Microbial Transformation of Bile Acids. a Unified Scheme for Bile Acid Degradation and Hydroxylation of Bile Acids

SR P T03 Form 3 CONDITIONAL

THE UNIVERSITY OF NEW SOUTH WALES

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Witness...

..e GENETIC MANIPULATION OF PSEUDOMONADS TO PRODUCE STEROID CATABOLITES FROM BILE ACIDS.

A THESIS SUBMriTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF NEW SOUTH WALES AUSTRALIA

JOHN ALTON IDE

SCHOOL OF BIOTECHNOLOGY MAY, 1989. UNIVERSITY OF N.S.W. 16 MAY 1990 CONTENTS

Page

ABSTRACT i

DECLARATION iii

ACKNOWLEDGEMENTS iv

LIST OF PUBLICATIONS v

LIST OF TABLES vi

LIST OF FIGURES viii

ABBREVIATIONS xi

1 INTRODUCTION 1

LI INTRODUCTION TO STEROID PRODUCTION. 1

L2. EARLY SYNTHETIC PROCESSES FOR THE

MANUFACTURE OF STEROID DRUGS. 4

L3. CURRENT PROCESSES 8

L4. MICROBL\L TRANSFORMATION OF STEROIDS. 12

L5. ALTERNATIVE BIOTECHNOLOGICAL PROCESSES. 18

1.6. FERMENTATION PROCESSES AND ALTERNATIVES. 18

1.7. MICROBL\L DEGRADATION OF STEROLS 24

1.8. CONVERSION OF STEROLS BY MUTANTS. 27

1.9. BILE ACIDS AS AN ALTERNATIVE SUBSTRATE. 28

1.10. MICROBIAL DEGRADATION OF BILE ACIDS. 29

1.11. GENERAL PROPERTIES OF TRANSPOSONS. 40

1.12. TRANSPOSONS AS MUTAGENIC AGENTS AND

VECTORS FOR THEIR INTRODUCTION INTO

RECIPIENTS. 44

continued.... Contents (continued) 1.13. USE OF TRANSPOSONS IN GENE CLONING. 46 1.14. AIMS OF THIS THESIS. 50

2. MATERIALS AND METHODS. 51 2.1. BACTERIAL STRAINS AND PLASMIDS. 51 2.2. GENERAL PROCEDURES AND CHEMICALS. 51 2.2.1. Water and sterilization. 51 2.2.2. Growth conditions. 51 2.2.3. Chemicals and reagents. 51 2.3 BUFFERS AND SOLUTIONS. 57 2.3.1. Amino Acids and Nucleotide Growth Factors. 57 2.3.2. 25% Glucose. 58 2.3.3. Antibiotics. 58 2.3.4. PAS Salts Concentrate. 58 2.3.5. Vogel-Bonner Salts Concentrate. 58 2.3.6. Saline plus 10% Nutrient Broth (SNB). 58 2.3.7. Citrate Buffer. 59 2.3.8. Tris-EDTA (TE). 59 2.3.9. Tris-Acetate-EDTA (TAE). 59 2.3.10. Saline Sodium Citrate (SSC). 59 2.3.11. Denhardt's Reagent. 60 2.3.12. Pre-Hybridization Buffer. 60 2.3.13. Hybridization Buffer. 60 2.3.14. Deoxynucleotide Triphosphates. 61 2.3.15. Nick Translation Buffer. 61 2.3.16. Nick Stop Buffer. 61 2.3.17. Restriction Buffer. 62 2.3.18. Ligation Buffer. 62 continued Contents (Continued)

2.3.19. Phenol. 62 2.3.20. Chloroform. 62 2.4. MEDIA. 63 2.4.1. Nutrient Broth (NB). 63 2.4.2. Nutrient Agar (NA). 63 2.4.3. Luria Broth (LB). 63 2.4.4. PAS Minimal Media. 63 2.4.5. VB Minimal Media. 64 2.5. METHODS 64 2.5.1. Curing of Plasmids. 64 2.5.2. NTG Mutagenesis. 64 2.5.3. Filter Mate Conjugation. 65 2.5.4. Transposon Mutagenesis. 66 2.5.5. TLC Identification of Steroid Products. 66 2.5.6. Small Scale Isolation of Plasmid DNA. 67 2.5.7. Large Scale Isolation of DNA. 67 2.5.8. Isolation of Genomic DNA. 68 2.5.9. Isolation of Mu Phage DNA. 68 2.5.10. Restriction Endonuclease Digests. 69 2.5.11. Dephosphorylation of Spliced DNA. 69 2.5.12. Ligations. 69 2.5.13. Agarose Gel Electrophoresis. 70 2.5.14. Electro-elution of DNA Fragments from Agarose Gels. 70 2.5.15. Transformation of Plasmid DNA. 71 2.5.16. Labelling DNA by Nick Translation. 71 2.5.17. Southern Transfer of DNA and Hybridization. 72 continued Contents (Continued)

2.5.17.1. Transfer of DNA from Agarose Gels. 72 2.5.17.2. Colony Blotting. 73 2.5.17.3. Hybridization. 74 2.5.17.3.1. Hybridization with Southern Blots. 74 2.5.17.3.2. Hybridization with Colony Blots. 75

3. RESULTS. 76 3.1. PRELIMINARY CHARACTERIZATION OF BILE-UTILIZING PSEUDOMONADS. 76 3.1.1. Plasmid Profile. 76 3.1.2. Catabolic Properties. 76 3.1.3. Antibiotic-resistance Properties. 79 3.1.4. Isolation and Identification of Auxotrophic Mutants. 79 3.1.5. Introduction of Catabolic Plasmidsby Conjugation. 81 3.1.6. Curing of the Resident Plasmids. 81 3.2. ISOLATION OF MUTANTS BLOCKED IN STEROID BIOCONVERSIONS. 84 3.2.1. Isolation of NTG-induced Mutants Affected in Steroid Utilization. 85 3.2.2. Transposon Mutagenesis with Tn5 to Isolate Steroid Catabolic Mutants. 88 3.2.3. Transposon Mutagenesis with Tnl and Tn7 to Issolate Steroid Catabolic Mutants 100 3.2.4. Transposon Mutagenesis with TnlO. 106 3.2.5. Summary of Transposon Mutation. 107 3.2.6. Double Mutation of Bile-utilizing Strains. 108 3.2.7. Fermentation Studies of Transposon-induced Mutants. 118 Continued Contents (Continued) 3.3. MECHANISM OF TRANSPOSITION BY TN5 FROM pJB4JI. 123 3.3.1. Selection for Clones Encoding Kanamycin Resistance from pJB4JI-derived Mutants. 124 3.3.2. Restriction Mapping and Southern Hybridization Analysis of Clones isolated from pJB4JI-derived Mutants. 127 3.3.3. Southem Hybridization Analysis of Tn5-induced Mutants Derived from the Vector pJB4JI. 135 3.4. MECHANISM OF TRANSPOSITION BY Tn5 FROM pSUPlOll. 152 3.4.1. Selection for Clones Encoding Kanamycin Resistance from pSUPlOll-derived Mutants. 152 3.4.2. Restriction Mapping of pND209. 153 3.4.3. Hybridization of pND209 with Fragments of pSUPlOll. 153 3.4.4. Southem Hybridization Analysis of Tn5-induced Mutants Derived from the Vector pSUPlOll. 158 3.5. CLONING OF THE STEROID CATABOLIC PATHWAY 161 3.5.1. Hybridization of DNA from PS5-1 and PS8-1 with the Clones pND200 and pND209. 164 3.5.2. Derivation of Gene Libraries of PS5-1 and PS8-1 using the Vector pKT230. 170 3.5.3. Cloning of Pseudomonas DNA into the //mdin Site of pKT230. 171 3.5.4. Screening of the Pseudomonas Gene Libraries for Clones Encoding the Steroid Catabolic Pathway using Colony Hybridization. 173 Continued Contents (Continued)

3.5.5. Preliminary Cloning of PS8-1 and PS5-1

DNA into the Cloning Vector pBR329. 174

4. DISCUSSION. 177

5. BIBLIOQRAPHY. 203 1.

ABSTRACT.

