatory roles (Hanson and Whiteheart 2005). While the ipfR full ORF was not
16 identified, the ready availability of a transposon mutant with an insertion in ipfR coupled
17 with its possible regulatory role made an initial screening of mutant activity fairly simple.
18 pFOS4F6 is one of the Ibu-2 pCC1 chromosomal library clones capable of
19 metabolizing ibuprofen and phenyylacetic acid all the way to the corresponding meta-
20 cleavage products. A transposon mutant A3 had already been created by random
21 transposon mutagenesis. E. coli epi300 pFOS4F6TnA3 was subjected to metabolic
22 analysis alongside E. coli epi300 pFOS4F6, E. coli epi300 pFOS3G7, and negative
23 control epi300. Because E. coli epi300 pFOS4F6 does not accumulate catechols, visual
245
1 ferric chloride assays were not useful. Phenylacetate disappearance and 2-
2 hydroxyphenylacetate were monitored via HPLC and quantified against standard curves
3 (Figure A3.14).
4 Surprisingly, the HPLC analysis showed that E. coli epi300 pFOS4F6Tn: ipfR
5 actually metabolized more phenylacetate than intact E. coli epi300 pFOS4F6. Whereas
6 E. coli pFOS4F6 has been known for years to be less active towards phenylacetic
7 substrates than E. coli epi300 pFOS3G7, E. coli epi300 pFOS4F6Tn: ipfR actually
8 reached substrate disappearance rates similar to that shown by E. coli epi300 pFOS3G7.
9 It is however important to note that this only represents the first steps of the pathway and
10 that pathway products were not quantified to any degree.
11
246
6
5
4
day 3 3 day 5 mM paa mM 2
1
0 4F6 4F6Tn-ipfR 3G7 4F6TnB1 1
0.3
0.25
0.2
day 3 0.15 day 5
mM 2OHpaa mM 0.1
0.05
0 4F6 4F6Tn-ipfR 3G7 4F6TnB1 2
3 Figure A3.14. the ipfR transposon mutant HPLC assay showing increased phenylacetate 4 disappearance (top) and increased appearance of 2-hydroxyphenylacetate (bottom). Also 5 included in the assay was the ipfY pFOS4F6 transposon insertion mutant pFOS4F6B1. 6
7 In order to make a more detailed statement, resting cell assay with 3-
8 flouorcatecholic meta-cleavage inhibition with measurement of catechol accumulation as
9 described in chapter 2 would be necessary. Strangely, while phenylacetate disappearance
10 in E. coli epi300 pFOS4F6Tn: ipfR was similar to that shown by E. coli epi300 pFOS3G7,
11 accumulation of 2-hydroxyphenylacetate was remarkably higher in the ipfR mutant. This
12 may indicate non-ideal expressional control, the result of which might be that the first
247
1 few steps of the pathway have been released from regulatory control by disruption of the
2 putative regulatory element ipfR resulting in over-production of the diol intermediate at
3 such a rate that more is lost to abiotic dehydration to yield the 2-hydroxy product (Figure
4 A3.14). These results led to the general hypothesis that ipfR serves a regulatory function.
5 Two further mutually exclusive hypotheses derive from this initial obvious one.
6
7 Hypothesis 1
8 Hypothesis 1 is that the higher rate of activity in E. coli epi300 pFOS4F6Tn: ipfR
9 might indicate that E. coli epi300 pFOS3G7 does not have an intact copy of ipfR which
10 might explain the difference in metabolic rates between the two intact fosmid clones.
11 Note that this hypothesis does not take into account the differences in 2-hydroxy
12 byproduct. However, the hypothesis was fairly simple to address by examining existing
13 sequence information and by testing primers that target the existing ipfR ATPase domain
14 gene fragment. Sequence information showed that one end of the pFOS3G7 fosmid
15 backbone appears to end approximately 1000bp downstream of the ATPase ipfR domain
16 making the question as to the presence of an intact gene unresolved (Figure A3.15).
17 AAA+ ATPase domain-containing proteins are of highly variable and totally
18 unpredictable sizes given that the C-terminal of these proteins are extremely divergent
19 and substrate/role-specific.
20
248
pCC1FOS
ipfR
1
2 Figure A3.15. SeqMan (DNAStar inc., Madison, WI) screencap of the fosmid library 3 sequence assembly project showing the proximity of the end of the pFOS3G7 DNA insert to 4 the vector; sequence “3G7 pCC1R yahong.seq” begins at the end of the vector. 5
6 To further address the question, primers flanking the domain were created (Table A3.2)
7 and used on E. coli epi300 pFOS3G7 and E. coli epi300 pFOS4F6 (Figure A3.15). In
8 accordance with the sequence data, E. coli epi300 pFOS3G7 responded to the ipfR
9 domain flanking primers. In order to determine whether pFOS3G7 has an intact ipfR
10 ORF, more sequence data would be required.
11
249
4F6 3G7 T R Y T R Y
1
2 Figure A3.16. PCR of ipfT , ipfR , and ipfY from fosmid pFOS4F6 or pFOS3G7 template. 3
4 Hypothesis 2
5 The second and exclusive hypothesis is that pFOS3G7 encodes an intact IpfR
6 regulatory protein that if knocked out, would lead to more rapid substrate utilization and
7 more rapid byproduct accumulation. To address this hypothesis, the ipfR ATPase domain
8 was targeted for deletion using the lambda red system (primers in Table A3.2).
9 Incorporation of the pKD4 cassette into pFOS3G7 was successful, but PCR results
10 continued to suggest the presence of both replaced gene and intact gene despite several
11 round of extra-copy fosmid curing (Figure A3.17). The presence of intact ipfR ATPase
12 domain suggests that there is an extra copy of ipfR on pFOS3G7.
13
250
1
2 Figure A3.17. PCR of E. coli epi300 pFOS3G7 with ipfR replaced by the pKD4 kanamycin 3 resistance cassette using primers that flank the region of ipfR targeted for deletion. Note 4 that the double bands correspond to both the pKD4 insertion cassette (large band) and 5 intact ipfR (small band). 6
7 Conclusions
8 Initial data suggests that ipfR AAA+ ATPase domain is part of an inhibitory
9 regulation apparatus. In order to address this properly, more sequence information is
10 required so as to more confidently identify the ORF. Next, the gene could be over
11 expressed in one of the fosmid clones in order to examine whether it has an inhibitory
12 effect on metabolic activity.
13
14 Acyl-CoA racemase ipfT and enoyl-CoA hydratase ipfY
15
16 Two more genes with conserved domain identity that suggests an involvement in
17 aromatic metabolism were located in the pFOS4F6 transposon mutant library. A single
18 E. coli pFOS4F6 transposon mutant was identified that had the tranposon insertion in a
19 region dubbed ipfY with high BlastX similarity to enoyl-CoA hydratases (Figure A3.18).
20 Upstream of this region was a second region with an acyl-transferase/racemase domain
21 dubbed ipfT. The sequence quality in this region was very poor, prohibiting confident
251
1 determination of the ORFs. ipfY appears to be very near the end of insert DNA on
2 pFOS4F6 (Figure A3.18).
3
ipfY ipfT
4
5 Figure A3.18. Map of partial ipfY and ipfT ORFs showing the location of the pFOS4F6 6 vector, the location of the pFOS4F6Tn: ipfY transposon isertion, and the targets of the ORF- 7 specific and lambda red pKD4 primers. 8
9 ipfY
10
11 The gene with highest blast-X similarity (30% identity and 86% coverage) to IpfY
12 was 3-hydroxypropionyl-CoA dehydratase (Teufel, Kung et al. 2009), while wide general
13 similarity was found to many general types of enoyl-CoA hydratases and dehydratases.
14 This general gene family is involved in several types of pathways including general fatty
15 acid oxidation and anaerobic aromatic metabolic pathways for cinnamoyl-CoA and
16 chlorobenzoates (Hamed, Batchelar et al. 2008). An initial screening of the pFOS4F6
17 ipfY transposon mutant suggested that general phenylacetate metabolism was unaffected
18 by inactivation of this gene (Figure A3.14). Screening of pFOS3G7 with primers specific
252
1 for an internal region of the gene revealed that it is not present on pFOS3G7 (Figure
2 A3.16), fairly strongly arguing against an essential role in the upper ipf pathway. Indeed,
3 the wide applicability of the enoyl-CoA reaction could reflect the involvement of IpfY
4 farther downstream in the lower pathway of the reaction.
5
6 ipfT
7
8 Upstream of ipfY, the sequence contains a putative ORF with a conserved acyl-
9 CoA transferase/racemase domain. CoA transferases generally catalyze the exchange of
10 a CoA group from one acyl-CoA to another carboxylic acid group, a reaction that is not
11 suspected to be relevant to the ipf pathway. However, the second general function of this
12 conserved domain in the racemation of chiral acyl-CoAs. Most intriguing is its high
13 similarity to alpha-branched acyl-CoA isomerases found in animals with which it shares
14 50-65% identity (see human 2-methylacyl-CoA racemase Q9UHK6.2 ). The methyl-
15 acyl-CoA racemases are responsible for isomerization of bile acids and pristanoyl-CoA in
16 particular (figure 3.8)(Setchell, Heubi et al. 2003) which is particularly interesting given
17 the related putative functions of IpfD and IpfE in the beta oxidatition of alpha-methyl-
18 acyl-CoA compounds. Perhaps most intriguingly, in humans this protein is responsible
19 for the conversion of (R)-ibuprofen-CoA to (S)-ibuprofen-CoA (Reichel, Brugger et al.
20 1997). Sphingomonas Ibu-2 growing on ibuprofen as sole carbon and energy source
21 depletes the R enantiomer more quickly than the S (figure 2.1), suggesting that a similar
22 chiral inversion system may be at work. Indeed, in the metabolism of bile acids and
23 pristanoyl-CoA, the beta-oxidative steps are highly enantiomerically-specific, suggesting
253
1 that such an inversion of ibuprofen-CoA would be necessary when taking into
2 consideration the analogies between the eukaryotic and Ibu-2 pathways.
3 There was no characterized pFOS3G7 or pFOS4F6 ipfT transposon mutant
4 available for screening. PCR screening of pFOS3G7 with internal primers was negative
5 (Figure A3.16), suggesting that pFOS3G7 does not have a copy of ipfT (Figure A3.16).
6 The fact that Sphingomonas Ibu-2 is able to metabolize both isomers of ibuprofen
7 (Chapter 2) suggests that there is an isomerase involved in the pathway. The complete
8 metabolism of ibuprofen by E. coli epi300 pFOS3G7 despite the apparent presence of an
9 invertase is somewhat puzzling; it may suggest that the true ibuprofen-CoA isomerase is
10 present elsewhere on the fosmid or that inversion is either taking place abiotically or is
11 being fortuitously catalyzed by an E. coli protein. Futher sequence analysis as expression
12 will be required so as to more fully determine whether or not ipfT does indeed code for
13 the first known bacterial 2-arylpropionyl-CoA, more specifically ibuprofen-CoA,
14 isomerase. This gene product could be of industrial interest given the fact that the
15 isomers of phenylacetyl-based NSAID drugs display remarkably different properties.
16
17 Conclusions
18
19 While the hypothetical missing gene was not found, several genes of interest were
20 sequenced and characterized to some degree. The failure of the electron transport
21 proteins ipfI and ipfH to complete the pathway along with pJ25 proved somewhat
22 suprising and may indicate that pJ25 does not express one or more of the ipfABDEF
254
1 genes correctly. It may also indicate that ipfIH is not the electron transport chain of the
2 ipf pathway but rather that the genes have yet to be located on pFOS3G7.
3 Initial data seems to indicate the IpfR plays a role in regulation of the ipf
4 metabolic system on pFOS3G7 and pFOS4F6. Further sequencing and subcloning of the
5 gene along with analyzing the different responses to substrate induction on the two
6 fosmids and on ipfR mutants will further elucidate what role IpfR might play. Given that
7 IpfR seems to be a repressor, shown by the fact that when it is disrupted by a transposon
8 insertion, metabolic activity actually increases, clearly indicates that it is not required for
9 metabolism and therefore not the elusive missing gene.
10 IpfT, which has similarity to an isomerase/transferase, may prove to be an
11 ibuprofen isomerase. Further sequencing, subcloning and expression along with ipfF
12 followed by enantiomeric analysis would reveal whether or not this is the case. Such an
13 alpha-branched phenylacetate chiral isomerase would be the first such bacterial protein
14 and could prove to be of commercial and industrial value.
15
16 Periplasmic binding protein IpfG
17
18 pFOS3G7Tn :ipfG was originally identified in the pFOS3G7 transposon library by
19 its slower rate of catechol accumulation. HPLC analysis showed that ibuprofen
20 disappearance was slower than E. coli epi300 pFOS3G7. The ipfG transposon mutant
21 was sequenced using the transposon-specific primers FP1 and RP1 (Table 3.2) and
22 characterized via blastx analysis (Altschul, Madden et al. 1997). and ipfG was
23 hypothesized to code for the periplasmic binding component of an ABC transporter
255
1 system. Ibuprofen disappearance assay using the the methods for E. coli epi300
2 pFOS3G7 transposon mutants described in Chapter 3 revealed that E. coli epi300
3 pFOS3G7Tn :ipfG demonstrates reduced catechol production activity (46% of the activity
4 of E. coli epi300 pFOS3G7, Figure A3.19). Uptake assays were performed on
5 pFOS3G7Tn: ipfG using the methods described in Chapter 4. While for the most part
6 uptake by pFOSTn: ipfG was no different than intact pFOS3G7, uptake by E. coli epi300
7 pFOS3G7Tn :ipfG was actually higher than E. coli epi300 pFOS3G7 at 2.5µM (p<0.05)
8 (Figure A3.20).
9
90%
80%
70%
60%
50%
40%
percent ipf percent disappearance 30%
20%
10%
0% 3G7 3G7Tn:ipfG epi300
10
11 Figure A3.19. Disappearance of ibuprofen in E. coli epi300 pFOS3G7Tn: ipfG cultures 12 compared to intact pFOS3G7 and vectorless epi300. 3G7tn: ipfG showed significantly 13 reduced ibuprofen disappearance as determined by student’s t-test. 14
15
256
100
10
1 TnG 3G7 0.1 TnF
0.01 nmol paa uptake / (mg protein * min) 0.001 0.1 1 10 100 1000 uM phenylacetate 1
2 Figure A3.20. Uptake of radiolabelled phenylacetate by E. coli epi300 harboring pFOS3G7 3 or pFOS3G7 with transposon in ipfG or ipfF. Uptake by pFOS3G7Tn: ipfG was higher than 4 pFOS3G7 at 2.5 uM phenylacetate as indicated by the star (p<0.05). 5
6 The role played by ipfG proved difficult to elucidate. While its interruption via
7 transposon mutagenesis reduced the rate of ibuprofen metabolism, the same ipfG mutant
8 showed no clear difference from the control strain in substrate uptake rate. However, any
9 characterization of ipfG would have wide implications. Paul Black and colleagues in their
10 pioneering work on fatty acid uptake mechanisms encountered a similar gene tsp (Azizan
11 and Black 1994). The putative Tsp protein had similarity to periplasmic binding proteins
12 of ABC transporter systems as well and was also found to adversely impact fatty acid
13 metabolism through random transposon mutagenesis tests. However, the role that tsp
257
1 plays was likewise difficult to characterize and no work regarding it has been published
2 since. It would be informative to confirm its projected localization in the periplasmic
3 space and also to investigate its affinity for the substrate compound, perhaps by purifying
4 large amounts of the protein and testing to see if radiolabelled substrate binds to the
5 protein using western blotting and autoradiography. If localization to the periplasm and
6 direct interaction of the protein with ibuprofen could be established, then the nature of its
7 role in uptake could be infered. Perhaps such proteins are crucial for uptake under certain
8 environmental or physiological states, such as particular substrate concentrations, stress
9 levels, or under particular ionic or pH conditions. Given the occurrence of such proteins
10 in bacteria and in animals, such work could have wide implications from both a purely
11 scientific and an applied point of view
12 The predicted protein product of ipfG has high similarity to a variety of
13 periplasmic binding proteins of ATP-binding cassette (ABC) transport systems. When
14 ipfG was knocked out, metabolism of ibuprofen was reduced. The proximity of a
15 putative ABC periplasmic binding protein to an aromatic acid CoA ligase is somewhat
16 unexpected given that ABC transport systems and vectorial acylation have not been
17 reported to be related to one another to any degree (Busch and Saier 2002). While ABC
18 systems always have a membrane-spanning protein that serves to shuttle the chemical of
19 interest across the membrane and an ATPase that powers the transport (Jones and George
20 2004; Szakács, Váradi et al. 2008) some, but not all ABC systems have periplasmic
21 components (Jones and George 2004). There are very few examples, however, of the
22 reverse: periplasmic binding proteins rarely occur without a cognate ATPase. The actual
23 role played by periplasmic binding proteins is the subject of some debate. In fact, it has
258
1 been demonstrated that ABC systems that have an associated periplasmic binding protein
2 do not require the periplasmic component for transport to function (Holland and Blight
3 1999). It has been suggested that the periplasmic protein merely serves to speed the
4 diffusion of the target chemical across the periplasmic space to the inner membrane-
5 spanning protein (Holland and Blight 1999).