The object of the project was to modify, by mutation, bile steroid catabolizing Pseudomonads so that they could accumulate steroid intermediates of the breakdown pathway. In the four Pseudomonas strains investigated, genetic information relevant to the steroid catabolic pathway appeared to be encoded on the chromosome. One NTG-induced mutant, PS5-7, accumulated secophenol and secocatechol compounds. Although speculated, the secocatechol had never previously been isolated as a degradation product. Transposon-induced mutants of PS5-7 were isolated which accumulated only the phenolic secosteroid. A total of 46 transposon-induced mutants of bile acid-catabolizing Pseudomonads (from 13000 transposed clones tested) were isolated which accumulated steroid pathway intermediates. Seven classes of intermediates were accumulated and although mutants varied in stability, individual mutants were obtained which enabled some products to be accumulated in yields approaching theoretical. The major products accumulated from cholic acid fermentations (and the yields) are as follows : 7a,12a-dihydroxy-3-oxo-l,4-pregnadiene-20-carboxylic acid (86%), 7a, 12a-dihydroxy-1,4-androstadiene-3,17-dione (97%), 7a, 12P-dihydroxy- l,4-androstadiene-3,17-dione (96%) and 3,7,12a-trihydroxy phenolic secosteroid (97%). The remaining product classes were mixtures and were not studied in detail.

Introduction of a second transposon into selected strains seldom altered the products they accumulated, but often altered the stability of the clone.

The mechanism of transposition by two Tn5-loaded vectors, pJB4JI and pSUPlOll, was investigated. Hybridization studies with DNA isolated from selected mutants showed that all of the mutants investigated had at least one copy u.

of Tn5 in the chromosome. Clones, containing the transposon and the DNA encoding the flanking regions about that transposon, were isolated from selected mutants and investigated. The transposon Tn5 is carried within Mu DNA in pJB4JI. In 14 out of 19 pJB4JI-derived mutants, Mu was inherited as well as Tn5. The remaining 5 mutants harboured only Tn5. The one pSUPlOll- derived mutant investigated harboured only Tn5.

Attempts to clone genes relevant to the steroid catabolic pathway into the cloning vector pKT230 were unsuccessful because of instability of DNA inserts in the vector. Preliminary cloning experiments have shown that DNA of the required size could be stably inserted into pBR329. Cloning of DNA encoding steroid catabolic genes appears practicable using this altemative vector. m

SR.P.T10

CERTIFICATE OF ORIGINALITY

I hereby declare that this thesis is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by an other person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of a university or other institute of higher learning, except where due aknowledgement is made in the text of the thesis.

(Signed) IV.

ACKNOWLEDGEMENTS. I would like to express my gratitude to my co-supervisors, Noel W. Dunn and Robert J. Park for their support and continuing advice throughout the project. Special thanks must go to Noel for his friendship and enthusiasm over the years. I would also like to thank the many colleagues in the School of Biotechnology and others who assisted me with their time and expertise and/or returned me to sanity with their laughs and friendship. In particular, I wish to thank Keith Brown, Steve Delaney, Amanda Goodman, Melissa Harvey, Liz Kirov, Peter Milic, Gwen Ng, Louise Opolski, David Park, Pamela Rickard, Ghislaine Samways, Aneta Strzelecki, Sharon Tandy, Janet Valentine, Rhonda Warr, Jeff Welch and Phil Wordsworth. My final thanks must go to Barbara and Jason, who kept the home fires burning and provided the tic (not chromatography) and the patient ear when necessary. V.

PUBLICATIONS

REVIEWED JOURNALS.

Ide J.A., RJ. Park & N.W.Dunn (1986). Bile acidcatabolites accumulated by transposon-induced mutants of Pseudomonas putida : Production of hydroxylated l,4-androstadiene-3,17-diones. BiotechnoL Letts. 8 : 763-768

Ide J.A., R.J. Park, R.A. Leppik & N.W. Dunn (1987). Pseudomonas mutants able to accumulate phenolic and catecholic 9,10-secosteroids from bile acids. AppL Microbiol. BiotechnoL 26 : 234-236

Park R.J., N.W. Dunn & J.A. Ide (1986). A catecholic 9,10-secosteroid as a product of aerobic catabolism of cholic acid by a Pseudomonas sp. Steroids 48 : 439-450

Park R.J., J.A. Ide, H. Motyka & N.W. Dunn (1987). Transposon induced mutants of Pseudomonas which accumulate catabolites from bile acids. Production of hydroxy-3-oxo-l,4-pregnadiene-20-carboxylic acids. J. BiotechnoL _5 : 149-155

NON-REVIEWED JOURNALS.

Ide J.A., R.J. Park, R.A. Leppik & N.W. Dunn (1986). Construction and characterization of strains of Pseudomonas able to accumulate steroid intermediates from bile steroids. Proc. 7th. Aust. BiotechnoL Conf. Univ. of Melbourne, pp 134-139

PATENTS.

Park R.J., N.W. Dunn & J.A. Ide (1983). Process for the Microbial Preparation of Steroid Drug Intermediates. Aust Patent Appl. No. 581183

Park R.J., N.W. Dunn & J.A. Ide (1984). Process for the Microbial Preparation of Steroid Drug Intermediates. French Patent No. 84/12711 VI.

LIST OF TABLES.

Page-

1 Reported Industrial Use of Microorganisms for Steroid Transformations. 17 2 Comparison of Biotechnological Processes with Chemical Methods. 19 3 Bacterial Strains and Plasmids. 52 4 Growth Responses of the Wild-type Pseudomonas Strains on Various Carbon Sources. 78 5 Antibiotic and Mercury Resistance of the Pseudomonas strains PS5-1, PS6-1, PS7-1 and PS8-1. 80 6 Transfer Frequencies of Catabolic Plasmids into Steroid Utilizing Strains. 82 7 NTG-Induced Mutants of PS5-4 and PS6-3 Affected in Bile Steroid Degradation. 86 8 Frequency of Km^ (Tn5) Transconjugants, No. of Colonies Tested and No. of Transconjugants Affected in Steroid Substrate Utilization. 90 9 Growth Properties of Tn5-Induced Mutants of Bile Acid-Utilizing Pseudomonas Strains and the Compounds Accumulated after Incubation in 2g/L Deoxycholic Acid. 92 10 Frequencies of Cb^ (Tnl) and Sm^ (Tn7) Transconjugants of PS5-1 and Control Strain PPl-2 Trp", No. of Transconjugants Tested and No. Affected in Steroid Utilization. 102

Continued Vll.

Table Page. 11 Growth Properties of Tnl-induced Mutants of PS5-1 and Accumulated Compounds after Incubation in 2 g/L Deoxycholic Acid. 103 12 Catabolites Accumulated from Deoxycholic Acid by Transposon-induced Mutants of Bile Acid Utilizing Strains. 109 13 Transfer Frequencies of Tnl Transposition into Strains 105(4), 105(6) and 119(2), No. of Transconjugants Tested and No. Affected in Steroid Utilization. 113 14 Growth Properties of Tnl-induced Mutants of 105(4), 105(6) and 119(2) and Accumulated Compounds after Incubation in 2 g/L Deoxycholic Acid. 114 15 Production of Steroid Intermediates from Bile Acids by Fermentation with Mutants. 120 16 Clones of Eco Rl-restriction Fragments of Tn5- Induced Mutants, their Approximate Size and Tetracycline Sensitivity. 126 17 pJB4JI-derived Mutant Strains Investigated by Southern Hybridization Analysis. 136 18 Estimated Sizes of Restriction Fragments of PS5-1 and PS8-1 which Hybridize to the Clones pND200 and pND209 Respectively. 169 Vlll.