6 This particular experiment was performed in order to determine if the product of
7 ipfG was involved in uptake. ABC transporters are fully dependent upon the hydrolysis
8 of ATP to drive transport. If both ipfF and ipfG were involved in uptake transposon
9 inactivation of ipfF should have helped to reveal the role this putative ABC transport
10 system was playing. The observation that the metabolic poisons, and thus a lack of
11 available ATP or membrane charge, had no effect on uptake by the ipfF mutant suggests
12 that the residual uptake was not dependent upon ATP or a chemiosmotic gradient, and
13 thus was unlikely to be encoded by a standard ABC transporter such as ipfG .
14 In fosmid E. coli epi300 pFOS3G7 from Ibu-2, no ORFs have been located to
15 date that correspond to the other component of an ABC system despite the fact that
16 approximately 6kb of DNA has been sequenced in either direction of ipfG . While ipfF
17 encodes ATP-dependent CoA ligase activity, and is therefore likely an ATPase, the ATP
18 binding cassette of ABC systems is of a very specific tertiary structure that is
19 characteristic of the group. IpfF shares no similarity with the ABC motif. The question
20 stands then as to how ibuprofen is transported into the cell and what role, if any, does
21 ipfG play in transport or metabolism of ibuprofen.
22 The clear inhibition of substrate disappearance (Figure 3.19) suggests a role in the
23 pathway, though the uptake data showed that IpfG is not involved in transport. While
259
1 IpfG does share identity with characterized periplasmic binding proteins of ABC
2 transporter systems, that similarity is low (20-25%). Additionally, Swiss-model was
3 unable to build a three dimensional homology model with any level of confidence.
4 Taken together, the paucity of information regarding IpfG-like proteins leaves wide-open
5 what role it might play in metabolism. Given that there over 100 putative genes with
6 over 40% identity in the NCBI database, none of which has been characterized to any
7 degree, further investigation of the role of IpfG may prove valuable to the scientific
8 community.
9 In the case of bacterial fatty acid metabolism, what happens to the substrate once
10 it reaches the periplasmic space is poorly understood. It seems that the high
11 hydrophobicity would discourage the fatty acid’s movement across the aqueous
12 periplasmic space, a situation exacerbated by the acidic nature of the periplasmic space
13 which would serve to protonate fatty acids, making them even more hydrophobic. A
14 gene named tsp has been shown to facilitate, but not be necessary for fatty acid transport
15 in E. coli (Azizan and Black 1994). The gene product is water soluble and localized to
16 the periplasmic space, but little else is known about it. ipfG is somewhat similar to tsp
17 (21% identity / 34% similarity), with analogous residues distributed across the entire
18 length of the sequences. The protein tsp was shown in one case (Azizan and Black 1994)
19 to speed fatty-acid transport but has not been the subject of any published research since.
20 The most similar protein in the Swiss-prot database was BraC, the periplasmic binding
21 component of a branched-chain amino acid ABC transport system in P. putida (Hoshino
22 and Kose 1990), with which IpfG shares 25% identity and 46% similarity.
260
1 Schuehle et al. (Schuehle, Jahn et al. 2001) described a similar situation to that
2 presented by ipfFG in Ibu-2: in Azoarcus evansii, a 2-aminobenzoate CoA ligase is
3 located adjacent to a putative periplasmic binding protein in the same gene order as that
4 of ipfFG (42% and 39% similarity respectively). The role of the periplasmic binder has
5 not been investigated in this system, but this example suggests that the co-localization of
6 these two types of genes may not be an accident, but rather suggests that the IpfG-like
7 protein might play some enigmatic role in the transport system.
8 In this system IpfG actually seemed to be working against the uptake process at
9 2.5 µM, leading to the possibility that IpfG might actually play a role in excretion at low
10 phenyacetate concentrations (<50 uM). However, these should be regarded as
11 preliminary results the role they play in the transport process is still unclear.
12
261
1 REFERENCES
2 3 4 Altschul, S., T. Madden, et al. (1997). "Gapped BLAST and PSI-BLAST: a new 5 generation of protein database search programs." Nucl Acids Res 25 : 3389 - 3402. 6 Azizan, A. and P. N. Black (1994). "Use of transposon Tn phoA to identify genes for cell 7 envelope proteins of Escherichia coli required for long-chain fatty acid transport: 8 The periplasmic protein tsp potentiates long-chain fatty acid transport." Journal of 9 Bacteriology 176 (21). 10 Busch, W. and J. Saier, M.H. (2002). "The transporter classification (TC) system." 11 Critical Reviews in Biochemistry and Molecular Biology 37 (5): 287-337. 12 Hamed, R., E. Batchelar, et al. (2008). Mechanisms and structures of crotonase 13 superfamily enzymes – How nature controls enolate and oxyanion reactivity. 14 Cellular and Molecular Life Sciences , Birkhäuser Basel. 65: 2507-2527. 15 Hanson, P. I. and S. W. Whiteheart (2005). "AAA+ proteins: have engine, will work." 16 6(7): 519-529. 17 Holland, I. B. and M. A. Blight (1999). "ABC-ATPases, adaptable energy generators 18 guelling transmembrane movement of a variety of molecules in organisms from 19 bacteria to humans." Journal of Molecular Biology 293 : 381-399. 20 Hoshino, T. and K. Kose (1990). "Cloning, nucleotide sequences, and identification of 21 products of the Pseudomonas aeruginosa PAO bra genes, which encode the high- 22 affinity branched-chain amino acid transport system." Journal of Bacteriology 23 172 (10): 5531-5539. 24 Jones, P. M. and A. M. George (2004). The ABC transporter structure and mechanism: 25 perspectives on recent research. Cellular and Molecular Life Sciences, 26 Birkhäuser Basel. 61: 682-699. 27 Reichel, C., R. Brugger, et al. (1997). "Molecular cloning and expression of a 2- 28 arylpropionyl-coenzyme A epimerase: a key enzyme in the inversion metabolism 29 of ibuprofen." Molecular Pharmacology 51 (4): 576-582.
262
1 Schuehle, K., M. Jahn, et al. (2001). "Two similar gene clusters coding for enzymes of a 2 new type of aerobic 2-aminobenzoate (anthranilate) metabolism in the bacterium 3 Azoarcus evansii." Journal of Bacteriology 183 (18): 5268-5278. 4 Setchell, K. D., J. E. Heubi, et al. (2003). "Liver disease caused by failure to racemize 5 Trihydroxycholestanoic acid: gene mutation and effect of bile acid therapy." 6 Gastroenteroloty 124 (1): 217-232. 7 Szakács, G., A. Váradi, et al. (2008). "The role of ABC transporters in drug absorption, 8 distribution, metabolism, excretion and toxicity (ADME-Tox)." Drug Discovery 9 Today 13 (9-10): 379-393. 10 Teufel, R., J. W. Kung, et al. (2009). "3-Hydroxypropionyl-Coenzyme A Dehydratase 11 and Acryloyl-Coenzyme A Reductase, Enzymes of the Autotrophic 3- 12 Hydroxypropionate/4-Hydroxybutyrate Cycle in the Sulfolobales." J. Bacteriol. 13 191 (14): 4572-4581. 14 15
263
1 APPENDIX 4
2
3 ADDITIONAL EXPERIMENTS AND LINES OF INQUIRY
4
5 Preface
6
7 Herein are described several experiments and pilot projects related to the Ibu-2
8 project. The rationales, methods, and results are presented in abbreviated form.
9
10
11 Screening, Metabolic, and Genetic Work done prior to the Fosmid library
12
13 Screening assays
14 Two colorimetric indicators had been shown to be useful in the detection of ipf
15 pathway intermediates; catechol polymerization which is possible to enhance by addition
16 of ferric chloride and p-toluidine and the yellow color consistent with metacleavage
17 product of catechols. Several additional chemicals were investigated for utility as
18 chromogenic reagents indicative of ipf pathway activity or the presence of ipf pathway
19 metabolites.
20 Napthalene dioxygenase acts on indole to create blue indigo (Ensley, Ratzkin et
21 al. 1983 ) while benzoate dioxygenases are able to act on indolecarboxylic acids to
22 produce pigments (Keener, Watwood et al. 2001). Ibu-2 was grown on ibuprofen in the
23 presence of these chemicals (indole, indole-2-carboxylic acid, indole-3-carboxylic acid)
264
1 at two concentrations in the case of the indolecarboxylic acids (150 and 600ppm) with no
2 detectable color change. Pseudomonas HK44, a naphthalene degrader, was used as a
3 positive control for color generation for indole.
4 Next, indoleacetic acid was screened for activity under the hypothesis that the ipf
5 pathway might be able to act upon this aromatic acetic acid. As no such activity has ever
6 been demonstrated, no positive control was available. A variety of indoleacetic acid
7 concentrations were tested (200, 300, 400, 500ppm). No ibuprofen-dependent color
8 change was detected in Ibu-2 cultures.
9 The nitro blue tetrazolium / 2,3,5-triphenyl-2H-tetrazolium chloride (NBT/TTC,
10 20ppm/25ppm) indicator described by Finette et al. (Finette, Subramanian et al. 1984) for
11 the detection of toluene dioxygenase electron transport chain activity was also employed.
12 The NBT/TTC indicator did not discriminate between ibuprofen grown and glucose
13 grown Ibu-2. All colonies turned slightly reddish-brown with no blue color indicative of
14 NBT reduction by a ferredoxin reductase.
15
16 Mineralization assays
17 In order to confirm the mineralization of ibuprofen and to gain insight into the
18 fate of the carboxyl group, 14 C-ibuprofen, labeled only on the carboxyl group, was used
19 in classic mineralization assays. A 5% inoculation of exponential phase Ibu-2 was added
20 to 5ml of 500ppm ibuprofen MSM media in a 40ml conical vial that had been spiked with
21 50,000 dpm of 14 C-ibuprofen. A small vial of 1ml 1M NaOH was placed in each sample.
22 The NaOH vial serves as a CO 2 trap. The NaOH trap was removed and replaced
265
1 periodically. Total dpm per time point was measured by scintillation counter (Figure
2 A4.1).
3
16% 14% 12% 10% Ibu-2 8% (-)control 6% 4% 2% % total radiolabel in NaOH trap 0% 0 10 20 30 40 50 60 Time (hours) 4 5 Figure A4.1. The percent of total radiolabel present in sodium hydroxide trap following 6 growth of Ibu-2 in 500ppm ibuprofen MSM in the presence of 50,000 dpm 14 C-ibuprofen. 7 This experiment was performed in plastic culture tubes. 8 9 The data demonstrated that Ibu-2 was mineralizing ibuprofen, but only 14% of the
10 radiolabel ended up in the NaOH trap, leading to the assumption that the remainder had
11 either been incorporated into biomass or remained in the media in some form, either as
12 unmetabolized substrate or as a waste product. However, measurement of the
13 radioactivity present in the total active culture samples revealed only 5.1% (+/- 2.2%) in
14 the biomass and 1.9% (+/- 0.2%) in the media, leaving a total of 79% of the radiolabel
15 unaccounted for. On the other hand, the negative control media still contained 65% (+/-
16 12%) of the radiolabel. In other words, the active culture was missing 44% of the total
17 radiolabel when compared to the negative control. Two hypotheses for where the
18 missing radiolabel might have gone to, CO 2 that escaped the reaction chamber or a waste
19 product that sorbed to the plastic culture vial, were addressed in a following experiment.
266
1 In order to prevent possible escape of radiolabelled CO 2 via a poorly sealed
2 reaction chamber, the 40ml plastic conical vials were replaced with 40ml glass vials, the
3 lids of which had a soft plastic seal. The use of glass vials instead of plastic also
4 addressed the possibility that some radiolabel was being tied up in a waste product with
5 affinity for the plastic vial walls. Furthermore a chloroform extraction of the glass tubes
6 upon termination of the assay in order to remove possible hydrophobic insoluble or glass-
7 sorbing radiolabel. The amount accumulating in the NaOH trap in the Ibu-2 samples
8 (14.2% +/-3.3%) was almost identical to the previous value (Figure A4.2).
9
20% 18% 16% 14% 12% Ibu-2 10% (-)control 8% 6% 4%
% radiolabel in NaOH trap 2% 0% 0 50 100 150 200 250 Time (hours) 10 11 Figure A4.2. The percent of total radiolabel present in sodium hydroxide trap following 12 growth of Ibu-2 in 500ppm ibuprofen MSM in the presence of 50,000 dpm 14 C-ibuprofen. 13 This experiment was performed in rubber-sealed glass tubes. 14 15 The culture and media of the Ibu-2 samples contained only 11.4% (+/- 6.8%) of
16 the total radioactivity added while the negative control media contained 80% (+/- 0.7%).
17 The choloroform extracts of the culture vials contained no measurable radioactivity,
18 indicating that all of the residual media radiolabel was water soluble. Again, most of the
267
1 radiolabel that was added to the Ibu-2 cultures, 57%, was unaccounted for when
2 compared to the negative control.
3 Both experimental systems resulted in almost identical trapping of radiolabel
4 (14.1% vs. 14.2%), though there were differences in the amount of missing radiolabel
5 versus the negative control; 44% for the plastic vial versus 57% for the glass. One
6 explanation might be that the periodic opening of the vials led to release of radiolabel that
7 had for one reason or another had not partitioned into the NaOH trap. The NaOH trap
8 was tested at termination of the assay to confirm that it was still strongly alkaline, which
9 it was. The remarkable difference in missing radiolabel between the Ibu-2 samples and
10 the negative controls indicates that the loss was biologically mediated in some way;
11 perhaps there was a volatile radiolabelled waste product that did not readily partition into
12 the NaOH trap.
13 It is interesting to note that the negative controls were also missing notable
14 amounts of radiolabel, 35% for the plastic and 20% for the glass, which seems to indicate
15 that there was a consistent leakage or measuring error in at least one point in the system.
16 The loss of radiolabelled ibuprofen from the negative control might be due to
17 volatilization of ibuprofen and loss via the gas phase and is somewhat consistent with the
18 hypothesis that the Ibu-2 cultures were releasing a volatile radiolabelled waste product.
19
20 Investigation of megaplasmids in Sphingomonas Ibu-2
21 Sphingomonas aromaticivorans F199 (Romine, Stillwell et al. 1999) has a 184
22 kilobase megaplasmid that harbors several aromatic catabolic clusters. In order to
23 address the possibility that Ibu-2 harbors the ipf pathway genes on a similar
268
1 megaplasmid, a two tiered approach was employed. Firstly, Ibu-2 total DNA was
2 subjected to pulsed field gel electrophoresis (PFGE), which separates chromosomal DNA
3 from large plasmid DNA. Three to nine possible megaplasmids of indeterminate size
4 were observed (Figure A4.3).