LIST OF FTGURRS

Figure. Page. 1 The Numbering of the Carbon Skeleton and the Rings for a Typical Steroid, Stigmastane (24-ethylcholestane) 3 2 Saturated Parent Hydrocarbons 3 3 Structures of Some Natural Raw Materials Used for Steroid Synthesis. 5 4 The Conversion of Stigmasterol to useful Steroid Products by the Upjohn Co. 9 5 General Process for the Manufacture of Steroid Drugs from Diosgenin and Analogues. 10 6 Common Pathway Intermediates in the Microbial Degradation of Sterols. 25 7 The Bile Acids and Analogues, Their Trivial Names and Structures. 30 8 Proposed Microbial Degradation Pathway of Bile Acids. 32 9 Proposed Deoxycholic Acid Degradation Pathway by Pseudomonas spp. 37 10 The Transposon-carrying Vectors pJB4JI and pSUPlOll 49 11 Plasmid Profile of the Wild-type Bile Acid-utilizing Strains, PS5-1, PS6-1, PS7-1 and PS8-1. 77

Continued.. IX.

Figure. Page. 12A Restriction Endonuclease Digestion of the Clones pND200, pND201, pND204 and pND209 with the Enzymes EcoRl, Hindm and Double Digests. 128 12B Restriction Endonuclease Digestion of the Clones pND200, pND201, pND204 and pND209 with Various Enzymes. 129 13A Hybridization of Digested Clones in Figure 12A with Mu DNA. 132 13B Hybridization of Digested Clones in Figure 12B with Mu DNA. 133 14 Restriction Digest Maps of the Clones pND200, pND201 and pND204. 134 15A Total DNA of PS5-1 and pJB4n-derived Mutants. 138 15B Total DNA of PS5-1,PS8-1 and pJB4JI- derived Mutants. 139 16A Hybridization of Total DNA of pJB4JI-Derived Mutants in Figure 15A with pKan2 141 16B Hybridization of Total DNA of pJB4JI-Derived Mutants in Figure 15B with pKan2. 142 17A EcoKl Digests of Chromosomal DNA of pJB4n-derived Mutants of PS5-1. 144 17B EcoKl Digests of Chromosomal DNA of pJB4JI-derived Mutants of PS5-1 and PS8-1. 145

Continued X.

Figure Page. 18A Hybridization of DNA Digests in Figure 17A to pKan2 Plasmid Probe. 146 18B Hybridization of DNA Digests in Figure 17B to pKan2 Plasmid Probe. 147 19 EcdRl Digested Chromosomal DNA of pJB4JI-derived Mutants of PS5-1 149 20 Hybridization of R1-digested Chromosomal DNA of pJB4n-derived Mutants of PS5-1 with Mu DNA Probe. 151 21 Restriction Enzyme Map of pND209 154 22A Restriction Endonuclease Digests of the Plasmids, pACYC184, pSUPlOll, pSUP202 and pND209, and Chromosomal DNA of PS8-1 and PS8-10. 156 22B Hybridization of Digested Component Fragments of pSUPlOll, PS8-1 and PS8-10 with Isolated Fragments of pND209. 157 23 Restriction Map of pKT230 163 24 PS5-1 Genomic DNA digested with Various Restriction Endonucleases. 167 25 Hybridization of Digested PS5-1 Genomic DNA with Flanking Regions of pND200. 168 26 Eco R1 Digests of Sample of Ap^CmS Transformants Containing PS5-1 and PS8-1 DNA Cloned into pBR329. 176 XI.

ABBREVIATIQNS

The following abbreviations are used in the text:

AD androstene-3,17-dione ADD androstadiene-3,17-dione Ap ampicillin arg argenine ATCC American Type Culture Colllection, Washington DC, USA ATP adenosine triphosphate bp base pairs CA cholic acid CAM camphor degradation Cb carbenicillin CCC covalentiy closed circular CDCA chenodeoxycholic acid Cm chloramphenicol cpm counts per minute CS catecholic secosteroid DCA deoxycholic acid DNA deoxyribonucleic acid EDTA ethylenediamine tetraacetic acid g gram Gm gentamicin Hg mercury PBDCA hyodeoxycholic acid his histidine continued xu

Abbreviations (continued)

HPLC high performance liquid chromatography IS insertion sequence SKA mixture of 3-oxo-cholanic and -bisnorcholanic acids 3KEA mixture of 3-oxo-4-cholenic and 4-bisnorcholenic acids kb kilobase pair Km kanamycin L litre LB Luria broth leu leucine met methionine mg milligram ml millilitre Mob mobilization N A nutrient agar NB nutrient broth NCIB National Collection of Industrial Bacteria, Aberdeen, Scotiand no. number NTG N-methyl-N'-nitro-N-nitrosoguanidine OD optical density OH hydroxy OPDC 3-oxo-l,4-pregnadiene-20-carboxylic acid PAS phosphate ammonium salts media phe phenylalanine Pip pipercillin pro proline PS phenolic secosteroid continued Xlll. Abbreviations (continued) psi pounds per square inch ipm revolutions per minute SDS sodium dodecyl sulphate Sm streptomycin SNB saline nutrient broth Sp spectinomycin ssc saline sodium citrate buffer TAE tris-acetate, EDTA electrophoresis buffer Tc tetracycline TE tris-HCl, EDTA buffer thi thiamine thr threonine TLC thin layer chromatography Tn transposon TOL toluene degradation Tp trimethoprim tra transfer tip tryptophan tyr tyrosine UV ultraviolet V volt VB Vogel Bonner salts media X unknown compound |lCi microcurie microgram microlitre lambda % per cent 1. INTRODUCTION.

1.1. INTRODUCTION TO STEROID PRODUCTION.

Since the late 1940's, the manufacture and use of steroid drugs has rapidly increased such that for 1983 steroid drug manufacturers used 2000 tonnes of raw materials for a final product market in excess of US$1.8 billion (Kieslich, 1985). Manufactured steroidal drugs have served as anti-inflammatory agents, contraceptives, blood pressure regulatingan d cardiotonic agents, progestional male and female sex hormones, sedatives and anti-tumour substances. Some are effective in the treatment of allergic and dermatological diseases and as veterinary products. Increased demand for steroid products was initiated by the discovery in 1949 by the Hench and Kendall group at the Mayo clinic of the anti-inflammatory properties of cortisone in the treatment of rheumatoid arthritis. Increased research in steroid biochemistry followed with thousands of novel steroid derivatives being synthesized and tested for different and improved therapeutic properties.

The major sources of steroid products have come from three general processes: A) direct isolation from animal extracts B) conversion of inexpensive plant and animal steroids to the desired product and C) total chemical synthesis from simple non-steroid starting materials. The size of the world market as well as the economics of extraction excludes direct isolation as a commercial proposition considering the vast amount of animal tissue or urine needed to isolate pure steroids such as estradiol, cortisone or progesterone. The therapeutic drug "premarin", a mixed estrogen preparation from pregnant mare urine is still commercially prepared. The method favoured by most of the major manufacturers has been chemical modification of inexpensive steroid raw material to produce either useful intermediates for general steroid production or the specific conversion to finished products. Most industrial processes have incorporated microbiological fermentations to bypass the more difficult chemical steps. The third production method, total synthesis, has been the preferred method by some companies (notably Roussel-Uclaf, Wyeth and Schering AG) for androstane and estrane derivatives (Briggs & Brotherton, 1970; Applezweig, 1974). The industrial total synthesis of C-19 norsteroids (mainly contraceptives and progestational agents) have also recently incorporated microbiological steps in the resolution of racemates. At present total synthetic methods are being challenged by the recent isolation of microbial mutants that accumulate androstane and pregnane derivatives by selective side-chain cleavage of sterols.