5 Ibu-2 Ibu-2 (-) control (-) control
6 7 Figure A4.3. PFGE of Ibu-2 total DNA (third lane) with two non-megaplasmid containing 8 E. coli controls in the first two lanes. Faint bands possibly representing megaplasmids are 9 outlined. 10
269
1 Each of these bands was cut out, subjected to three freeze thaw cycles, and centrifuged
2 for fifteen minutes at 15,000 G in order to remove them from the agarose matrix. Each
3 sample was then subjected to PCR analysis with the dmpB and dmpC primers (Table
4 A4.2, Figure A4.4).
5 6
7 8 Figure A4.4. Gel electrophoresis of PCR screening reactions of the nine putative 9 megaplasmid gel purifications. “1” employed the largest band as template, “2” the next 10 largest and so forth. “+” is positive control reactions (diluted Ibu-2 PCR products) and “-“ 11 is non-template controls. “c” is dmpC primed reactions while “b” is dmpB primed 12 reactions. 13 14 These results suggested that, assuming that each triad of bands represented a single
15 megaplasmid in three different secondary structures, the largest of the megaplasmids had
16 a dmpC -like catechol metacleavage gene.
17 In order to investigate whether or not the megaplasmids are necessary for growth
18 of Ibu-2 on ibuprofen, Ibu-2 was subjected to plasmid curing via two methods. Firstly a
19 method adapted from Crosa et al. (Crosa and Falkow 1981) in which Ibu-2 was streaked
270
1 on 1/10 LB plates in the presence of a range of acradine orange concentrations (75, 100,
2 125, 150, 175, 200, and 600 ppm). Acradine orange inhibits DNA replication and
3 selectively inhibits the replication of plasmids. Each resulting treatment was still able to
4 grow on ibuprofen as sole carbon and energy source. Second, Ibu-2 was streaked six
5 successive times on 0.2% glycerol MSM plates on the assumption that after so many
6 generations in the absence of selective pressure, any specialized catabolic plasmid would
7 be lost. Ibu-2 remained able to grow on ibuprofen as sole carbon and energy source after
8 this treatment.
9 While it did appear that Ibu-2 carries up to three megaplasmids and that one or
10 two of them have genes associated with aromatic metabolism, neither plasmid curing
11 methods led to loss of ability to grow in ibuprofen.
12
13 Sphingomonas Ibu-2 antibiotic resistances and attempts to create knockouts
14 Ibu-2 was screened for antibiotic resistances by plating on LB solid media with
15 the presence of standard concentrations of ampicillin, kanamycin, tetracycline, and
16 rifampicin. Ibu-2 was able to grow in the presence of ampicillin and rifampicin, but not
17 tetracycline or kanamycin.
18 In order to create a strain of Ibu-2 less prone to contamination during long growth
19 incubations, plasmid pBBR1mcs-3, which carries a tetracycline resistance marker, was
20 cloned in via triparental mating with E. coli S17 pBBR1mcs-3 and E. coli BH101
21 pRK2013, a helper plasmid for the transmission of non-transmissable plasmids (Vallaeys,
22 Albino et al. 1998). Negative selection against the E. coli strains was achieved by plating
271
1 the mating on minimal media. Mating success was confirmed by plasmid purification.
2 Sphingomonas Ibu-2 pBBR1mcs-3 was placed in the culture collection.
3 An attempt was made to construct an Ibu-2 transposon library using the plasposon
4 pTn Mod OTC (Dennis and Zylstra 1998) was unsuccessful as no tetracycline resistant
5 tranposon mutants were recovered from transformation of chemically competent Ibu-2.
6 Additionally, an Ibu-2 dmpC PCR fragment was cloned into the suicide vector
7 pLD55. The resulting vector was maintained in E. coli BW21037 and an attempt to clone
8 into Ibu-2 via triparental mating with E. coli HB101 pRK2013 was unsuccessful.
9
10 Generating an isobutylcatechol standard
11 An attempt to create large quantities of isobutylcatechol, which is not
12 commercially available, was undertaken by incubating E. coli JM109 (DE3) pDTG141
13 (Gibson, Resnick et al. 1995) with isobutylbenzene. JM109 (DE3) pDTG141 has the
14 capacity to deoxygenate naphthalene and indole. The strain was incubated with 50ppm
15 isobutylbenzene in LB media with 100ppm p-toluidine and 1.5mM ferric chloride to
16 detect any accumulating catechols. No catechols were detected.
17 There are also three point mutations in pDTG141 that alter substrate specificity
18 (Gibson, Resnick et al. 1995). These constructs might prove useful for generating
19 isobutylcatechol, but were not screened for activity.
20
21 Early attempts to gain a foothold in the Ibu-2 ibuprofen catabolic genes
22 Prior to the creation of the fosmid library as described in Chapter 3, attempts to
23 identify Sphingomonas Ibu-2 genes involved in the metabolism of ibuprofen revolved
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1 around the use of degenerate primers for the amplification of general aromatic-type
2 genes; the dmpC primers amplify a small conserved fragment semimuconic acid (meta
3 cleavage product) dehydrogenase, while the dmpB primers amplify an internal fragment
4 of catechol 2,3 dioxygenase meta-cleavage genes. Both primer sets yielded fragments of
5 expected size from Ibu-2 (Figure A4.5). Two dmpB amplicons and a dmpC amplicon
6 were cloned into pGEMt-easy. The dmpB amplicon was sequenced and specific primers
7 named ”Ibu2cat23” F/R were designed. Sequences of these fragments were consistent
8 with the putative gene identity. This project was later abandoned in light of the rich data
9 provided by the fosmid library; the amplicons were never compared to Ibu-2 fosmid
10 library sequences.
11
12 13 Figure A4.5 Purified PCR products using the dmpB and dmpC primer sets on Ibu-2. 14 15 16 In order to determine whether the dmpB-like gene was upregulated by, and
17 therefore presumably involved in, ibuprofen metabolism, an RNA dot-blot was
18 performed. RNA from LB and ibuprofen grown Ibu-2 cultures was isolated and spotted
19 onto membranes. The membrane was probed with P 32 -labelled Ibu2cat23 amplicon. The
20 results were somewhat suggestive of Ibu2cat23 up-regulation but were unclear, with only
273
1 one of three triplicate spots of RNA from ibuprofen-grown Ibu-2 showing hybridization
2 (Figure A4.6)
3
4 5 Figure A4.6. RNA dot blot comparing induced and uninduced Ibu-2 probed with ibu2cat23 6 radiolabeled PCR product. 7 8 The primers were not specific enough so as to yield any useful information via
9 sequencing directly from Ibu-2 DNA. An attempt to obtain additional larger fragments
10 by PCR amplification using one dmp primer and a random hexamer was unsuccessful.
11 Because direct sequencing approaches were fruitless, digestion/probing methodologies
12 were employed.
13 Firstly Ibu-2 DNA was digested with a concentration of Sau3AI that
14 methodological development assays had shown would yield an average of 10kb
15 fragments (Figure A4.7). The digested DNA was cloned into pBBR1mcs-3 and cloned
16 into E. coli, resulting in an approximately 5000 clone a chromosomal library. The library
17 was transferred to membranes and probed with a P 32 -labelled Ibu2cat23 amplicon. The
18 probing identified six clones (Figure A4.8). The plasmids from the six dmpC-positive
19 library clones were isolated, but determination of their sizes revealed only 1-2kb insert
274
1 size. PCR with both the Ibu2cat23 and dmpC primer sets revealed that each clone had
2 both genes (Figure A4.9) and sequencing with Ibu2cat23F/dmpCR revealed that the two
3 genes were adjacent to one another in each case (Figure A4.10). A sequencing project
4 was undertaken but put on hold in lieu of fosmid library generation.
5
6 7 Figure A4.7. Dilution restriction enzyme method development. Fraction of one unit of 8 SmaI (shown above the lanes) incubated with Ibu-2 DNA for four hours. 9 10 11
12 13 Figure A4.8. Representative pBBR1mcs-3 Ibu-2 chromosomal library probed with 14 ibu2cat23 radiolabelled PCR product. Positive hybridization is at the bottom of the 15 membrane. 16
275
1 2 Figure A4.9. ibu2cat23 and dmpC primed PCR reactions using the six ibu2cat23 positive 3 pBBR1mcs-3 Ibu-2 DNA chromosomal library clones. Products match the expected sizes. 4 5
6 7 Figure A4.10. PCR products using ibu2cat23F and dmpCR primers on the six ibu2cat23 8 positive chromosomal library clones. The amplicon is approximately 1400bp. 9 10 Second, Sphingomonas Ibu-2 chromosomal DNA was isolated and overdigested
11 with EcoRI, SmaI, and BamHI alone and in combinations, separated by gel
12 electrophoresis, and transferred to a membrane (Figure A4.11a). The membrane was
13 probed with a P 32 -labelled Ibu2cat23 amplicon (Figure A4.11b). A project was
14 undertaken to clone a 2kb EcoRI / SmaI fragment into a pBBR1mcs vector but put on
15 hold for the generation of the fosmid library.
16
276
AB
1 2 Figure A4.11. B; BamHI, E; EcoRI, S; SmaI. A: Overnight overdigestion of Ibu-2 3 chromosomal DNA using the enzymes indicated above the lanes. B: Result of probing with 4 radiolabelled Ibu2cat23 PCR product. 5 6 7 Activity of ibuprofen-induced aerobically grown Ibu-2 under anoxic conditions
8 Given that coenzyme A ligase mediated aromatic acid metabolic pathways have
9 been more commonly associated with anaerobic metabolism, a small experiment to
10 demonstrate the oxygen-requirement of the capacity for aerobically ibuprofen grown
11 Sphingomonas Ibu-2 to degrade ibuprofen was designed and executed.
12 Sealed 10ml glass bottles were filled with 2ml of concentrated washed resting
13 ibuprofen-grown Ibu-2. Nitrogen gas was bubbled through three samples for thirty
14 minutes, and three were left undisturbed. 200ppm of ibuprofen was added from a 1%
15 methanol stock solution and the samples were shaken for two hours. The samples were
16 killed with 0.1% sodium azide and samples prepared for ibuprofen quantification via
17 HPLC.
277
1 During the two hour incubation, the aerobic samples turned yellow in a manner
2 consistent with metabolism and accumulation of meta-cleavage product. In the aerobic
3 samples, 42.4% (+/- 5.8%) of the ibuprofen remained while in the anaerobic samples,
4 98% (+/- 2%) remained. These results are consistent with the oxygen requirement of the
5 ipf catabolon and support the involvement of the putative IpfAB dioxygenase in the
6 pathway. If Ibu-2 does possess the ability to metabolize ibuprofen under anaerobic
7 conditions, which was never tested, it would seem that any such pathway is active only
8 when induced. The presence of an anaerobic pathway is somewhat suggested by the
9 utilization of a coenzyme A ligase (IpfF), which has been hypothesized to be employed
10 particularly in facultative anaerobic bacteria as a universal dual-purpose reaction
11 intermediate in terms of general metabolic strategy (Fuchs 2008).
12 13 14 Constructs and Cell-Free Assays
15
16 Meta-cleavage product degradation assay
17 Ibu-2 has been shown to grow on the methylated phenylacetic acids 4-tolylacetic
18 acid (4TAA), 3-tolylacetic acid (3TAA), and (2-(4-tolyl)propionic acid (24TPA), but not
19 on the non-ring substituted phenylacetic acid (PAA) and 2-phenylpropionic acid (2PPA)
20 (Chapter 2). Additionally, it has been shown that the two non-growth substrates were
21 metabolized via the same catechol intermediate. One possible explanation of the failure
22 of growth would be that the meta cleavage product (mcp) of catechol is not
23 metabolizable. The first step in metabolism of mcp is typically via an NAD+ dependent
278
1 dehydrogenation of the terminal aldehyde group; indeed, the product of this reaction was
2 detected via GC/MS of Ibu-2 culture supernatant (Chapter 2).
3 In order to investigate whether or not such a dehydrogenase was at work, cell-free
4 extract was incubated with fresh mcp with and without the addition of NAD+ and the rate
5 of disappearance of the mcp was measured via kinetic spectophotometric reading.
6 Firstly, mcp was generated by ibuprofen-grown resting 40-fold concentrated Ibu-2
7 preparations by adding 5 mM of either catechol, 3-methylcatechol, or 4-methylcatechol
8 for 30 minutes. The supernatant was removed and the mcp characterized via UV-vis
9 spectral analysis (figure A4.12).
10 catechol
4-methylcatechol 3-methylcatechol
11 12 Figure A4.12. UV-vis spectrum (350-400nm) of the supernatants of Ibu-2 resting cell assays 13 exposed to catechol, 4-methylcatechol, or 3-methylcatechol. 14 15 The mcp preparation was then combined with cell free extract from ibuprofen-grown Ibu-
16 2 with and without the addition of 5mM NAD+. A negative control without addition of
17 cell-free extract was also included. The samples, prepared in triplicate, were immediately
18 placed in a KCjunior plate reader for kinetic measurement of absorbance at 382nm, a
279
1 wavelength at which all three mcps absorb strongly. The disappearance of the mcp over
2 15 minutes was plotted and a slope calculated.
3 4 Table A4.1. The rate of the disappearance of metacleavage product of the indicated 5 chemical when exposed to Ibu-2 cell free extract and NAD+. Rate of dissappearance of mcp, OD382/min. (standard dev.) meta -cleavage product of: extract extract plus NAD+ NAD+ catechol -4.09 (1.11) -25.55 (0.94) -8.2 (0.17) 4-methylcatechol -10.6 (2.3) -21.50 (6.24) -3.32 (0.29) 6 3-methylcatechol -19.68 (3.12) -17.98 (6.84) -1.69 (0.15) 7 8 The disappearances of catechol mcp (5-fold increase, p<0.05) and 4-methylcatechol mcp
9 (2-fold increase, p<0.05) were both strongly enhanced by the addition of NAD+ while the
10 disappearance of the mcp of 3-methylcatechol was not affected by NAD+ addition (table
11 A4.1).
12 While the results did not confirm the hypothesis that Ibu-2 fails to grow on PAA
13 and 2PPA due to a dead-end mcp product of catechol, the results were fully consistent
14 with what is generally known regarding lower pathways and ring substitutions (figure
15 A4.13).
16
280
OH OH OH OH 2,3 diox COOH COOH
+ COOH O NAD NADH
OH OH OH OH 2,3 diox COOH COOH COOH + O NAD NADH
OH OH OH
OH 2,3 diox COOH COOH
H2OC2H5O2 1 O 2 Figure A4.13. Widely distributed bacterial strategy for the metabolism of metacleavage 3 products of catechol (top), 4-methylcatechol (middle), and 3-methylcatechol (bottom) 4 (Murray, Dugoleby et al. 1972). 5 6 In many lower pathways studied to date (Murray, Dugoleby et al. 1972) employ two
7 different partial pathways depending on the ring substitution. Notably, while non-
8 substituted and 4-substituted catechols tend to be dehydrogenated with NAD+ following
9 ring cleavage, 3-substituted catechols are instead subjected to non-NAD+ dependent
10 hydrolysis.
11
12 Catechol 2,3 dioxygenase
13 As described elsewhere in this section, a catechol 2,3 dioxygenase was identified
14 during early degenerate primer PCR on Sphingomonas Ibu-2. During analysis of fosmid
15 transposon libraries as described in Chapter 3, additional sequencing on non loss of
16 function mutants described the DNA sequence upstream of ipfA (figure A4.14).
17 Immediately upstream of ipfA, an ORF with high similarity to catechol 2,3 dioxygenases
18 was identified and cloned into pGEMt-easy. The E. coli JM109 pGEM:Ibu2cat23 clone
19 proved to readily degrade catechol to ring cleavage product (Figure A4.15). The
281
1 Ibu2cat23 ORF has a strong conserved ribosomal binding site seven bases upstream of
2 the putative start codon (Figure A4.16).
3 Partial evidence of at least one other catechol 2,3 dioxygenase gene was found on
4 other locations of pFOS3G7 and pFOS4F6. The sequence information can be found in
5 the GeneQuest sequence assembly projects on the attached data DVD.
6
7 8 Figure A4.14. Location of the putative catechol 2,3 dioxygenase relative to ipfA and ipfB . 9 10
11 12 Figure A4.15. UV-vis spectrum of the supernatant of E. coli JM109 pGEM:Ibu2cat23 with 13 500ppm catechol. Maximum optical density = 377nm. 14 15 16
282
TCGAATGTCGATTATATTATCGAGATTATGACGAGACACACGTTGCTGAGCCTTGAT TCGGTCCATGCTGAAGTGTGGCGAAATCTTCTGTAATGACCAAGTGAAAAATACAAT GAATGCGAGAA CTTCAAA ATGAGTATCAAAAGCTTGGGTTACATGGGATTCGCGGTC AAGGATGTGCCCGCCTGGCGTTCTTTCCTGGCCCAGAAATTGGGCTTGATGGAAGCA GGCGTCACCGATGACGGCGCCTTGTTTCGCATCGATTCGCGCGCCTGGCGGATTGCC GTCCAGCAGGGCGAGGCAGACGATTTGGCCTACGCTGGCTACGAGGTGGCCGATGCA GCGGGTCTGACGCAGATGACAGAAAAACTGCGGCAGGCCGGAATCGAAGTGGTCACG GGCGATGTCGAACTGGCCAAGCGGCGCGGCGTGATGGGCTTGATTTCGTTTGCCGAT CCGTTTGGTCTGCCACTGGAAATTTATTATGGCGCCAGCGAGGTGTTCGAAAAACCG TTCATGCCCGGCGCGGCGGTGTCGGGTTTTCTGACCGGCGAGCAGGGGCTGGGTCAT TTTGTGCGCTGCGTGCCGGATTCGGACAAGGCGCTGGCGTTTTATACCCAGGTGCTC GGCTTCCAGTTGTCGGACGTCATCGACATGAAAATGGGACCGGACGTGACGATTCCG GCGTATTTTCTGCACTGTAACGAACGCCACCACACCCTCGCGATCGCAGCTTTTCCG CTGCCCAAGCGCATTCACCACTTCATGCTCGAAGTCTCGTCGCTCAATGACGTCGGT TTTGCGTTTGACCGGGTTGACGCTGACGGCTTGATCACCTCCACGCTGGGGTGCCAC ACCAATGACCATATGGTGTCGTTCTATGCAGCGACCCCGTCCGGAGTCGAGGTCGAG TACGGCTGGGGCGCCCGCACCGTTGACCGCTCCTGGGTGGTGGCACGGCACGATAGC CCGAGCATGTGGGGCCATAAGTCGGTGCGCAACAAAGCATAA ACAACAAGCTGACCC ACACAGGAGTTTTTGAATGAAGCTTTACTACAGCCCTGGCGCCTGCTCCCTGTCCCC GCACATCGCCTTGCGCGAGGCGGGCCTGAACTTCGAGCTGGTACAAGTCGATCTGAC AT 1 2 Figure A4.16. Sequence of Ibu-2 ORF (shown in bold type) consistent with catechol 2,3 dioxygenase. 3 Putative ribosomal binding site (underlined). 4 5 6 Phenylacetyl-CoA disappearance when incubated with Ibu-2 cell-free extract
7 Partially in the hopes of identifying novel reaction intermediates, ibuprofen grown
8 Ibu-2 cell free extract was incubated with 3mM phenylacetyl-CoA (PAACoA) with the
9 addition of 3mM NADH. For this pilot assay, a no-enzyme treatment was used as a
10 negative control. The general cell-free protocols described in Chapter 4 were employed.
11 PAACoA was monitored via HPLC, which was performed using 84% 30mM phosphate
12 buffer pH 7.4, 7% acetonitrile, 9% methanol running buffer with detection at 254nm.
13 After two hours of incubation, 69% (+/- 1.5%) of the PAACoA was absent from the
14 samples while none had degraded in the no-enzyme control. No PAA was detected via
15 HPLC, indicating that the PAACoA disappearance was not simply due to hydrolysis of
283
1 the PAA-CoA thiol bond. However, no notable novel peaks were detected in this HPLC
2 analysis.
3 While this line of research suggested that cell-free extract has the capacity to
4 degrade PAA-CoA to an unknown product, it was not investigated further. An
5 investigation of cofactor requirement, inducibility, or the utilization of denatured cell-free
6 extract might have led to insights regarding whether the PAA-CoA disappearance was
7 actually being catalyzed by the ipf catabolon.
8
9 Cloning ipfFG
10 Briefly, ipfF and ipfG were cloned adjacently by pGEMt-easy ligation to the PCR
11 amplicon of ipfFF/ipfGR primers. The size and orientation of the construct were
12 confirmed by PCR of the construct (successful amplification with M13R/GR primers). A
13 successful construct was stored in the culture collection.
14
15 Cloning the ferredoxins
16 Two ORFs with similarity to ferredoxin encoding genes were found during the
17 fosmid sequence assembly project. One, a plant-like ferredoxin encoding ORF, was
18 described in Appendix 3. The other was found 3600 bp downstream of ipfF . Cloning
19 primers were generated for both (Table A4.2) and both were cloned into pGEMt-easy
20 (and stored in the culture collection) (Figure A4.17). While directional PCR screening
21 revealed that the insert in both constructs was in the reverse orientation in respect to the
22 lac promoter, the plant-like ferredoxin (ipfI ) turned red, suggesting the presence of a
23 cloned promoter region.
284
1
2 3 Figure A4.17. T7F / gene-specificR primed PCR product of pGEMt-easy ferredoxin 4 constructs. 5 6 Table A4.2. Sequences of primers described in this appendix. primer name sequence dmpBF CGACCTGATCTCCATGACCGA dmpBR TCAGGTCAGCACGGTCA dmpC333F CCGCACGTTTCGACGAGTTCC dmpC890R AGACGATGCCGGCGTTCTTGC ibu2cat23F CCTGATCTCCATGACCGA ibu2cat23R ACGGTCAGGAAACGTTCG ipfFeDox2F AGCAGGAATATTACAGCCGCGACA ipfFeDox2R AGTCCTCAACCTCCTGGTGAAGAT ipfFeDox4F GCGAGATCCCGGATGCGAATTT 7 ipfFeDox4R ACCGGATAGGCACGCGTTATTGAT
8
9
10 In the hope of identifying fosmid transposon library mutants with transposon
11 insertions within the ferredoxin ORFs, the fosmid transposon librariers were screened via
12 PCR using combinations of the tranposon-specific FP1 and RP1 primers with one of the
13 ferredoxin cloning primers. A single positive was found, pFOS3G7TnD2 (Figure
14 A4.18), but sequence analysis of the clone revealed that the insertion was not inside of
15 the ORF.
16
285
1 2 Figure A4.18. PCR screening of the fosmid transposon libraries for mutants with a 3 transposon inside of a ferredoxin-endocing ORF. The single positive result is shown. 4 5 6 Cloning pJ25 into Novosphingomonas aromaticivorans F199
7 E. coli epi300 pJ25 showed very poor ibuprofen and phenylacetic acid
8 metabolism despite having every ipf ORF shown to be necessary for activity in Ibu-2.
9 pJ25 was cloned into the aromatic acid degrader Novosphingomonas aromaticivorans
10 F199 in the hopes that it would provide a missing factor of some sort that would allow for
11 the expected pJ25 activity. pJ25 was cloned into J199 by chemical transformation and
12 incubated with or without 500ppm PAA in the presence of 100ppm p-tolylacetic, 1.5mM
13 ferric chloride, 1mM IPTG, and 10ppm chloramphenicol. After a week of incubation,
14 growth of F199 pJ25 was very poor. It would also prove to grow very poorly in the
15 absence of test substrates. This strain would prove difficult to resurrect from the culture
16 collection. This line of inquiry was dropped.
17
18 ipfF toxicity assay
19 Observations of impaired growth in ipf clones led to the general hypothesis that
20 ipfF has a growth inhibitory effect. Specifically, it was hypothesized that the putative
21 ligase IpfF leads to the accumulation of reactive coenzyme A thioesters which can react
286
1 non-specifically with vulnerable intracellular targets. A simple toxicity assay was
2 designed in order to test this hypothesis. E. coli JM109 with pGEMt-easy with and
3 without the ipfF insert was grown and then exposed to a variety of ibuprofen or 2-(4-
4 tolyl)propionic acid concentrations. The ability of the strains to grow in the presence of
5 the test substrates was quantified by dilution plating. Specifically;
6
7 The strains were grown overnight on LBamp. Eight dilutions of the culture were created,
8 the first at 1/1000x, which was sequentially diluted 1/10x. Three 10ul of each dilution
9 was spotted onto seven different plates. The plates were LB with 150ppm ampicillin
10 with 1mM IPTG with;
11 -nothing
12 -5, 50, or 500 ppm ibuprofen
13 -5, 50, or 500 ppm 2-(4-tolyl)acetic acid
14 All compounds were added to the LB prior to pouring the plates. The fifth quadrants
15 were counted because they had sufficient colony numbers for statistical testing while
16 offering sufficient colony distinction so as to allow clear counting.
17
18 The presence of ipfF in the proper orientation was screened for via PCR using
19 ipfF774F / T7 primers (figure A4.19).
20
287
ladder kb 1 pgGEMipfF JM109 pGEM JM109
1 2 Figure A4.19. PCR amplification of JM109 with the indicated plasmid using the 3 T7F/Ipf774F primers. 4 5
50
40
30 pgem ipfF 20 pgem microliter) 10 (1x10^6 viable cells / / cells viable (1x10^6
colonies in fifth quadrant quadrant fifth in colonies 0 0 5 50 500 ppm 2,p-tolylpropionic acid 6 7 Figure A4.20. Millions of colonies per microliter of overnight JM109 harboring the 8 indicated plasmid incoculated onto LB with the indicated 2-(4-tolyl)propionic acid 9 concentration. Significant inhibition of growth as determined by t-test is indicated by a 10 hashed bar. 11
288
60 50 40 pgem ipfF 30 pgem 20 microliter) 10
(1x10^6 viable cells / cells viable (1x10^6 0
colonies in fifth quadrant quadrant in fifth colonies 0 5 50 500 ppm ipf 1 2 Figure A4.21. Millions of colonies per microliter of overnight JM109 harboring the 3 indicated plasmid incoculated onto LB with the indicated ibuprofen concentration. 4 Significant inhibition of growth as determined by t-test is indicated by a hashed bar. 5 6 The results clearly show a reduction in viability of the ipfF-expressing strain
7 when exposed to any 2-(4-tolyl)propionic acid concentration (A4.20) or 50ppm ipf
8 (A4.21). High variability in the pGEM: ipfF control (LBamp) spotting likely masked
9 significant differences in the 5 and 500 ipf plates. There was no apparent dose-
10 dependency.
11 These results seemed to suggest that about half of the ipfF-expressing cells were
12 unable to grow when exposed to the supposed toxin. It seems more likely that either
13 growth is slowed by the stressor or that most cells are unable to grow without a mutation.
14 Neither of these reasonable hypotheses were supported by these results. One explanation
15 that is in line with the results might be that the observed data is simply due to what stage
16 of growth each individual cell was at first exposed to the toxin. For example, a young
17 cell might be unable to survive the initial exposure while a mature cell could.
18 Regardless, this data was inconsistent with the hypothesis that ipfF is generally
19 toxic whether or not substrate is present. However, this assay did not address growth
20 rate, which could still be severely impacted by ipfF expression.
289
1 Environmental Work
2
3 Spiking sewage sludge with ibuprofen and 3-fluorocatechol
4 The capacity for 1L activated sewage sludge to metabolize ibuprofen to
5 isobutylcatechol was explored by spiking in 500ppm ibuprofen, incubating for six days,
6 then splitting the sample into two; with and without presence of 0.1% 3-fluorocatechol
7 (3FC). The supernatant analyzed via GC/MS. 50 ppm 3FC is sufficient to inhibit meta-
8 cleavage enzymes in pure cultures. An initial assay was performed with 50 ppm 3FC.
9 However, an HPLC analysis of the supernatant failed to detect any 3FC, leading to the
10 hypthesis that 3FC was being sorbed to the extensive solid suspended phase of the
11 sewage sludge or was being otherwise chemically modified. In order to find a
12 concentration of 3FC that would stay in solution for the duration of the assay, increasing
13 amounts were added until approximately 50ppm was detectable in the supernatant via
14 HPLC. Via this approach, it was found that 0.1% was required. At the termination of the
15 30 minute assay, culture supernatant was recovered by centrifugation, filtered, and
16 subjected to aqueous acetylation and methylation derivitiation.
17 While several novel peaks were found in the 3FC treated sample, none of them
18 corresponded to any previously hypothesized ibuprofen metabolites, neither
19 isobutylcatechol nor 2,3-dihydroxyibuprofen, nor were they recognizable by comparison
20 to the NIST database. However, many of the novel peaks had the two-loss-of-42-mass-
21 units signature of diphenolic hydroxyl groups. All had masses above that which would
22 be expected for an ibuprofen metabolite. Some representative catecholic ion mass
23 fragment patterns included 350/308/266 (21.4 and 21.75 min.), 380/338/296 (21.7 min.),
290
1 366/326/282 (which occurred in four separate GC peaks; 21.8, 21.9, 22.3, and 22.4 min.),
2 and 382/340/298 (20.5 min.). Both samples contained a peak with a fragmentation
3 pattern consistent with the human ibuprofen metabolite carboxyhydrotropic acid
4 (222/191/ 163 /132/104).
5
6 Spiking bacterial fractionated sludge with ibuprofen
7 Taken altogether, the sludge 3FC assay results suggested that the Ibu-2 style
8 metabolic pathway was not overwhelmingly employed by bacteria in the sewage sludge
9 sample but that rather some unidentified ring-hyrdoxylation leading to meta-cleavage was
10 occurring. However, if the apparently high capacity for the sludge to sorb or react with
11 3-fluorcatechol also applies to isobutylcatechol (K ow = 2.997, KOWWIN), then any trace
12 amounts of isobutylcatechol produced would not be expected to be detected. In order to
13 address this hypothesis, a project was designed in which the bacterial fraction would be
14 removed from the sludge in order to decrease the threshold of detection for catechols. A
15 protocol employing acid-washed polyvinylpyrolidone and differential centrifugation was
16 applied to sludge and Cayuga lake water. However, the putative bacterial fractions
17 showed no ibuprofen metabolism and streaks onto LB solid media suggested that very
18 little biomass was recovered from the fractionation protocol, leading to the adjusted
19 experimental design described below
20
21 Isolation of additional ibuprofen-growing organisms
22 Furthermore, twelve additional isolates were streaked from an enrichment culture
23 and subjected to 16S DNA amplification using the 1055F/1492R primer set and
291
1 sequencing with 1055F. BLAST analysis of the colonies revealed that many of the
2 isolates were also Sphingomonads (Table A4.3).
3
4 Table A4.3. Nearest characterized NCBI nucleotide sequence to the 1055F/1492R amplicon 5 of each of the twelve isolates as determined by BLAST analysis. species %identity range of identity NCBI accession
Sphingomonas sp. 83 403 X94099
£¤¥£¦ ¢ Delftia acidovorans 77 304 ¡ Sphingomonas sp. KMG425 90 406 X94099
no similarity, degraded sequence n/a n/a n/a
¦
¨© ¨ © Sphingomonas sp. KMG425 92 56 §
Sphingomonas sp. KMG425 88 403 X94099
¥¥ © ¨ Pandoraea sp. G3307 86 403 ¡ no similarity, degraded sequence n/a n/a n/a
no similarity, degraded sequence n/a n/a n/a
¢¢© ¨¨
Burkholderia sp. MN101 85 402 ¢
¦
¨© ¨ © Alphaproteobacterium S-A(3)-(A) 87 41 § 6 no similarity, degraded sequence n/a n/a n/a 7 8 9
10 Incubation of environmental bacterial fractions with ibuprofen and m-tolylacetic acid
11 Partly because the biomass recovery from bacterial fractionation of environmental
12 samples had proven too low for rapid metabolite generation, bacterial fractions were
13 incubated with ibuprofen and 3-tolylacetic acid (3TAA) for much longer periods of time
14 and were monitored for accumulation of ring cleavage metabolites.