The nomenclature of steroids can be confusing as many steroids have a trivial name, an lUPAC systematic name and alternative names used by workers in the field with a host of descriptions for substituents and saturations. Steroids are a class of tetracyclic organic compounds containing 3 six-membered rings (A, B and C) and 1 five-membered ring (D). The carbon atoms are numbered as shown for tiie 29-carbon sterol, stigmastane (Figure 1). The nomenclature relies on the number of carbon atoms present in the saturated parent hydrocarbon as shown in Figure 2, with estrane (C18), androstane (C19), pregnane (C21), cholane (C24) and cholestane (C27) these days being the only parent hydrocarbons used. Unsaturation is assumed in sequential numbering with exceptions shown by a bracketed number (eg: 9-ene implies a double bond between C9-C10 whereas 9(ll)-ene denotes between C9 and Cll). The lUPAC unsaturation system places tiie numbers in the middle of tiie name of tiie parent hydrocarbon tiius making the name unwieldy eg: androsta-l,4-diene while tiie more commonly FIGURE 1. The Numbering of the Carbon Skeleton and the

Rings for a Typical Steroid, Stigmastane (24-ethylcholestane).

FIGURE 2. Saturated Parent Hydrocarbons.

Estrane ( CI 8) Androstane (C19) Pregnane (C21)

Cholane (C24) Cholestane (C27) used method retflins the integrity of the name, eg: 1,4-androstadiene. The substitutions are applied, ie: only one suffix is permitted with an order of preference (acids, aldehydes, ketones, alcohols) and all other substituents are attached as prefixes. Halogens and hydrocarbons are always prefixes. The terms "nor" prefixed by a carbon number indicates that carbon is missing in the molecule and "seco" prefixed by 2 carbon numbers indicates ring fission between those carbons with the addition of hydrogen atoms at each terminal group. The junction between rings A and B can be either trans giving the 5a series of compounds or cis giving the 5P series. Most biologically-active steroids belong to the 5a series and unless otherwise stated (eg: 5p-cholanic acid) are assumed to be in the 5a-orientation. The prefix "epi" refers to the inversion of a substituent in the normal molecule.

1.2. EARLY SYNTHETIC PROCESSES FOR THE MANUFACTURE OF STEROID DRUGS.

Over the past 60 years, raw materials for the synthesis of pharmaceutical steroid products has shifted from animal extraction to the modification of animal and plant steroids and sapogenins. As chemical and biological processes were introduced and improved, certain classes of sterols were favoured as the starting material only to be replaced by other sterols for technical and/or economical reasons. Conmionly used starting compounds and their structures are presented in Figure 3.

The first synthetic process, developed in 1933, was the chemical synthesis of progesterone from 3P-hydroxy-23,24-bisnor-5-cholenic acid which had been prepared by ozonolysisof stigmasterol. The process was commercially used with modifications until 1952 when the more efficient Enamine method was 3. Structures of Some Natural Raw Materials Used for Steroid Synthesis.

COOH

Deoxycholic acid (Animal bile) Cholesterol (Wool grease)

\ \

HO HO

Stigmasterol (Soybean extract) Sitosterol (Soybean extract)

Diosgenin (Dioscora spp) Hecogenin (Sisal plants)

Ergosterol (Yeasts) Solasodine {Solanum spp ) developed. This method requires only 4 chemical steps to produce progesterone from stigmasterol with an approximate yield of 60% and resulted in the price of progesterone dropping from approximately US$80/g in 1944 to $0.20/g (Briggs and Brotherton, 1970). The Upjohn Co. used this chemical method in all processes which required progesterone as an intermediate and as far as is known curently still uses the process (Djerassi, 1976). From 1935, Schering AG commercially produced dehydroepiandrosterone by a route involving the oxidation of cholesterol with chromic acid. The final yield was approximatiy 10% (Chamey and Herzog, 1967). A by-product of this process was 3p-hydroxy-5-cholenic acid. In 1946, an efficient method for the degradation of the bile acid side chain was reported by Sarrett. The method was used with the above by-product for conversion to progesterone in an overall yield of 33% (Briggs and Brotherton, 1970). Cholesterol was replaced in the early 1950's due to difficulties in converting the long side-chain to the required two-carbon cortical side-chain and the multiple side-products formed in the chemical oxidative processes, however alternative procedures using cholesterol have recentiy started which circumvent many of these problems.

Cortisone was first synthesized by Sarrett under a group research scheme to obtain sufficient quantities for clinical evaluations by the Mayo clinic (Chamey and Hertzog, 1967). The process was developed by Merck and Co. in 1949 and involved 37 chemical steps from deoxycholic acid to cortisone-21-acetate with a yield of 0.16% (Sebak, 1977). The major task in the procedure was the displacement of the 12-hydroxy group and the incorporation of an 11-keto group. The 12-hydroxy group was oxidized with chromium trioxide and the C9-C11 carbons dehydrogenated with selenium dioxide in chlorobenzene-acetic acid. The resulting 12-ketone was reduced with platinum, then methylated and chlorinated after displacement witii HCl in chloroform. Bicarbonate formed a 3a,9a-oxide which on bromination formed an 11,12-dibromide, subsequentiy converted by oxidative hydrolysis to the 12-bromo-l 1-ketone. Reduction with phenyhnagnesium bromide followed by dehydration with hot acetic acid led to the successful transfer of the oxygen group from the C12 to die Cll. The whole process was later simplified by the Upjohn Co. to 11 steps, including a microbial lla-hydroxylation, but the synthesis of corticosteroids from bile acids was viewed as uneconomic and unable to satisfy a growing world demand (Briggs and Brotherton, 1970).

Synthetic procedures using phytosterols were investigated due to the potential greater supply of raw materials. Initially hecogenin was explored as an alternative, being derived from the fermentation waste liquor of Agave spp. in sisal manufacture. Glaxo use hecogenin for corticosteroid production but costs are high due to the C12-oxygen function. The real commercial success came from Marker at the Pennsylvania State College, who by 1947 had developed an efficient process for the chemical conversion of diosgenin to progesterone in good (60%) yields. Diosgenin is the product of the uncontrolled fermentation of dioscin, by soil bacteria attached to the tubers of yams of the Dioscorea species (Kieslich, 1980a). Marker established the firm Syntex in Mexico in the late 1940's which began the commercial conversion of diosgenin to 16-dehydropregnenolone and 16a,17a-epoxypregnenolone in 4 chemical steps (Briggs and Brotherton, 1970; Djerassi, 1976). Selective hydrogenation and Oppenauer oxidation produces progesterone from the first compound while the second is an important starting material for cortisone production (see Figure 5). 8

1.3. CURRENT PROCESSES

The early processes using cholesterol and deoxycholic acid were mainly discontinued due to the number of chemical steps and the many side products. From the 1950's onwards, because of very efficient chemical methods and the discovery of oxidative fermentation processes, the preferred raw materials have been diosgenin and stigmasterol. Both are easily converted to C21-intermediates from which most CI 8-, C19- and C21-steroids are manufactured today (Smith, 1984). Although most manufacturing details are undisclosed or kept obscure, general pathways have been reported in some instances. For example, a basic outline of the use of stigmasterol by the Upjohn Co. is shown in Figure 4, including the chemical and microbiological steps used for each product (Sebek & Perlman, 1979). The company purchases soy bean seed oil and seperates stigmasterol from the noajor steroid component p-sitosterol.