15 Three 1L samples were taken from both the Ithaca municipal sewage treatment
16 plant and from Cayuga lake (at the Stewart Park pontoon bridge) and subjected to
17 bacterial fractionation. The fractions, suspended in MSM, were spiked with 500ppm of
18 either R/S-ibuprofen, S-ibuprofen, or 3TAA and incubated for six days total.
19 Disappearance of substrate was monitored via HPLC. At six days, the sludge sample had
20 degraded 100% of the S-ibuprofen and 3TAA while only degrading 21% of the R/S-
292
1 ibuprofen. After spiking again with 500ppm of substrate and incubating overnight, each
2 sample supernatant contained a slight acid-labile yellow color consistent with meta-
3 cleavage product. The UV-vis spectrum was characterized against an MSM blank, but
4 the color was too weak to determine a definitive λ max (Figure A4.22).
5 S-ibuprofen R/S-ibuprofen
m-tolylacetic acid
6 7 Figure A4.22. UVvis spectra of the supernatants of sludge bacterial fractions exposed to 8 500ppm of the indicated aromatic acid for six days. 9 10 11 The lake bacterial fraction did not degrade ibuprofen, though a slight amount of
12 3TAA was degraded. The supernatant of the 3TAA-exposed sample accumulated an acid
13 labile yellow color which was concentrated by back extraction (acidification, ethyl
14 acetate extraction, and re-extraction with a smaller amount of neutral pH water). The
15 λmax of the concentrated extract was 388nm, while the λ max of the meta-cleavage product
16 of 3-methylcatechol, the metabolite generated by Ibu-2 when it grows on mtaa, is 380nm,
293
1 suggesting that the predominant mcp was not consistent with Ibu-2 type metabolism. The
2 supernatant absorption spectrum showed a fairly wide hump ranging across the
3 wavelengths commonly associated with many other meta-cleavage products though, so
4 the presence of the mcp of 3-methylcatechol cannot be ruled out (Figure A4.23).
5 6 Figure A4.23. UVvis spectra of the supernatants of Cayuga lake bacterial fractions exposed 7 to 500ppm of m-tolylacetic acid for six days. Raw supernatant is shown on the left, while 8 back extracted concentrated supernatant is shown on the right. 9 10 The identities of the meta-cleavage products or catechol substrates was not further
11 investigated, though this experiment demonstrated that environmental samples have the
12 capacity to degrade ibuprofen and 3TAA via metacleavage pathways over a fairly short
13 period of acclimation when compared to traditional enrichments, which frequently go
14 through three cycles of acclimation and incubation with the compound of interest.
15
294
1 REFERENCES
2 3 Crosa, J. H. and S. Falkow (1981). Plasmids. Manual of methods for general 4 bacteriology . P. Gerhardt, R. G. E. Murray, R. N. Costilowet al. Washington, 5 D.C., American Society for Microbiology : 269-270. 6 Dennis, J. J. and G. J. Zylstra (1998). "Plasposons: Modular Self-Cloning Minitransposon 7 Derivatives for Rapid Genetic Analysis of Gram-Negative Bacterial Genomes." 8 Appl. Environ. Microbiol. 64 (7): 2710-2715. 9 Ensley, B., B. Ratzkin, et al. (1983 ). "Expression of naphthalene oxidation genes in 10 Escherichia coli results in the biosynthesis of indigo." Science 222 (4620 ): 167- 11 169 12 Finette, B. A., V. Subramanian, et al. (1984). "Isolation and characterization of 13 Pseudomonas putida PpF1 mutants defective in the toluene dioxygenase enzyme 14 system." J. Bacteriol. 160 (3): 1003-1009. 15 Fuchs, G. (2008). "Anaerobic Metabolism of Aromatic Compounds." Annals of the New 16 York Academy of Sciences 1125 (1): 82-99. 17 Gibson, D., S. Resnick, et al. (1995). "Desaturation, dioxygenation, and 18 monooxygenation reactions catalyzed by naphthalene dioxygenase from 19 Pseudomonas sp. strain 9816-4." J. Bacteriol. 177 (10): 2615-2621. 20 Keener, W. K., M. E. Watwood, et al. (2001). "Use of selective inhibitors and 21 chromogenic substrates to differentiate." Journal of Microbiological Methods 46 : 22 3. 23 Murray, K., C. J. Dugoleby, et al. (1972). "The Metabolism of Benzoate and 24 Methylbenzoates via the meta-Cleavage Pathway by Pseudomonas arvilla mt-2." 25 European Journal of Biochemistry 28 (3): 301-310. 26 Romine, M. F., L. C. Stillwell, et al. (1999). "Complete Sequence of a 184-Kilobase 27 Catabolic Plasmid from Sphingomonas aromaticivorans F199." J. Bacteriol. 28 181 (5): 1585-1602. 29 Vallaeys, T., L. Albino, et al. (1998). "Isolation and characterization of a stable 2,4- 30 dichlorophenoxyacetic acid degrading bacterium, Variovorax paradoxus , using 31 chemostat culture." Biotechnology Letters 20 (11): 1073-1076.
295
1 2 APPENDIX 5 3
4 IBU-2 DNA SEQUENCES AND ADDITIONAL PUTATIVE PROTEIN
5 CHARACTERIZATION
6
7
8 This appendix contains large DNA sequences that were assembled from
9 individual sequencing reaction results using primarily fosmids 3G7 and 4F6 as template.
10 Three contigs that contain the putative genes described in this dissertation are included.
11 The contigs were assembled using SeqMan (DNA Star, Madison, WI); the alignments for
12 each contig were studied by eye for discrepancies and repaired manually. A brief list of
13 putative ORFs and BLAST gene homologies precedes each of the three contigs listed
14 here. Additional sequences and small contigs are contained in an attached data DVD.
15 16 Assembled Sphingomonas Ibu-2 DNA sequences 17 18 19 Contig 1, 11741 bp 20 21 11>526 Transposase 22 840>1718 Catechol 2,3 dioxygenase 23 2074>3378 ipfA 24 3485>4021 ipfB 25 4030>5229 ipfD 26 5242>5640 ipfE 27 5665>7246 ipfF 28 7342>8481 ipfG 29 9337>8543 Transposase 30 9800>10809 Catechol 2,3 dioxygenase, incomplete 31 10818>11150 Ferredoxin 32 11740>11209 AAA+ ATPase 33 34 1 AATTCGGTGA ATGCTGCACC AAAGCTTGGC GCTTTTGTCG GGTATTGAGC CGTGGCAGCT 35 61 TTTTTCCCAA GTGTTTCAGG CTTCGCAAGG GCCTGGCCCA GGAACCGCTT CGTCTTGGCG 36 121 CTGGGGGTCG GCACAGGTAG AAATCGATCG TGTCGCCCCG CTTGTCGACT GCCCGGTACA 37 181 GGTAGGTCCA CTTGCCCCGC ACCTTGACGT AGGTTTCATC CAGGCGCCAG CTCGGATCAA 38 241 AGCCACGCCG CCAGAACCAG CGCAGCCGCT TCTCCATCTC CGGGGCGTAG CACTGGACCC 39 301 AGCGATAGAT CGTCGTATGG TCGACCGAAA TGCCGCGTTC CGCCAGCATT TCCTCAAGGT
296
1 361 CGCGATAGCT GATCGGATAG CGACAATACC AGCGCACCGC CCACAGGATC ACATCACCCT 2 421 GGAAATGGCG CCACTTGAAA TCCGTCATCG TTCCGTCCGT CCAATCTCCG CCAAGCATGC 3 481 TCAAGCTTCA CGATTTTTGC AACAGAGCCC GGCCTGGGAT TCATGAATCT CAGCACGCGG 4 541 TGCGGTGGGG TAGTGCGTGC CATCGGATGG ACCATACTCA TGATGCGTTC TCCTCAGTAC 5 601 CAGAGGCCAA AGGCCCGCCA ACAGGGGCCC GAAACAACGG CCAGCTGCGA CGGGGTACAT 6 661 AATCGCACGG AACTCGACAC CTAGGGTTTT TCCCTATCGT AAAAATGTCG AATGTCGATT 7 721 ATATTATCGA GATTATGACG AGACACACGT TGCTGAGCCT TGATTCGGTC CATGCTGAAG 8 781 TGTGGCGAAA TCTTCTGTAA TGACCAAGTG AAAAATACAA TGAATGCGAG AACTTCAAAA 9 841 TGAGTATCAA AAGCTTGGGT TACATGGGAT TCGCGGTCAA GGATGTGCCC GCCTGGCGTT 10 901 CTTTCCTGGC CCAGAAATTG GGCTTGATGG AAGCAGGCGT CACCGATGAC GGCGCCTTGT 11 961 TTCGCATCGA TTCGCGCGCC TGGCGGATTG CCGTCCAGCA GGGCGAGGCA GACGATTTGG 12 1021 CCTACGCTGG CTACGAGGTG GCCGATGCAG CGGGTCTGAC GCAGATGACA GAAAAACTGC 13 1081 GGCAGGCCGG AATCGAAGTG GTCACGGGCG ATGTCGAACT GGCCAAGCGG CGCGGCGTGA 14 1141 TGGGCTTGAT TTCGTTTGCC GATCCGTTTG GTCTGCCACT GGAAATTTAT TATGGCGCCA 15 1201 GCGAGGTGTT CGAAAAACCG TTCATGCCCG GCGCGGCGGT GTCGGGTTTT CTGACCGGCG 16 1261 AGCAGGGGCT GGGTCATTTT GTGCGCTGCG TGCCGGATTC GGACAAGGCG CTGGCGTTTT 17 1321 ATACCCAGGT GCTCGGCTTC CAGTTGTCGG ACGTCATCGA CATGAAAATG GGACCGGACG 18 1381 TGACGATTCC GGCGTATTTT CTGCACTGTA ACGAACGCCA CCACACCCTC GCGATCGCAG 19 1441 CTTTTCCGCT GCCCAAGCGC ATTCACCACT TCATGCTCGA AGTCTCGTCG CTCAATGACG 20 1501 TCGGTTTTGC GTTTGACCGG GTTGACGCTG ACGGCTTGAT CACCTCCACG CTGGGGTGCC 21 1561 ACACCAATGA CCATATGGTG TCGTTCTATG CAGCGACCCC GTCCGGAGTC GAGGTCGAGT 22 1621 ACGGCTGGGG CGCCCGCACC GTTGACCGCT CCTGGGTGGT GGCACGGCAC GATAGCCCGA 23 1681 GCATGTGGGG CCATAAGTCG GTGCGCAACA AAGCATAAAC AACAAGCTGA CCCACACAGG 24 1741 AGTTTTTGAA TGAAGCTTTA CTACAGCCCT GGCGCCTGCT CCCTGTCCCC GCACATCGCC 25 1801 TTGCGCGAGG CGGGCCTGAA CTTCGAGCTG GTACAAGTCG ATCTGACATG ATTGAAGGTC 26 1861 GCACCGAAGA GCAAAAACGC GCGGTGATCG AAAAGGTCAC GCAGGCGCTG GTTGACGCTG 27 1921 TCGGCGCGCC CAAGGAAAAC GTCAGAGTCT GGATCCAGGA CGTGCCCAAG GAGAACTGGG 28 1981 GCATTGCCGG GGTGAGCGCC AAGGATCTCG GACGCTAGCT GGCGCGCGGC CCGGGAAGGA 29 2041 AATACTTTCA GCGCCAGTTT TTACACTATT TTTATGGAGA ATCAAAATGG CAAGTACACA 30 2101 ACGATCACCA TCACACGCAA GGCTCCTGAC GTTGATGTCG GTGCCTTGAT AGAGCGAGAC 31 2161 CGTATTCACG GCAGCTTGTA CGCGAACGAG TCGATATTCG AGCTCGAAAT GAAGAAAATA 32 2221 TTCTACGACG GCTGGGTTTT CGTTGGTCAC GACTCCGAAG TGCCAACGGC CGGCGAATAC 33 2281 GTGCGCCGCA CGCTGGGCCG AGAAGAGGTC TTGATGGTGC GCCAGCGCGA TAGCTCCATC 34 2341 GCCGTCATCG CCAACCGTTG CGCGCATCGC GGCAACATGA TGTGCATCGC CAACCATGGC 35 2401 AAGGAAAAAT ACTTCACCTG CACCTACCAC GGATGGGTCT ATGATTTGGC CGGAAACCTG 36 2461 AAGGACGTTC CCTATCCCGG CGGGTTTGAC AAGGACAAGT CGGAGCTGAA GCTGCAGCCC 37 2521 TTGCGCACTG AAGTCTATCG TGGCTTCGTG TTCGCCACCT TCAATGCTTC GGCGCCACCC 38 2581 TTGATGGAAC AACTGGGGCG AGGAAAAATC CTGATCGACC GCGCCTGCGA CATGTCGCCC 39 2641 ACCGGTCGCC TTCAGCTCAC GGCGGGCTGG ACCAAGCAGC GTTTCGGTGC CAACTGGAAG 40 2701 ATGCTGCCCG AGAACGACAC GGACGGCTAC CACGTCAACG ACGTGCATGC TTCCTTTGCC 41 2761 CAAGTGATCG ATTCGCACTA TGACAGCGCC GCGATTGCTG CAGAAGACAG CCTGCGCTCC 42 2821 CAAGCCAAGG ATTGGGGTAA CGGGCATACA GAATTGTATC TCTCCCCGAC CTACACCGAG 43 2881 TATCTCAAGT GGTTCAACAC CACGCCGAAT CGTTTTCCGG AGTACATCGC GCAGATGAAG 44 2941 GCCGCCTACG GCGAGGAAAA GGGCGACAAC ATTCTGCGAG ATGGCCCGCC CCACGCCACG 45 3001 ATCTTCCCTA ACCTGTTCCT CGGCGAGATG AACATCATCA TTTTTCTGCC AATCAACGCC 46 3061 CATGAGTGCG TGCAATGGCA CACCCCGATG CTGCTTGAAG GCGCTCCTGA CGAAGTCAAC 47 3121 CAGCGCATTA TCCGCAACTC CGAAGCGGCG ATGGGCCCCT CGGCCTTCCT GCTTGCCGAC 48 3181 GACAGCGTGA TTTCGGAGCG CCAGCAAATC GCCTTGCGCG ACCGTGCCGA CTGGCTGGAC 49 3241 GTGTCGCGCG GTCTGAACCG AGAGCATGTC GATGAGATGG GCGTGGTGGT GGGGCACGTG 50 3301 ACCGACGAGT GTACCAACCG CGGCTTTTGG CAGCACTACA AAAAGGTGAT GACAGCCCCT 51 3361 TCGCCGTCCC CTGTTTGAGC GTATTGATCA CCACCTCATC CGCCCCCCTT CGGGGGCGGC 52 3421 GGATTCACTG AAATTTCGTG TTTAAAAAAG CGTCCTGCGA GCAGGACGCG CAAGGAGATA 53 3481 CATCATGACA AGCATCACAG CCGCTGTCGG CACCAAGCAA AAGACTGACG TGATCGGGCT 54 3541 GGAAGAGCAC CGCGAAGTCT GCGACTTTTT GTACCGAGAG GCGCGCCTGG CTGACGAATC 55 3601 GCGTTACGCC GAATGGGAGG CGTTGGTTGA GGACGACATG ACCTATTGGG TTCCGCGCGG 56 3661 CGAGGGTGAT TACGACATGA ACAAACATGT GTCGATTACC GCGGACAACC GTTCCCGCTT 57 3721 GCGCGTCCGC ATCGCGCAGT TGATGACAGG CAAGCGCCAT GCCCAACTGC CGGTGTCGTC 58 3781 GATGCGGCGG ATCGTCAGCA ACATTGAGGT AGAGCATCAT GCGCAGGGCG GCTACCGCGT 59 3841 ACTGTCGAAC TTTGTGCTTT ACGAATTGCG GCGCTCATCG ACAGGTCAGA TAGAAGTTTG 60 3901 GCCGGGGCGG GTGGAGCACC ATCTGCGCCG CCGAGCGGAT GGCTCGCTGG GCATGTTCTT 61 3961 CAAGAAAGTG GTGCTGATCC ATGGGGACGA GGCTGTTCCC AGTCTGGCAT TCATCATCTG 62 4021 ACGCGCACCA TGGTCACGCG AGTGAAGAAA AAGACCAGGG CCAGAGCCGC GATTGCCGGG 63 4081 CTGGGTTTCA GTGCGATGTC ACGCCAGCCC GTCGGCACGA TCCGCGAGTT GGCGGCCACT
297
1 4141 GCGGTGGCCG CCGCTGCCGC TGATGCGGGT CTGCGCCTGC AAGACATCGA TGGCTTGTTG 2 4201 CTCAACAAAA GCCCGGCAGC AGAGCCTGAA GAGTTGCCCT TGCGTCTGCA AAACGACCTT 3 4261 GGGCTGCGCG ACCTGGGGCT GCTGGCTGCG ATGGATTCCG AGGGCTCGAC AGCGGTGCAG 4 4321 ATGGTGCAGT ATGCAGCGAT GGCGGTGCGT GAAGGACTGG TCAAGTCGGT GGCCTGTGTG 5 4381 TTTGCCGACA CCCCGCTCAA AGGATCGGGG GCAGGTGGGG GTGATGCCTT TGCTCTGGCG 6 4441 ATGCCGCTGA CCGGTGTCGA GGGTTGGGAG GCCCAGCAAG GCTTCCTGGG GGCCACCGCC 7 4501 GCCTATGCGT TGGCCGCGCG CCGTCACATG GCGCTCTATG GCAGCACGGC CGAGCAGCTC 8 4561 GGCGCCTATG CGCTTGCTTG CCGGCAATGG GCGGCGCTGA ACCCGCAGGC CTTCCTGCGC 9 4621 AAGCCGATGA CGATGGACGA CTACCTCGCA TCGCCTTTCG TCGTGGAGCC GTTTCGCGTG 10 4681 TTCGACTGCT GCTTCCCGGT CAACGGCGCT GTCGCGCTCA TCGTCACCTC CGCCGACCGT 11 4741 GCTGTTGATG GGCCTCAGCC GCCCGTTTTC ATCCACGGCA TGGGCCAGGG GCACCGCGGC 12 4801 CGCAGTGGCC TGAGCGGCGA CGATCCAGAG GTCTTCACGG GTGCCATCCA GGCCGGCCAG 13 4861 ACCGCTTACC GTAGTGCCGG GGTGAATGCC AGTGATGTGA CTCAGTGCCA GTTTTACGAC 14 4921 GCTTTTTCTT ACGCCGGCAT CCTGGGTCTG GAGGCATATG GGTTGTGTCC CCGGGGTGAG 15 4981 GGCGGGGCCT TCGTCGCGCA GGGCCACACC GCGCCCGGCG GCAAGTTGCC GGTCAATACC 16 5041 GGTGGTGGGC ACCTGTCGGG CTTTTACCTG CAGGGCATGA CGCCGCTGTC GGAGGCGGTG 17 5101 ATCCAGGCAC GCGGTGCCGG CGGCGCGCGG CAGGTCGTGC GCAACGACTT GATTCTGGTG 18 5161 ACCGGCAATG GTGGCTGCCT TGACTACCAC ACCTGCGTAC TGGTCAGCCC ACACCGCACC 19 5221 CTCGCCTGAC TGGAGCCATT GATGGATAGC ACCTTTTTTC ACCGCTACGA CGAGGCATTT 20 5281 CTCGCATTCA TCGCTGCTGG CGAATTGCGC ATTCCCGTGC ATACCGAAAC CGGCCGGGCG 21 5341 CTGGGTCTGC ATCAGCGTGC CTGGTGTGTC GCTGGAGATC ACGGCGTGCA ATGGAGGCCC 22 5401 GCGTCTGGCC GCGGGGCTGT TCTCTCTTTT ACCGTGACGC GTCGGCCCTA CACGCCCGAA 23 5461 TTTCCCGTGC CTCTGGTGCA TGGATTGATC GAGCTGGCCG AGGGGCCGCG ACTGATATGC 24 5521 CGGCTTGACG GCGTCACGCC CGAGGCGGTC GCCGTCGGTC AGGCGGTCCA GGCGCATTTC 25 5581 GACCGACAAG GTCTGGTGTT CCGACCGGCC CTTGACGACG GCAATAAGGC CGACAAGTGA 26 5641 AACTTAAAAC CAAGGAGACA AGTTATGTTG GCAAGAGACC TGGTTAAGCG CTGTGCGCGT 27 5701 AACTACCCGA CCAAGACCGC CTACCTGTGC GGCGAACGCT CGCGTAGCTG GCGCGAGATG 28 5761 GATCAACGCT CCGACCGGTT CGGCGTGGCC TTGCAACAAC TCGGCCACCG TCCGGGCGAG 29 5821 GCGGTGGCCA TCCTGACGCA AGAAAGCATC GAGGTGTATG AGCACTTCTT TGCCTGCATG 30 5881 AAGATCGCGG CGCCGCGGGT CGGGCTCAAT ACAGGTAGTT GGCCCGAGAT GCTGCACGTG 31 5941 CTGAAGGACA GCGAGGTCAA GTTCCTGCTG TTGGGATACG CGCTGCCGGC ATCTCTCGCC 32 6001 GAGCGGCTGG GCGAGCTCAA GGCACTGGGC ATCACGCTGA TTGGCTATGG TGCAGGCCAT 33 6061 GGTCTGGAGC GCGACTACGA AAGCTTGCTG GCGACAGCCG AGGGTGAGCC GCACTGGCCA 34 6121 GCGTTGGCGC CCGACGACAT CCTTTTTGTC AGCTACACCT CCGGCACGAC CGGTGTACCC 35 6181 AAGGGCGTAA TGCTCACGCA GGAGGGCGGT GTCAACTGCA TCCTCCACTC GCTGATTTCT 36 6241 TTTGGTTTTG GACCTGACGA TGTCTGGTAC ATGCCGGCGG CATCGGCCTG GGTGGTCGTG 37 6301 ATACTGAACG CATTCGGGCT CGGCAACGGG ATGACGACCG TGATTCCGGA CGGCGGATAC 38 6361 CAATTACAGG CTTACCTGCG CGACATCGAG CGTTTTCGCG TTACGGTCGG GATGCTCGTC 39 6421 CCGACCATGC TGCAGCGCGC GATTGTTGAA ATCCAAACCA ATCCAGTTTA CGACCTGTCC 40 6481 TCTCTGCGCA TGGTGGTGTA CGGCTCGTCG CCCGCCACGC CCAAATTGAT CCGTGATGCG 41 6541 AGGGCGACCT TCAAGGGGAT CAAGCTGCTG CAGGCCTACG CGATGACAGA AGCCACCGGC 42 6601 GGCTGGATCA GCTACCTGAC CGATGCCGAC CACGAGCATG CGTTGCGCGA GGAGATCGAG 43 6661 CTGCTCAAGT CGGTCGGCCG CATCGGCATT CACTACGACT GTTCGATTCG CGACGAGTCG 44 6721 GGCCAGCCAG TGCCGATCGG TCAATCCGGC GAGATCTGGC TGCGTGGCAA TACCATGATG 45 6781 AAGGGTTACC GCAACCTGCC CGAGGCTACG GCGGAAGCGA TGCCGGACGG CTGGCTGCGC 46 6841 ACCAACGACA TCGGGCGGCT CGACGAGCGC GGCTATCTGT ATCTGCTGGA TCGGCAGAAG 47 6901 TTCCTGATCA TTACCGGCGC GGTCAATGTG TTTCCAACCA CGGTCGAGGC GATTCTGGTC 48 6961 GAGCACCCGG CGGTGGAGGA GGTGGCCGTG GTCGGCGTGC CGCACCCGGA ATGGGGCGAG 49 7021 GCGGTAGTCG CTGTGGTCGT CCGAAAACCT TCGCACCGCG ATGTGACGGT GCAGGCGCTT 50 7081 ATCGATTTTT GTCACGGCAA GCTCAGTCGC CCCGAAACGC CCAAGCATGT GGTGTTTGTC 51 7141 GATGAACTGC CCAAGACATC CAACGCCAAG CTGAAGAAAG GCGAGTTGAA GAAATGGCTG 52 7201 TCTGGCGGTG CAGTGCCCCT TCCCTGGCAA CTCGAAGTTG CTTGACCTTG TAGTGCCCCG 53 7261 TCAAGCACCT CTAAAAATTC CCCCTGCTTA AAACTTGTAA CCCTAGGAGA CAAAAATGTT 54 7321 GAAACTACGC AACCCCATGA AATGTTTGAT GGCTACCGCC GTCGTGTGTG TGTCCGTCTT 55 7381 GTTGCCGGCG GCAGCTCAAA CCAAGCCGCC GTTGAAAATC GGTGCCTATC TGTCCGTCAC 56 7441 TGGCCCTGCT TCTTATCTGG GCGCGGTGCC ATGAAAACCG TCGAGATGTA CGTCGAAGCC 57 7501 ATCAACGCCG CTGGCGGCAT CGATGGCCGC AAAGTCGAGT TCCAGGGCTA CGACGACGAA 58 7561 TCGGATGCAT CGCGCGCCAA TACGCTGGTC AAGCGTCTGA TCGAGAACGA CAAGGTTCAC 59 7621 GTCATCATTG GCGGCTCTAC GACAGGCGCG ACGATGTCAG CCGTCCCGCT GGTGGAACGG 60 7681 GCCGGTATCG CCATGCTGTC GCTGGCCGGT GGCAGTGTGA TCACCGACCC TGTCAAGAAG 61 7741 TTCGTTTTCA GACTGGCCCA CAACGATGCG ATGGTAGTCA GTCGTCTGTA CGACCACATG 62 7801 CTGGGTCGCG GTATCAAGAG CATCGGCATC ATTGCCGGTA GCGATGCGTT CGGTCGATCC 63 7861 TGTCTCTCAT TCGCGCAGAA ATTGGCACCG GGAAAAGGCG TCAAGATTCT GCGCGATGAG
298
1 7921 TCCTACAACG CCAAGGACAC GGACATGACC GTACAGCTGA CCAAACTGCG ACAGGAACCG 2 7981 GGTTTGCAGG CGATCTTCAA TTGCGGCTTC GGCGAGCCGG CGGCGATTGC CACCAAGAAC 3 8041 TACAAGCAAC TCGGCATCAC CGTGCCGCAC TACGGAACCC ATGCGCTGGC GTCGGACGCT 4 8101 TTCGTCAAAT TGGCGGCCGG CGCGGCGGAG GACATGATCA TGGCCAACGG CCCCATCCTT 5 8161 GTGTGGGATC AATTGCCGGC CAGCGACCCC CAGCAACCGG TGGTGCAGTC CTATGTGAAG 6 8221 GCCTACCGCG CCAAATTCAA CGAAGCCCCT TCCTTCATTG CGGGTATTGC ACATGACTCG 7 8281 TTCTTCGCCA TCCGGGAGGC GGTGCGGCGC AGTGGCAGTA TCGAGCCCGC CAAAATTCGC 8 8341 GATGCCATCG AAAAAGGCAA CGCCTTTGTG GGTGTGACAG GGCATTTCCG GATGATGAAC 9 8401 GAGACCGACC ACATTGGCTT GACCCCCGAC TCCTTGCGGA TCGTACAGGT CAAGAATGGT 10 8461 CGATGGAAAC TCGTTGAATA AGTGTAATGG CTCTGTTGCA AAGATTGGCG GCAGTCAGAG 11 8521 GTAGGCTGTC GCTCTGCGCC GATCAGGCGG CTGCTGCGAA ATGGTGGTTG AGCATGCCCA 12 8581 TGGCCTCCGT CAGCGCCGAG GGCCCAATGC CAAAAGCTCT CTCCACAAGG CGCACCTCGC 13 8641 CCCTGATGCC GGGCTGCAGG CACCAGGGGC GAGCCTGTCC TTTGCGCAGG GCTCGCATGA 14 8701 CTTCGAATCC CTTGATCGTG GCATAGGCCG TGGGGATCGA TTTGAAACCG CGCACCGGCT 15 8761 TGATCAGTAT CTTGAGCTTT CCGTGATCGG CCTCGATCAC GTTATTGAGA TACTTCACCT 16 8821 GCCGGTGGGC CGTCTCCCGG TCCAGCTTTC CTTCGCGCTT CAATTCGGTG ATCGCTGCAC 17 8881 CATAGCTCGG CGCTTTGTCG GTATTGAGCG TGGCAGGCTT TTCCCAGTGC TTCAGGCCTC 18 8941 GCAGGGCCTT GCCCAGGAAC CGCTTCGCTG CCTTGGCGCT GCGGGTCGGC GACAGGTAGA 19 9001 AATCGATCGT GTCGCCCCGC TTGTCGACTG CCCGGTACAG GTAGGTCCAC TTGCCCCGCA 20 9061 CCTTGACGTA GGTTTCATCC AGGCGCCAGC TCGGATCAAA GCCACGCCGC CAGAACCAGC 21 9121 GCAGCCGCTT CTCCATCTCC GGGGCGTAGC ACTGGACCCA GCGATAGATC GTCGTATGGT 22 9181 CGACCGAAAT GCCGCGTTCC GCCAGCATTT CCTCAAGGTC GCGATAGCTG ATCGGATAGC 23 9241 GACAATACCA GCGCACCGCC CACAGGATCA CATCACCCTG GAAATGGCGC CACTTGAAAT 24 9301 CCGTCATCGT TCCGTCCGTC CAATCTCCGC CAAGCATGCT CAAGCTTCAC