To further illustrate the complexity of current industrial processes, a general pathway for Syntex and other companies which use diosgenin, or its nitrogen analogue solasodine, is outiined in Figure 5 (Smith,1984). All processes revolve around the requisite intermediates, 16-dehydropregnenolone, progesterone or Substance S, which are either used by the manufacturer in their own processes or sold as a process raw material to other manufacturers.

In the early 1970's, the Mexican government nationalised the diosgenin industry raising fears of a steroid raw material shortage or considerable price increases. To further compound the situation, the solvents required for the Enamine process were in short supply, therefore many companies looked for alternative raw materials (Applezweig, 1974). Most current reports indicate diosgenin is still the major basic raw material, but now is partially replaced by 8

1.3. CURRENT PROCESSES

The early processes using cholesterol and deoxycholic acid were mainly discontinued due to the number of chemical steps and the many side products. From the 1950's onwards, because of very efficient chemical methods and the discovery of oxidative fermentation processes, the preferred raw materials have been diosgenin and stigmasterol. Both are easily converted to C21-intermediates from which most C18-, C19- and C21-steroids are manufactured today (Smith, 1984). Although most manufacturing details are undisclosed or kept obscure, general pathways have been reported in some instances. For example, a basic outline of the use of stigmasterol by the Upjohn Co. is shown in Figure 4, including the chemical and microbiological steps used for each product (Sebek & Perlman, 1979). The company purchases soy bean seed oil and seperates stigmasterol from the major steroid component p-sitosterol.

To further illustrate the complexity of current industrial processes, a general pathway for Syntex and other companies which use diosgenin, or its nitrogen analogue solasodine, is outlined in Figure 5 (Smith,1984). All processes revolve around the requisite intermediates, 16-dehydropregnenolone, progesterone or Substance S, which are either used by the manufacturer in their own processes or sold as a process raw material to other manufacturers.

1.3. CURRENT PROCESSES

The early processes using cholesterol and deoxycholic acid were mainly discontinued due to the number of chemical steps and the many side products. From the 1950's onwards, because of very efficient chemical methods and the discovery of oxidative fermentation processes, the preferred raw materials have been diosgenin and stigmasterol. Both are easily converted to C21-intermediates from which most C18-, C19- and C21-steroids are manufactured today (Smith, 1984). Although most manufacturing details are undisclosed or kept obscure, general pathways have been reported in some instances. For example, a basic outline of the use of stigmasterol by the Upjohn Co. is shown in Figure 4, including the chemical and microbiological steps used for each product (Sebek & Perlman, 1979). The company purchases soy bean seed oil and seperates stigmasterol from the major steroid component p-sitosterol.

To further illustrate the complexity of current industrial processes, a general pathway for Syntex and other companies which use diosgenin, or its nitrogen analogue solasodine, is outlined in Figure 5 (Smith,1984). All processes revolve around the requisite intermediates, 16-dehydropregnenolone, progesterone or Substance S, which are either used by the manufacturer in their own processes or sold as a process raw material to other manufacturers.

Steroid Products by the Upjohn Co. ^^^

Enamine Degradation. 4 chemical steps.

CH. C=0

Progesterone

1 Microbiological 2 Microbiological 2 Microbiological 4 Chemical steps 4 Chemical steps T 9 Chemical steps

CHÔH CHÔH CHÔH I ^ I ^ c=o c=o

0 CH3

I 1 Chemical step 1 Chemical step 6 Chemical steps

CH^OH CH-OH I ^ I ^ I ^ c=o c=o c=o OH HQ OH

F 0 0 CH3

(a) from Sebak and Perlman (1979) 10

FIGURE 5. General Process for the Manufacture of Steroid Drugs from Diosgenin and Analogues.

0 Diosgenin

Mariner Degradation 4 Chemical steps.

® 16-deliydropregnenolone.

CH, I ^ c=o iXT

Megestrol 19-nor-4-AD 16a-17aEpoxypregnenolone Progesterone (Contraceptive)

CH^OH CH^OH CH2OH OH C=0

9a-Fluorocortisol Norethisterone Substance S Cortisol (Oral contraceptive)

CH^OH CH-OH

Triamcinolone ( & other Prednisone Cortisone Prednisolone glucocorticoids) 11

solasidine. Total synthesis of steroids such as epiandrosterone and estrone, from non-steroidal raw materials, have been possible since the 1940's, but more economic processes from phytosterols restricted its use. The anticipated natural steroid raw material shortage led some companies to expand their synthetic facilities such that by 1978, ahnost 20% of total world steroid production was by total synthesis (Sebek, 1977; Smith, 1984). This method is particularly attractive as these companies are not dependent on steroid raw materials.

Other steroid raw materials which recently have found favour are P-sitosterol and cholesterol. The former is the main steroid found in soy bean seed oil and tall oils, a waste product of paper manufacturing, and is considered a cheap starting material. The latter is considered one of the most cosdy raw materials because its purificationfrom woo l grease is an expensive process. For a long time, 4-androstene-3,17-dione (4-AD) and its 1-dehydrogenated derivative l,4-androstadiene-3,17-dione (1,4-ADD) have been the starting materials for the preparation of androgens, estrogens and anabolic drugs. Both cholesterol and p-sitosterol are commercially converted to 4-AD, 1,4-ADD and 9a-hydioxy-4-AD by degradation processes using a one-step fermentation process with microbial mutants. Schering AG and G.D. Searle convert androstane derivatives produced from p-sitosterol to the diuretic spironolactone, an important drug in the treatment of hypertension. Mitsubishi Chem. Ind. is believed to produce estrone, an intermediate in the synthesis of 19-norsteroid contraceptives, by a pyrolytic aromatization of 1,4-ADD which is derivedfrom th e fermentation of cholesterol by microbial mutants. Although estrone production by this process would seem feasible, most large companies which possess similar mutants (especially the Upjohn Co.) do not appear to use this process because chemical reduction steps downstreamfrom estron e are cumbersome and low-yielding (Martin, 1984). Data of world steroid production indicates that Japanese companies are the only major 12

users of cholesterol whereas p-sitosterol is mainly used in the U.S.A. and today rivals stigmasterol as a raw material source (Sebak, 1977; Smith, 1984). Degradation is mainly by Mycobacterium and Arthrobacter strains which, due to their taxonomic relatedness with pathogens, increases production and recovery costs (KiesUch, 1980a).

The advantage of a one-step process from raw material to a desired product is obvious and therefore attracts considerable research. Recent improved procedures for the reconstruction of the pregnane side-chain have resulted in improved alternative processes for corticosteroid production. The above mutants could be used to produce the required 17-ketosteroid followed by chemical reconstruction of the side-chain, thus yielding corticosteroids from either raw material in 3 to 4 steps. Other interesting mutants, patented recently by Upjohn and Henkel, accumulate 20-carboxyl pregnenes from cholesterol and p-sitosterol (Kieslich, 1985). The 20-carboxyl side-chain can easily be chemically converted to the required progesterone side-chain (Martin, 1984). At present, these latter mutants are not used commercially but the possibility for future use exists.

1.4. MICROBIAL TRANSFORMATION OF STEROIDS.

Progesterone is a female sex hormone with no corticosteroid activity. The structural differences between progesterone and active corticosteroids are the C21-hydioxy group, the C17a-hydroxy and the CI 1-oxygen function. The first two can be introduced economically by chemical processes, but the introduction of oxygen at the C-11 position, as shown previously, is a laborious and low-yielding procedure.

Hecter et al, in 1949 (cited by Sebek and Perlman, 1979) reported the 13 llp-hydroxylation of 11-deoxycorticosterone (cortexone) to corticosterone by perfusion of beef adrenal glands. Although being non-microbial, this is deemed to be the first successful application of biotechnology in the preparation of a useful steroid. The process was taken up by G.D. Searle and Co. for the preparation of adequate supplies of hydrocortisone for clinical evaluations. The report stimulated other leading steroid manufacturers to investigate biological hydroxylations as an alternative to existing chemical processes.

Since 1913, it has been recognised that microorganisms of diverse genera such as Norcadia , Pseudomonas , Corynebacteria mdArthrobacter were capable of utilizing the natural steroids cholesterol and p-sitosterol as a sole carbon source. Research in this area was slow with the only notable reports being the Mamoli and Vercellone (1937) paper discribing the reduction of 3- and 17-keto steroids by yeasts, two papers by Horvath and Kramili (1947 and 1948) on the 7-dehydrogenation of cholesterol by Azotobacter and the 7-hydroxylation by Proactinomyces roseus (all cited by Chamey and Herzog, 1967). The first microbial process, patented by Schering AG in 1937 following Mamoli's work, claimed both the reduction of 17-keto groups by yeast and the oxidation of 3-hydroxy steroids (Kieslich, 1980b). The method was never used commercially as chemical conversions gave greater yields. Turfitt (1944) investigated the degradation of steroids by Proactinomyces sp. and noted the breakdown products of cholesterol and bile acids included 3-oxo-4-cholenic acid and 3-oxo-4-androstene-17-carboxylic acid. These results demonstrated that useful steroid intermediates could be produced by the exploitation of the microbial metabolism of steroids.

The first commercially important microbial transformation to be reported was the lla-hydroxylation of progesterone and Reichstein's Substance S by 14

Rhizopus arrhizus (Peterson and Murray, 1952). This biotransformation and subsequentiy many others were by co-metabolic processes, ie: neither the substrate nor the product were used as an energy or carbon source by the catalytic organism. R. nigricans was later reported to possess a similar capability by the same authors, but gave faster rates with yields as high as 90%. The process was immediatly patented by the Upjohn Co. and the chemistry developed for converting the lla-hydroxy group to the required llp-hydroxy or 11-keto. Shortly thereafter, Upjohn developed processes involving llp-hydroxylation of Substance S with Streptomyces fradiae (low yield) and Cunninghamella blakeleeana (60% yield), although the llp-hydroxylation by Curvularia lunata (SchuU & Kita, 1955) was the process developed for commercial hydrocortisone production from Substance S (Sebek and Perlman, 1979; Smith, 1984).

Corticosteroids have profound effects on carbohydrate, protein and mineral metabolism in humans and when applied at dose levels to suppress symptoms of rheumatoid arthritis, adverse side effects are apparent. By altering the functional groups of cortisone and hydrocortisone, the structures essential for biological activity were found to be the C3- and C20-ketones and the C4-double bond. The C21-hydroxy group affects carbohydrate metabolism, but also enhances sodium retention. The introduction of oxygen at Cll and a 17a-hydroxy group is necessary for the anti-inflammatory activity (Turner and Bagnara, 1976). It was desirable to seperate the adverse effects from the anti-riieumatic activity. In the early 1950's, it was discovered that the introduction of a halogen, in particular fluorine or chlorine, into the 9a position of hydrocortisone enhanced by over 10-fold the glucocorticoid and anti-inflammatory activity, but was also found to be 50-times more effective in controlling sodium excretion (ie: retention). Further research revealed that 1-dehydrogenation or 6a-methylation also enhanced the above activities but caused a reduction in the 15 sodium retaining activity. An even greater reduction without affecting the anti-inflammatory activity was attained with 16a-hydroxylated steroids (Fried and Borman, 1958). The 16a-hydroxylation offluorocorticoids pave d the way for the manufacture of extremely active synthetic corticosteroids, however large-scale production of compounds with some of these substituents proved uneconomic by the available chemical methods.

In 1952, Perlman and associates at E.R. Squibb reported the 16a-hydroxylation of progesterone by Streptomyces argenteolus although the reaction was not considered important until 4 years later. The same group also reported the 11a- hydroxylation of progesterone by Aspergillus niger and were first to describe the 1-dehydrogenation of progesterone by S. lavendulae (Sebek and Perlman, 1979). The latter strain and others also degraded the side-chain leaving a 17P-hydroxy or 17-keto steroid, or expanded the D ring to a 1-dehydrotestolactone. The side-chain could be protected by use of analogues but a more practical organism, Arthrobacter (Corynebacteriwn) simplex, reported by Nobile in 1955, could 1-dehydrogenate certain pregnanes with no side reactions. Use of this organism led to a simplified process for the synthesis of prednisone and prednisolone, both drugs having a 4-times greater therapeutic activity than cortisone or hydrocortisone with no drug-induced salt retention at clinical levels (Chamey and Herzog, 1967).

Many other potentially useful biotransformations have since been discovered which not only include the reactions mentioned above but many other oxidations, reductions, isomerizations, saponifications of esters, acetylations and esterifications. The number and types of reactions and the microorganisms used are too numerous to be listed here and two reviews, Chamey and Herzog (1967) and lizuka and Naito (1967) are recommended. Biotransformations have 16

simplified the laboratory production of numerous steroid derivatives. Many have been tested for improved therapeutic properties but few have been commercially developed. The reasons for this can be many, but the main considerations would be cost-effectivness. Some biotransformations, potentially useful but not developed, are the side-chain cleavage of progesterone to 4-AD or 1,4-ADD, ring A aromatization of 19-nor steroids to estrogens and the 17a- and 21-hydroxylation of pregnane derivatives (Miller, 1985).

The impact of microbiological fermentation has revolutionized the synthesis of steroid hormones and their derivatives from natural raw materials. Approximately 10 are reportedly used in commercial operations (Table 1). The first 4 reactions tabled are commonly used by the whole industry in corticosteroid manufacture and are considered the classical bioconversions. The 1-dehydrotestolactone product is used in the treatment of mammary cancer and is a breakdown intermediate of some strains capable of the l-dehydrogenation of steroids. It is not known by the author if this intermediate is accumulated by a controlled fermentation or the use of a biochemically blocked mutant. The 17-ketone reduction is used for the resolution of racemic seco-steroid products in the total synthesis of estrogens as well as the production of testosterone from 4-AD. The 3P-alcohol-5-dehydrogenation reaction replaces the Oppenauer oxidation favoured by most companies. The reasons for the preference for the chemical process are unknown by the author. All companies are secretive with regards to processes involving mutants and the partial degradation of sterols, however considering ^-sitosterol and cholesterol rival stigmasterol as a steroid source, one could assume that other major manufacturers have similar processes. 17

TABLE 1. Reported Industrial Use of Microorganisms for Steroid Transformations.

Transformation Typical reaction Microorganism Manufacturer lla-Hydroxylation Progesteronella-hydroxyprogesterone Rhizopus nigricans Upjohn Co.

llp-Hydroxylation Substance S^ Cortisol Curvularia lunata Pfizer Inc. Gist-Brocades. Substance S fluoro-,hydroxyl- and methyl- derivatives-> Cortisol synthetic C. lunata Schering AG. analogues Merck

16a-Hydroxylation 9a-fluorocortisol9a-fluoro- Streptomyces Lederle, 16a- hydroxycortisol roseochromogenes E.R.Squibb

1-Dehydrogenation Cortisols prednisolone Arthrobacter simplex Schering AG.