GATTTTTGCA 25 9361 ACAGAGCCAT AATTGGTACG AACGCCGTTC ACCGAGACGG TCTTGCCGAT CTCGGGGATC 26 9421 GTAGCTTGTT TTTCCATTAC TGCGTCCGAC ATGGAGATGA CAGGAATTGA CCTACAAGGG 27 9481 CGCGCCACCA GGAGGTCGTC TCACGGAATT CGCCGGAAAG GACCGGCTGT GCGCTCTTGT 28 9541 TTTAGTTGCA CATAGAGGAA CTCCTTACAA GGCGAATCTC GCCGTTCAGG CCCTCGAACA 29 9601 CGTCGATAAT CAGCGCTTGC GTGCTTCGCA AGCGCCGAGC CTGAAGCCGC GTTTCGACAT 30 9661 TTTTCCCGAT CTGGCTGATC TACGCTGTCG TTCAACCACT CCAAAAGCGG TTCGGGCCGG 31 9721 GGCGTTATCA GGCGAATCCC GTGCAAACCG TTGTCCTGAT GCGGTTGCGG AGGTGGTCGA 32 9781 GTTGGACTAA GGCGCGGGGG GCGGGACCGG GCTGGGGTGC CGGGCTTGAA GTAGAGACAA 33 9841 GAGAGAGGAT GAGCGCGATG TCGCAAGTGA CCGAGCTGGG TTATATCGGT CTGTCCATAT 34 9901 CGACGTTGAG GCGTGGAAGG ATTATGCGGC GTCGATCGTC GGCATGGAGA TCGTCGACGA 35 9961 GGGTGAGGGC GACCGCATTT ATCTGCGGAT GGACAAGTGG CATCACCGCC TCACGCTGCA 36 10021 CATCGATGGC GGCGACGACC TGGCCTATAT CGGCTGGCGG TTGCGGGTCC GTCCGAGTTC 37 10081 AACGAGCTGG TCGAGAAGCT GCGGCTCAAC AACCTCGACA TTCGCGTGGC GTCGTAGGAG 38 10141 GAATGCGCCG AGCGGCGTGT TCTGGGCCTG GCCAAGACGG TCGATCCGGG CGGCAACCCG 39 10201 ACCGAGATCT TCTATGGTCC GCTGGTCGAC AACTGGCGTG CGTTCCACCC GGGGCGGCCG 40 10261 ATGTTCGGCA AGTTCATGAC CGCCAAGGAA GGCATCGGCC ACTGAATCTT GCGCCAGGAC 41 10321 GACATTCCTG CCGCGGTGCG CTTCTACGAG ATGCTCGGCC TCACCGGCTC GGTCGAGTAC 42 10381 AAGCTTCCGC TTCCCGGCGG CATGGTGGCG CAGCCTTATT TCATGCACGT CAACGGGCGC 43 10441 CAACATTCGG TGGCGTTCGG CGCTTGGGCC GATGAAGAAG CGGATCAAGA TGCTCGAATA 44 10501 TACCGATCTC GACGATCTGG GGGTGGCGCA CGACATCATC CGCCAGCGCA AGATCGACGT 45 10561 TGCGCTGCAG CTGGGCAAGC ATTCGACCGA CGAGGCTTTG ACCTTCTACT GCCAACCTCG 46 10621 GCGCTGGAGC TGGGCTGGGT GCCAAGAAGG CGGACAACCA GCAGGAATAT TACAGCCGCG 47 10681 ACATTTTCGG CCACGGCAAC GAGGCCGCCG GCTACGGGAT GGACATCGAG CTCTAACCGA 48 10741 GCGTTTGAAG TCTTGGGCGA TGGACGCCGG GGTCTTGATG ATCCCGGCGT CCCAGCGACG 49 10801 ATTGCGAGAA GGATGATATG AGCGAGCTGA TTCGCCTGTG CCGAGTGGAT GAGGTGAAGG 50 10861 AGGGCGAGCC GGTGGCGGCG CATGTCGCCG GGCTGCCGCC GTTTGCGGTC TATGATGTCG 51 10921 GGGGCACTTA TTATGTGACC GACAACATCT GCACGCACGG CAATGCGATG CTGACCGACG 52 10981 GCTATCAGGA CGGCGGCACG ATCGAATGCC CGTTCCATGG CGGCGCGTTC GACATTGCGA 53 11041 GCGGGGCGGC GACGGTGTTC CCGTGCCAGA TCCCGCTCAA GACCTATAGT GTCGAGGTCG 54 11101 ACGACGGCTG GATCGCGATC CGGCTGGCAT CGCCCGAAGC GGGCGCCTGA AATGGGAGCT 55 11161 GAGATGGGGG CACGGACGGG GTTCGAGGAA GCTGGGGCGC GCGATCTTCA CCAGGAGGTT 56 11221 GAGGACTGGT GGCGCGCCAT GCCGAGCTGA TCACGAGGAC CGTCTGGAGG AATGGCCCGA 57 11281 GCTGTTCACC CAGGAGTGCG TCTACAAGAT CATCGCGCGC GAGAATGTCG ACCGCGGCCT 58 11341 GCCGATCGCG GCGATCTTCT GCGACAGCCG CAAGATGCTG GTGTGAACCG ACAATTTAAG 59 11401 CTGCAATCTG CTTTGAACGC GGAATGCTGC CAACCGACAG ATTATGCTGC AGCGGACGAG 60 11461 ACTTCACACG CACCGTGATT GCCAGCCCGG GCGCAATTCT CATGATGGTT TTGAAGTCCC 61 11521 GGCCACCAGC TGCTCCAGCG CCTGCACCAG AGGCATTGCC GTGATGCCGT GCGGCTTCGG 62 11581 GTAGGCCATT TCTCCGGGGT AAATCAGCCA GCGCTTCTCG ATCTCGAGAT CGTCACAGGC 63 11641 AAGGTGAAAC CCGCGCTCAA CGCTGGGCGC CGTCGACCGC TTCACTTCGA TGGCCACTTC
299
1 11701 GGGAACGCCG CCGGACACGA GGATGAGGTC TATCTCCGCA C 2 3 4 Contig2, 8229 bp 5 6 450>1274 4-hydroxy-2-oxovalerate aldolase 7 1231>2028 4-oxalocrotonate decarboxylase 8 2032>2244 4-oxalocrotonate isomerase 9 2226>2558 Plant-like ferredoxin (ipfI ) 10 3636>2593 Transposase 11 3798>5052 Ferredoxin reductase (ipfH ) 12 5533>5102 xylL -like dehydrogenase ( ipfL ) 13 5561>6502 Transposase 14 7044>8229 Transposase 15 16 1 TTCGGATCGA ACAACCCGGT CAAGATCCCC GGTTATGGNG AGTTCGCGGG GGCTCAAGAC 17 61 GAGCGTCTTC CTNGAGGTCG AAGGAGCGGG CGACTATCTC CCCAAATATG CGGGCAATCT 18 121 NGACATCATG ACCGCGGCCG CCAAGGCCGC CGGCGAGATG CTGGCGGCCC GCATGCTCAA 19 181 TCGGAAGGCT GCAGCATGAC GCTCTATCCC ACCCAGACCA AGCTCTACAT CCAGGACGTG 20 241 ACCTTGCGTG ACGGCATGCA CGCGATCCTG CACAATTATG GCACCGAAAG TGTCCGCACG 21 301 ATCGCCAAGG CGCTCGACGA TGCCGGTGTC GACGCGATCG AAGTGTCGCA TGGCGACGGG 22 361 CTCAACGGCA GCTCGTTCAA CTATGGCTTC GGCGCGCACT TAAATTGCAC TGAAATCTAG 23 421 AAATATTTTA TCTGATTAAT AAGATGATCC CCGGGTACCG AGCTCGAATT CATCGATGAT 24 481 GGTTGAGATG TGTATAAGAG ACAGCTTATG CGCTCGGCGT GCGCTCGGTG CGCGTCGCGA 25 541 CGCACTGCAC CGAAGCCGAC GTCGGCAAGC AGCATATCGG CATCGCGCGC GATCTCGGCA 26 601 TGGACGTGTC GGGCTTCCTG ATGATGAGCC ACATGCTCGA GCCCGAGGCG CTCGCCCAGC 27 661 AGGCGTTGCT GATGGAAAGC TATGGCGCGC ATTGCGTCTA TGTGACCGAC AGCGGCGGTG 28 721 CGCTCGACAT GGATGGCGTC CGCGCCCGCC TGCAGGCCTA TGACCGGGTG CTCAAGCCCG 29 781 AGACGCAGCG CGGCATGCAC GCCCACCACA ATCTCTCGCT CGGCGTCGCC AACTCGATCG 30 841 TTGCAGCGCA GGAAGGTGCG ATCCGCATCG ACGCCAGCCT CGCTGGCATG GGCGCGGGCG 31 901 CCGGCAATGC GCCGCTCGAG GTGTTCATCG CCGCGATCGA CCGCAAGGGC TGGAAGCACG 32 961 GCTGCGATGT GATGGCGCTG ATGGACGCCG CCGAGGATCT GGTCCGGCCG CTTCAGGATC 33 1021 GCCCAGTTCC GCGTCGACCG CGAGACCCTG AGCCTGGGTT ATGCGGGCGW MTAYTCGWCG 34 1081 WTCMTGCRCC ACGCCGAAAA GGCCGCCGAA CAATACGGAC TCGACACACG CGAAATCCTG 35 1141 GTCGAACTTG GCCGGCGCCG GATGGTCGGC GGACAGGAAG ACATGATCGT CGACGTCGCT 36 1201 CTCGATATCC TGAGCGCGCG CAAGGGGTGA GACCGAGCCC AAGGGGGAAA TGGCCGCGTG 37 1261 ACTTCGCTTG CTGAATATGC CGAGATTCTC GACCGTGCCG CGCACGAAGC GCATGCCACG 38 1321 CCTCAGATCA CCCACAGCAA TGACAAGCTG ACCGTCGCCG ACGCTTATGC GATCCAAAAG 39 1381 CTGTCGGTCG AGCGCCGCCT GGCGCGCGGC GAGAAGCGGA TCGGCGTCAA GATGGGCCTC 40 1441 ACCAGCCGCG CCAAGATGCA GCAGGTCGGG GTCGACGAGG TCGCCTGGGG GCGGCTCACC 41 1501 GACGCGATGC TGCTCGAGGA GGGTGCGGCG CTGTCGCTGT CGCGCTTCGT CCATCCGCGG 42 1561 ATCGAGCCGG AGATCGCCTT TCTGATGAAG GCGCCGCTCG CGGGCAAGGT CACGGCGGCG 43 1621 CAGGCGCTCG CCTGCGTCGA GGCCGTCGCC CCAGCGATGG AAGTGATCGA CAGCCGCTTC 44 1681 GAGAATTTCA AGTTCGCGTT GGTCGACGTG GTCGCCGACA ACACCTCGTC TTCGGGCCTG 45 1741 GTGGTCGGCG GCTGGGGCGA TCCGATGCAG GATCTCTCGA ACCTCGGCGT GATCCTGGAA 46 1801 ATCAACGGCG AGGTCGTNGA GGTCGGCTCG ACTGCCGCGA TCCTCGGCCA TCCGTTGCGC 47 1861 TCGCTGGTCG CCGCCGCGCG GTTGATCGGC GAGGCGGGCG AGACGATCAA CGCCGGCGAC 48 1921 ATCGTCATGG CCGGCGGCAT CACGGCGGCG CCGACCCTCA AGGCCGGCCA GACCATCCGC 49 1981 AACACCGTCC AGAACCTGGG ATCGGTCAGC ATCACGGTGG AAGCATAAAT GCCGATCATG 50 2041 GAAGTCACTT TGGTCGAGGG CCGCACGACC GAGAACAAAA AGGCGTTCAT CCTCGCGCTG 51 2101 ACCGATGCCG CGGAGCAGTC GCTCGGCGTG CCTCGCAGTC ATCCGCGTGR TCCTGCGCGA 52 2161 GATCCCGGAT GCGAATTTCG CGGYGGCGGG CGTGWCYTTC GCCGCGCGCA AAACCAATAC 53 2221 TTTGAAAGCG GCAAGCTGAC CACGATGATC AAGCATCAAG TCCAGATCGT CGATGGCAGC 54 2281 CGCTTCGAAT GCCCCAAGGA GGAACGGGTC CTGATCGCGA TGGAGCGCTT GGGGCTGAAT 55 2341 GACATTTTCG TCGGCTGCCG CGGCGGCGGG TGCGGCGTGT GCAAGGTGAA GGTGACCAGC 56 2401 GGAAATTATC GCACCGGTAA AATGAGCAGG CTACAAGTGA GCGAGGCGGA AGAGGCGGGG 57 2461 GGTTATGCGC TCGCCTGCAA GCTGTTCCCG CTCGACAATC TCGTCATCGA ATTGCCCAAC 58 2521 TAAAAACTTT TTGTCAGAAG ACGGAAAATG CAGCCTAAGC AATCTCGACG ACCGCAAGAT 59 2581 CGGCATGTTC GATCAATAAC GCGTGCCTAT CCGGTTAACG CAGACACCCA CTACATGTGG 60 2641 GGTGCGCCCA GCCACCGGAA TATAGCTCGG CGTAGGTCGA GCTTTCACCT CGCCCGCAAT 61 2701 TCCGAGGAGC GAGCGGAAGG CGTTGAACGG GTAGAACCGT CTGTTGAAGC GGAACGTGAA 62 2761 CTCGTTGAGG TAGGCCTGCA AATGCTTTGC GCTCACGCCG TGGTGGATGC CGTTGAGCCA 63 2821 AGTCTTCAGG TTGCTGAAGA CGAGGTGGAT GATCGGCATG AACTCCTCGG CCACCTCGGG
300
1 2881 GTCGCCACAT TCGGCGATTG CGTGGTGGTC ATAGCCGCGT GTCCGCAGGT TGCCATAGCC 2 2941 GCTCCAATCG TCGGTGACGA TCAGCGACCC CGGCATGACG GCACTCCCTA CGAAGCCGCA 3 3001 CAGCGAATCG GCGCTGCGGT CGGCGGCGAT AGACAGCCGC ACCCGACCGG CAAAGCGGCC 4 3061 ATCCTTACGC TTGTCCTGGG CTGTACCCGG CTCCCGGTGG CGGACTTCGA CGGCGGCAGC 5 3121 CACCAGCGTC TTATGATGAA CACCCCGGCC TTCGCCGCGT GTCTTTCCAC CGACATAGGT 6 3181 TTTCGTCCAC CTCGACATGA CCGTCCGTCT TGCCGCCGAT CCGGTCCTGC TCGGGCCGTA 7 3241 CCATGCCCGC GCGGAGCTTG TGCAGGATTC CGAAGGCGGT TTCGTACCGT GTCAGCCCAA 8 3301 GCTGCTGGAA CTGAACCGCC GACATGCCTG CGTCTGGCTG GCGACCAGAT AGGCTGCCCA 9 3361 GAACCATGTG CTCAAGGGCG TGTGGCTGCG TTCCATGACC GTGCCGACGG TCAATCCCGT 10 3421 CTGGCGGCGG CAAGCGCGAC AGGTCAGGAC GCCGGGCCGC GTAGCGATGC GGAAAGGCTC 11 3481 ACCCACAATG CCACAATGCG GGCAGGCGAA CCCGTCACCC CAGCGAATCC TTTCCAAGTA 12 3541 GGCGGCGCAG GCCCCATCGT CGGGAAAAAG CCGCTGAAAC TCGGGGAGCG ATTGCGGGAA 13 3601 GGGCAGATCG GTGCGGCCGA GAACGTCCAT AACCCACTAC CCGCTTCACG CACACAATCT 14 3661 ATCCCTAGTG GGTGTATGCG TCAACCGGAT AGGCACGCAA TAACGATCGT CACGAAAAGC 15 3721 AGAGGAAAGT TAGGCCAATC CCGGTTCAAT CAAACCGGGA TTGAGTCTAA GTTCGGTTCG 16 3781 AAGGGTACGG AGGCTAATTT ATGATCGCTT CGGTTGTGAT TGTCGGCGCC AACCTTGCAG 17 3841 GGGGACGCGC SGCCGAAGCG CTCCGGCTCA ACGGCTATGA AGGACGGATC GTCCTGATCG 18 3901 GGGAGGAGCG GTGGCTGCCC TATGAGCGGC CGCCGCTCTC CAAGGAATGC CTGTGGGGAC 19 3961 AGGGACCAGC TTCCCGAGAA TTTCTTCCTC CACGACCAGC AATGGTATGA AGACAATAAG 20 4021 ATAGAGCTGG AGCTCGGCGT GCGCGCCGAA RCACTGGAGC TTTCCGGCCG AGGGGTTCGG 21 4081 CTGGCCTCCG GCAAGGAAAT CCCGGCCGAT CGCATCCTGC TCGCGACCGG CGGCAAGGCG 22 4141 CGCTTGTTGC CGCTCGATGG TGCGACCGCC GCCAATGTCC ATCATTTGCG GACCAAGGAT 23 4201 GACGCCGACC GGCTCGCGGC AGATCTCAAG CCGGGTGCGC GTATCGTCGT CATCGGCATG 24 4261 GGCGTGATCG