Bacillus lends

6a-fluoro-16a-methyl-corticosterone fluorocortolone. II It

dienediol-> trienediol Septomyxo ajflno Upjohn Co.

Lactonization(^) Progesterone-^ 1-dehydrotesto- Cylindrocarpon lactone radidcola E.R.Squibb

17-ketone reduction 4-AD-> testosterone Saccharomyces sp. Schering AG rac-secosteroid secosteroid Saccharomyces uvarum "

3P-alcohol-5- 21-acetoxy- 17a-hydroxy- Corynebacterium dehydrogenation pregnenolone Acetylated mediolanum Soviet Union Substance S

3,17,21-triacetoxy-5-pregnene- Flavobacteriwn Schering AG 20-one^ Acetylated Substance S dehydrogenans Gist-Brocades.

Partial Degradation (d) ^-sitosterol ^ 4-AD,l,4-ADD and/or Arthrobacter G.D.Searle 9a-hydroxy-androstenedione Mycobacterium fortuitum Upjohn Co. Af. spp. Henkel P-sitosterol 4-AD M. spp. Schering AG. Cholesterol 4-AD and 1,4-ADD M. spp. Mitsubishi

(a) From Sebak & Perlman (1979), Smith(1984) and Martin(1984) (b) Precursors in production of Medrol and similar compounds (c) See text (d) Mutants blocked in degradation of sterols 18

1.5. ALTERNATIVE BIOTECHNOLOGICAL PROCESSES.

Not all biotechnological processes to be investigated in recent years have been conimercially developed although some have shown promise at a laboratory scale. The biosynthesis of steroid starting materials using plant cell cultures of Solanaceae and Dioscorea has proved to be uneconomic to date, but this may change as the technology improves. The production by yeast cells of ergosterol, a by-product in the preparation of vitamin D, could also be used as an alternative steroid source. Ergosterol can be degraded to progesterone by adding an Oppenauer oxidation and a hydrogenation step to the existing Enamine process, however extraction costs and the extra steps exclude its use (Kieslich, 1985). The biotransformation of steroids by plant tissue cultures is another biotechnological area receiving much attention. For example, callus tissue of Nicotiana tobaccum (tobacco) and Sophora angustifolia can reduce 3-keto-4-pregnene compounds to 3p-hydroxy-5a-pregnanes and Digitalis purpurea can 16p- hydroxylate certain pregnanes (Furuya et al, 1972). More recentiy, suspension cultures of D. lanata have been shown to be capable of glycosylation of the cardiotonic steroid, digitoxigenin with the product excreted into the medium (Vanek et al, 1986). The glycosylated derivatives are more water soluble, a useful property for an intravenous drug.

1.6. FERMENTATION PROCESSES AND ALTERNATIVES.

A biotechnological process generally would require highly technical equipment and a range of expensive measurement and control instruments (Kieslich, 1980a). As with any other process, there are advantages and disadvantages which must be taken into account, some of which are summarized in Table 2. 19

TABLE 2. Comparison of Biotechnological Processes with Chemical Methods.(^)

Advantages Disadvantages

1) Stereospecificity of enzymatic Often lower volume/time efficiency. reactions. 2) Introduction of chiral centres.

3) Coupling of several reactions in one fermentation.

4) High yield of complicated structures. Sometimes needs expensive purification procedures.

5) Mild reaction conditions. Sterile operation.

6) Low temperature (except for Energy required for sterilization and sterilization). aeration. 7) Use of cheap and easily available Yield variations due to biological components as media and substrate. fluctuations or component differences in batches of media.

8) Operations in aqueous media (except Sewage contamination with consumed for product recovery). media.

9) Low substrate concentration

10) Reduced chemical waste and recycling Waste biomass (especially with of extraction solvents. Mycobacterium).

11) Products not available by chemical Control and regulation parameters, synthesis. safety controls by health authorities.

(a) From KiesHch (1980a) 20

Most commercial processes are stirred vegetative cell cultures in an optimized batch fermentation. Substrate concentrations generally range between 0.2-2.0 g/L due to the low solubility of most steroids although higher concentrations are sometimes achieved. Each process has its own characteristics but the general procedure for commercial operations is the seeding of the production fermentor (up to 70,000 L size) followed by optimized cell growth (induction may be needed), then the addition of the substrate (Smith, 1984). Process times range from 48-100 hours depending on a variety of parameters. Improvements to processes are constantiy being explored, most notably by strain improvements, media optimization and fermentation control. Close monitoring of the process is necessary with continuous steroid analysis usually performed by Thin-layer chromatography or HPLC.

Various methods have been employed to overcome the low solubility of many steroids (Schoemer & Martin, 1980; Smith, 1984; Miller, 1985). The methods mostiy used are: a) dissolving the substrate in a water-miscible organic solvent such as ethanol, acetone, dimethyl sulfoxide or ethyleneglycol prior to addition to the medium. Often this method is limited by the toxicity of the solvent to the organism b) use of an emulsifying agent such as Tween or Span c) continuous slow feeding of the substrate d) pseudo-crystallofermentation where the substrate is finely ground before addition to the medium e) formation of water-soluble salts such as steroid-21-acetates f) addition of polymers into the fermentation broth, for example: Amberlite XAD-2 reportedly enhances the yield of 17-ketosteroids, presumably by selective adsorption of these products owing to 21

micelle formation of the sterol substrate.

One major problem encountered in fermentations where steroid concentrations are above their solubility is the formation of substrate/product aggregations in the aqueous solution. This often drammatically lowers the product yield as much of the substrate becomes "bound" in mixed crystals.

Higher substrate concentrations are often achieved by the use of one or more of the above methods with little effect on yields. For the 1 la-hydroxylation of progesterone, substrate levels can be increased to 0.5 - 5.0 g/L if added as an acetone solution or as a finely ground powder and to levels as high as 50 g/L if added as a powder wetted by Tween 80 (ICieslich, 1985). Normal substrate levels for l-dehydrogenation processes are 0.2 - 0.3 g/L added as a dimethylformamide solution, yet by a pseudo-crystallofermentation process, substrate levels of 400 -

500 g/L have been reported (Smitii, 1984).

Recovery of products is achieved by solvent extractions of the broth

(usually methyl isobutyl ketone) followed by evaporation of the solvent. In fermentations with very high steroid substrate levels, most of the product is out of solution and it is more cost effective to collect the steroid material and mycelia by filtration. The filtrate is discarded and the filter cake extracted with an organic solvent. Generally the yields are between 70 - 90% with the residual either unaltered substrate or unwanted by-products. The required degree of purity is dependent on how near the process is to the finished product. For example; stringent purification would be required for 1-dehydrogenated products, which are used in therapy, whereas the total purification of 11-hydroxylated products would not be necessary since these are raw materials in subsequent conversions.

The desired product is usually purified fromtiie crude product by recrystallization or fractional crystallization. Other methods such as the extraction of 22

17-ketosteroids from fermentation broths by co-precipitation with p-naphthol have been reported (Martin, 1977) as have the use of polymeric absorbant columns for low-volume fermentations. Additional costs are incurred with solvent recovery (particularly from tiie fermentation broth) and the disposal of the fermentation waste (Smitii, 1984).