GCGCTGAGGT CGCCGCGAGC GCGCGCAAGA GCGGCTGTGA GGTCACCGCG 25 4321 ATCGASVCGG CGCCGGTGCC GATGATCCGC ACGCTCGGTG CGCATTTTGG CGCCTGGCTC 26 4381 GGCCGCGAGC ACGACATKCG GGGGGTGAAG GCGCGTTACG GCATCGGCGT GACCAGGCTG 27 4441 TATCTTGACG GCGGCCTGGT CCGCACCGTC GAGCTCGACG ATGGCACGCG CATCGATTGC 28 4501 GACGCGGTGG TGGTCGGGAT CGGCATCGTG CCCTCGACCG AGCTTGCCGC CAACGCCGGG 29 4561 CTAGCGATCG GCAACGGCAT CGTCGTCGAT CGCCAGGGGC GCACCAGCCA TGAGGCGGTG 30 4621 TTCGCGGCGG GCGACGTCNC CGACCAGCCC AATTTCTTCG GCGGCCGGGT GCGGCTCGAA 31 4681 ACCTATCAGA ATGCCGCCGA TCAGGGCATG GCCGCGGCGC AGGCGATGAT CGGCCGCGAG 32 4741 GTCGATTATC TGAAGCCCTG CTGGTTCTGG TCCGACCAAT ATGACATCAA CATCCAGGTC 33 4801 TCCGGCCGGA TCGACGACAG CCTGCCGGTG GTGATGCGCG GCGAGCTCGA CAGCAGCCAG 34 4861 TTCACCGCTT TCTTCCTCGA CGGCAACGTC GTCGCCGGCG TGCTGACCGC AAATCGCGCG 35 4921 GTCGACATGG GCKTCGGTSA AGAGGATGGT CGAGCGTCGG CTCGAGGTCG ACCCGGGTCA 36 4981 GCTCGGCGAC GCCTCGATTC CGCTCCGCGA GTTCTTGAAG CCCAAGGCGC GGGCGGCATA 37 5041 GGCCTGCGGT GAAACCCTCT CCCGTACGGA AGAGGGTGGG CGCCTTGGAC GCCCCCTCAG 38 5101 ATCAGCCTAT CCCGCCGCCT GCCACGTACA GCGTCTGGCC GGTGATGTAG GACGCCTCGG 39 5161 GCGAGGCGAA AAAGCAGATC GCGGCGGCCA ATTCTTCCGG CGCCGCGAAG CGCTTCATCA 40 5221 AGGTGTCGCG CAACGTCTGC TCGCCGACTT CGCCGAAACC CTTGATGTCG GCCTCGGTCG 41 5281 GCGGCTTGGG ATTGCGCGGC GTGACCCGCT CGACATTGAC TCCGCCNGGG AGCGACGCAA 42 5341 TTGACCCGGA TCGGCCGGTC GTCGARGTCG AACGACAGCG CCTTGGGTAA GCGCCGACAC 43 5401 TCCGCCTTTG GCCGCGGCAT AGGGGACCCG GTTGATGCCG CGCGTTGCGA CCGGGCCGAT 44 5461 GTTTACGATC GCGCCGCTGT TCGCTTCGAG CATCAGCGGC AGCACGGCAT GGCAGCACCA 45 5521 CATTGTCGGC CATAAGGAGC GCTGGATCTC GGCCTCTCAG GCGCGCCGTA TGAAGGATCT 46 5581 GGAGAAGGAG AATCTGCGGC TTGGCCGGGC GATATCCAAC CTGACGCTGG ATAAGCTCAT 47 5641 TCTCCAGGAG GCCGCCCGGG GAAACTTCTG AGCCCCGCGC GGCGACGGCG CTGTATCGAC 48 5701 CAGCTGTGGC GAGAGCTACC GGTGTCCGAA CGACGGATTT GCCAGGTGCT CGGACAGCAT 49 5761 CGGTCGACCC ATCGCAAGGT AGCGCGCGGG GCGGACGACA AGCAGGCGCT GACCGAGGAC 50 5821 ATCATCGCTT TGGCCAAGCA ATATGGTCGC TATGGCTATC GACGTGTCAC CGCCCTGCTG 51 5881 CGCGACGCGG GGTGGACAGT GAACCGCAAG CGGGTCGAGC GGATCTGGCG ACGCGAGGGG 52 5941 CTCAAGGTGC CGCAGCGGCA GCCGAAGCGC GGGCGGCTGT GGCTCAACGA TGGCTCCTGT 53 6001 ATCAGGCTCC GGCCGGAATA TCCGGGCCAT GTGTGGGCCT ATGACTTCGT TCAGGGCCGC 54 6061 ACTCACGACG GGCGCAAGTT CCGGATCCTG ACCATTATCG ATGGGGCCAG CCGGGAATGC 55 6121 CTGGCGCTCA TCGTCGCCAG GAAGCTACGT CACGAGGACG TGCTGGCCGC GTTGGCCGAG 56 6181 TTGTTCGTCA TGCGCGGGCC TCCGGCACAC ATCAGGTCCG CTAATGGTCC TGAATTTATC 57 6241 GCTACGGCCG TCCAGAAGTG GCTCGGCAAG ATCGGCGTGA AGACGCTCTA CATCGCGCCG 58 6301 GGATCACCTT GGGAGAATGG CTACAATGAG AGCTTCAACG GGTCGCTGCG CGACGAGCTG 59 6361 CTCAACGGCG AGATCTTCTA TCCTCCGAGC GCTCCCAGCT GATATAACCT TCATGCGAGC 60 6421 CGGGAATCAG CGCCAACCAC TCATCCCGGG CCTTGCGACG ACTCCTCGTC CGCAGGGCGG 61 6481 CGCCATCATA TCCAGCTACA ACGTGGGTTT TACCATAGGC ATACAGGATC GCCTTGCGCT 62 6541 CAAGATGCTG GGGACCGATC TTCTCACAGA TCATCGCTCT TCTCCTCCCG TGCCGCGCAG 63 6601 GGGGCGCCGG TGGCGTGGTC GAGCAGTAGA CGCACGATCA GGGGCATCAA CTTCAGCCCG
301
1 6661 CGAAGGCCGT TCTTCGCCCC TACGTCGGCG AACAGCTTGT CGAGTTCCCG CGCGATGTAG 2 6721 GGCACGTGCG CGAAACGCGC ACCGCCCTTG GCGGTGTCGA CGGTGCGGTC CTGGCCAGCC 3 6781 CATTCGTAGA GGTCTTGGAA GAGATGCTTA TGCAGGGCAC GGTAGCCGTC GGCCGTTGCC 4 6841 GGAAAGGTCA GGCGCGCGGC TTCCGCGCCG CGCGCCTGAG TAAGTCGCCG CTCGGCCTCT 5 6901 GTGAGCGTCT TATCATCGGT GATCCCAAGC CGGTTCCGAA GCGTGTCGGT GCCGGGATAG 6 6961 GTATAAGGCC CAGTCGGCCC CAAGCTGCGA TTTTGGCCAA GTCGGAGAAC GTTTTGGGAA 7 7021 CGTGGCATTC GGTATTGACG ATGGAAATCA TAGAAGAGGA CAGTTCGCTC CATCATAGGG 8 7081 AGTGGGTTGA TGGATCGGAG TATTCCGGGC GGGGATTTGA TCGGACGATG GAGCCTGAGC 9 7141 TTTGCCGATA TTGATTTTGT AAATTCGAAG CCGGCCCTGA CGCGCCTTGG CCTCGCCGCG 10 7201 CAGCTGAAAT TCTTCGCTTC CCTGGGGTTT TTCGCGATCG ATCCCGGCTC AATCCCTACC 11 7261 GATGGCCTCT CGTATCTGGC CGAGCAACTC GGTGTCGAGG CTGGCGAGAT AGCCGGTTAT 12 7321 GACTTTTCCA GTCGGACAGC ACGACGGCAT TGTGCGGAGA TCCTGATCCA TCTTGGATAT 13 7381 CACCGCATGA AGCGGGTGGA TCGCGCGCAA TTGACGGAAT GGATTGCTGG CGAGCTGTGC 14 7441 CCGGGCGGCC AGTCGATCAA TGCCATGCTT GAGCATGTTT TCCTGTGGTG CCGGGACCGG 15 7501 CGTATTTATG GGCCGTCGCG CAAGGAGCTT GAACGTGTCG TTCGCTCACA ACGGCAAGAT 16 7561 TATCTGGACA CCTGGCTGAT CGGAGCCAGT GATCGGCTTT CGTCAGATGC GGTGGCGTTA 17 7621 TTGGAAGCCT CGCTTGCCGA TCCGGACAGC TCGACCGGAT TCAACAGGAT GAAGGGTGAC 18 7681 GCCGGACAGG CAACGCTCGA CAACATTCTC GACGTGACCG AGAAACTCGC CTTTATCCAG 19 7741 AGACTTGATC TTCCCCATGA TCTCCTGACG GCTACGGGCA AGCCATGGGT CGATCAGATT 20 7801 GTTCGCCGCG TTGCCGGTGA AAAGGCCTCG GAGATGCGCC GGCATGCGCC GGCGCGACAG 21 7861 CTCGGCCTTT ATGCGATTTA TCTAATGTCG CGGGAGGCGC AACTCACTGA CGCGATGATC 22 7921 GACCTGCTGA TCGAAACCGT TCACAAGATC GGAACGCGCT CGAAACGCAA GGTGGTGGGC 23 7981 GATATCGCGA AAGACATCGA GCGGGTCTAT GGAAAGGAGC GCCTGCTGGT CGAGATCGCC 24 8041 AGCGCCTCGA TCAATGAACC ATCGGGGCGC ATCTGCGATG TCATTTTCCC GATCGCCGGT 25 8101 AAGGCCAAGC TGGCGGCGAT CGTCAAGGAG AGCCATGCGA AGGGCGCTCT GGACCKGCGC 26 8161 ATCTACAAGG TGATGTGTCA ACGGCGGAGC AAAAGTCGGC CATTCGGCGG CGTAAAACCA 27 8221 GGCCATCGT 28 29 30 Contig3, 1954 bp 31 32 1047>247 Cinnamoyl CoA-hydratase 33 1954>1104 Acyl-CoA transferase / racemase 34 35 1 GGGCAGTGAG CGNAACGCAA TTAATGTGAG TTAGNTCACT CATTAGGCAC CCCAGGCTTT 36 61 ACACTTTATG CTTCNGGNTN GTATGTTGTG TGGAATTGTG AGCGGATAAS AATTCACCGG 37 121 AAAGCTATGA CCATGATTAC GCCAAGCTAT TTAGGTGAGA CTATAGAATA CTCAAGCTTG 38 181 CATGCCTGCA GGTCGACTCT AGAGGATCCC ACGATATTCA TTCAATATTC CTATTTCCTA 39 241 TTGTCGTATA GTTTTCTAGC GCCGGTTTGA ATTTTCGATT AGAGAACCCC TGAAGTGCGA 40 301 CGTCAAGCCA TTTCTGACGC GATGTATACG AAAGTTCAAA GAATTTTGCT GATTCCCATT 41 361 CAACTGCTTC GTCGAAATCC ATTTCGACGG ACCGCTCGAA TACACGCTTG ATCGCGACAG 42 421 CCGCGTGCCT GTCTTTTGCT GCCAACTCGG TGCTCAGCCT GTCAATCTCG CCGCTCAGAT 43 481 CTGCAACCGG AACCACATGA TTGACCAATC CGATCTCCGC CGCCTTGTGA CCGTCGAACA 44 541 CCTGCCCCGT CAGGAGGTAA TAGAGCGCCA CTTTGCGTGG CAAGTGCCGG GCGACCATCC 45 601 AGGTCAGACC TGCACCGGGG ATGACGGCGA AGTTGATTTC CGAAAGGCTG AAACGCGCGT 46 661 TCGAGGCAGC GACCGACAAA TCGCAGAGCC CCGCGACCAA AATGCCTCCG CCCACGCACC 47 721 AGCCCTGAAC CGCCGAGATC GTGATGCCGT TAAAGTGGCG AAGACGCTTC ATCCACGACA 48 781 GCGCCAGATC CATCGTTCGC ATGAACATGT CGGAGTCCCC GAAATTCCCA AGCAGGAACT 49 841 CCTCAAGATC CATGCCTGCG CAGAAGTTGT CACCTCTCCC GACAATAACG ATAAGCCGTA 50 901 CATCGCCCCG GCGCTCCACC TCGTCCAGCG CGGAGTGCAT GTCGGCATGW ACTTGCGGAG 51 961 ACATCGCGTT GCGCTTTTCG GGGCGATTGA ACKCGATCGT TGCTCGCGCT CMATCGAAAC 52 1021 ACAGGTCGAC GGTCGGAAAA GTTACGGCGT TCATGTCGTC TCTCCCGTTG CGGCCCGGCC 53 1081 GACCCCGGTC TCTTCGCGTG CCCGCGCTTC ATCCGGTTCA CCGAAATAGC GATGATTATG 54 1141 CTCACCCAGA GCGGGTGAAG ATGCCGTGGA GTCGGGCGTG TATCCGCTGA TCTGTATCGG 55 1201 GAGTTCGAAT ATCACGTCTG ACGACGCTAT CCCGGCACAG CCCGCTTCCG CCTTTCCGTT 56 1261 CGCCATGGCT TCGGCTACGC TAAGCACGGG AGCGAAACAA CAGTCGGCCT TCACCAGCAG 57 1321 GTTGCACCAG TGATCAAGTG GGTGCGACCC AAAGATTCCT GCAAGCTCGG ACTTCAGCCA 58 1381 GGCTTGGCGA GTCTCGTCGA ACTGGAAGGG CTGCAAGTCG GGCCTCTGCA GCGCCGCACA 59 1441 CAGCCTCGCC CAGAACTTGG GTTCACCGGC CCCGACGGTC AGATGGCGAT AGTCAGATGT 60 1501 CTGATATATC TGGTAGCAGG CAAAGCTCCC GTCGAGAAGC GAAGGCCCGC CGGCCGACAT 61 1561 CGTTAGCGGC CCGATGTTCA GCGGCTTGCA GCATTCGGTC ATGGACACGC CGTAGAACCG 62 1621 TCCCCGGCCC GTCCGTTGCC GCTCGACGAT GCCGGTCAGC ACCGCGATCA CGGCGTGAAG 63 1681 AGATCCACCC ATGACGTCCG CGATCTGGAA ACGCGGCAAA ACCGGCCCTC CGGTATCATC
302
1 1741 ACGCAACTGA TCCAGAAGCC CCGCGGTATG CAAGGTAATT CAGATCGTGG CCCGCGAGAT 2 1801 CGCTGAAGGG AACCAGACTG ACCATAACCG GTTAATCGAG ACATAGACGA NCGCGGGCTT 3 1861 GATANCCNAA AAGANTCGCG TAATCNATTC CAANGCTTCG CTGCTAACGC CCGGCCTAAA 4 1921 GCTTTTCCAN NAAAAAAATC CGCTTCGCGC AACA 5 6 7 8 Phylogenetic analyses of putative gene products of ipfA and ipfB 9 10 The figures in this section represent additional results of phylogetic analyses
11 performed on IpfA and IpfB as described in Chapter 3.
12 13
14
15 Figure A5.1. Conserved domain architecture of the predicted protein encoded by ipfA 16 obtained via CDD analysis.
303
Benzene 1,2- dioxygenases Biphenyl 2,3- dioxygenases 3- phenylpropionate dioxygenases
Napthalene dioxygenases
p- cumate 2,3- dioxygenase
IpfA
Indole 3- acetic acid dioxygenase IacC e s a Benzoate 1,2- dioxygenases n e g y x 2,4D dioxygenase io d - 2 , 1 e t a il n a r h t n A 1
2 Figure A5.2. Phylogenetic tree of the predicted amino acid sequence of ipfA and 3 characterized dioxygase large subunits found in the Swiss-prot database and listed in Table 4 3.4. 5
304
Biphenyl 2,3- dioxygenases Benzene 1,2 dioxygenases
3- phenylpropionate dioxygenases
Napthalene dioxygenase
P- cumate 2,3- dioxygenase
Benzoate 1,2- dioxygenases IpfB
1 Indole 3- acetic acid dioxygenase
2 Figure A5.3. Phylogenetic tree of the predict amino acid sequence of ipfB and characterized 3 dioxygase small subunits found in the Swiss-prot database and listed in Table 3.5.
305