To minimize solubility problems, conversions in organic solvents or multiphase systems have been explored with the ultimate aim being to design a continuous flow process in the presence of a solvent or a second phase. Steroid conversions were first performed in organic solvents (eg: CCI4) by Buckland et al., (1975) and although the reaction rate of cholesterol to 4-cholestene-3-one was increased 100-fold over the aqueous system, enzymes for further degradation had been inactivated by the solvent. An improvement has been the two-phase system, either liquid-liquid with water and a poorly-water miscible solvent with a high solubility for the substrate and product, or a solid-liquid system (Leuenberger, 1984). An interesting variation recently reported was the 1-dehydrogenation of hydrocortisone by A. simplex in an aqueous two-phase system with the cells held in a dextran-rich bottom phase (Kaul and Mattiasson, 1986). Studies to date show possibilities but improvements in multi-phase processes would be needed to attract commercial interest

The second area of recent intensive investigation has been the use of resting cells, spores and immobilized cells and enzymes. Although some of these variations offer process simplification, most are not sufficientiy developed to compete with large-scale vegetative cell cultures.

Biotransformations with resting cells or spores offer the attraction of decreased conversion times and increased stability. The cells or spores are simply 23 collected and resuspended in water, or buffer containing glucose for energy, and the substrate for bioconversion added. For spores, no enzyme induction is needed even if the relevant vegetative cells require a steroid inducer (usually cholesterol). Ayerst have a process for the lla-hydroxylation of progesterone using Aspergillus ochraceus spores. An alternative process involves the suspension of acetone-dried cells resuspended in buffered water containing the substrate and a synthetic electron acceptor. Neither process is competitive with standard vegetative cell fermentations, but are considered useful for special preparations (Kieslich, 1985; Miller, 1985 and Smith, 1984).

Much attention has recently been given to biotransformations with inmiobilized cells or enzymes. The use of entrapped A. simplex cells in the industrial production of prednisolone from Cortisol has been reported by Cheetham (1980). The attraction of avoiding unwanted side-reactions has spurred research in the use of free or immobilized enzymes, but due to the cost of enzyme isolation, the rapid loss of activity and the need for expensive electron acceptors and co-factor regeneration, the commercial use of enzymes is not seriously considered (Kolot, 1982). Most research has centred on the immobilization of vegetative or dried cells or spores. Results are promising enough to warrent further investigations. Steroid bioconversion with immobilized cells (and enzymes) was first demonstrated by Mosbach and Larsson (1970). Immobilized C. lunata converted Substance S to Cortisol which was subsequently oxidized to prednisolone by immobilized A. simplex cell-free 1-dehydrogenase. The immobilizing agent in both cases was polyacrylamide. In later experiments, A. simplex was co-entrapped in polyacrylamide with C. lunata to enable a single-stage process (Larsson & Mosbach, 1976). Generally cells are entrapped in a porous polymer such as polyacrylamide or alginate (Ohlson et al, 1979) or adsorbed to a water-insoluble solid support such as DEAE-cellulose (Atrat et ai. 24

1980c). The entrapped cells have a far longer half-life (Kolot, 1982). Other researchers have employed modifications such as the use of immobilized cells or spores in the presence of organic solvents, thus developing a continuous flow process. Solvents impose an additional variable to the system with effects on both the cells and the support matrix. The interactions between the cell, support and solvent suggests a great deal of research would be needed to optimize each process (Leuenberger, 1984).

Besides single enzyme reactions, the selective degradation of sterols to required products using immobilized cells has been investigated. Atrat et al, (1980a; 1980b; 1980c; 1981) and Bohme and Horhold (1980) have explored different immobilizing techniques for the transformation of cholesterol esters by Mycobacterium and Norcadia spp. Current batch processses using free cells have a five times higher product turnover rate than processes with immobilized cells suggesting improvements are necessary for the latter processes to be competitive (Kieslich,1985).

1.7. MICROBIAL DEGRADATION OF STEROLS.

An area which has received a great deal of attention as an alternative to chemical methods is the microbial degradation of natural sterols such as p-sitosterol and cholesterol. Processes for the conversion of these sterols to 17-ketosteroids or 20-carboxyl pregnene derivatives have been developed and are considered commercially important. The degradation pathway of various sterols has been elucidated by a variety of authors with Mycobacterium, Norcadia, Pseudomonas mdArthrobacter (Arima^M/., 1969; Dodson and Muir, 1958; Sih and Whitlock, 1968; Hayakawa, 1973). Their efforts led to a general patiiway for the degradation of natural sterols (Figure 6). The primary reaction 25

Figure 6. Common Pathway Intermediates in the Microbial Degradation of Sterols.

HO XCr

Cholesterol P- sitosterol

COOH K\ COSCoA

COOH COOH 24-Carboxyllc acids I

17-Ketosteroids

Unstable 9a- hydroxy OH steroid

9(10)-secophenol HO

0 HO OH Hexahydroindan derivative 0 HOOC^ 3,4- Diol 26

in all cases was the oxidation of the 3p-hydroxy group followed by the isomerization of the 5-double bond. The terminal carbon of the side-chain, be it C27, C28 or C29 was oxidized to a carboxyl acid, then shortened to a common intermediate, a 24-carboxylic acid. Further degradation to a 17-ketosteroid is by a mechanism believed to be similar to the P-oxidation of fatty acids. Oxidation of the steroid skeleton involves 9a-hydroxylation followed by l-dehydrogenation or vice versa. In either case the introduction of a 1,2-double bond to a 9a-hydroxylated steroid causes instability to the integrity of the ring system. The resulting metabolite undergoes cleavage and aromatization of the B ring via a non-enzymatic reverse aldol type reaction to produce a 9(10)-secophenol. This compound is believed to be hydroxylated at C4 and the resultant 3,4-diol is opened at the A ring by a meta cleavage reaction. In subsequent enzymic reactions, ring A is degraded to yield a hexahydroindan propionic acid which is further metabolized to CO2 and water.

Most microorganisms investigated to date degraded the 4-AD metabolite through the 9(10)-seco pathway. It is thought that in most systems side-chain degradation and ring cleavage occurs independentiy and simultaneously and that reaction rate differences and other unknown factors cause accumulation of specific intermediates. If the side-chain degradation is blocked, the ring system attack will proceed and accumulate partiy-oxidized ring metabolites. By the same token, 17-ketosteroids will accumulate if ring cleavage such as 9a-hydroxylation or l-dehydrogenation is blocked. The latter compounds were seen as commercially useful since several chemical procedures are known for the construction of the 17a-hydroxyprogesterone side-chain. Methods employed to inhibit one or both of these enzymes have been: a) structural modification of the substrate thus preventing the enzyme attack of tiierin g system 27

b) inhibition of the 9a-hydroxylase with complexing agents c) mutation of the microorganism.

Dodson and Muir (1958) and Levy and Talalay (1959) reported the conversion of 19-hydroxy-4-AD into estrone by Pseudomonas sp. The same reaction was reported with Norcadia sp. by Sih and Whitlock (1968) who also demonstrated the aromatization of 19-nor steroids with the concommitant side-chain degradation. Organisms which use the 9(10)-seco pathway are unable to metabolize estrone. The same investigators reported the introduction of a 6p,19-oxido bridge blocked the 1-dehydrogenation reaction. Thus a means to produce estrones and 4-AD derivatives in one fermentation step by the use of modified substrates became available. An approach developed later was the fermentation in the presence of iron-chelating agents such as 2,2'-dipyridyl or 8-hydroxyquinolone. Studies of the 9a-hydroxylase of Norcadia sp. have shown the enzyme to be a mono-oxygenase consisting of several proteins forming an electron transport chain (Chang and Sih, 1964; Strijewski, 1982). The presence of ferrous ions is essential for the enzyme's activity. This method was taken up by Japanese companies to improve yields of 4-AD and 1,4-ADD (Schoemer and Martin, 1980).

1.8. CONVERSION OF STEROLS BY MUTANTS.

The degradation pathway was elucidated by the isolation and ordering of catabolic intermediates, some of which were seen as useful precursors for the manufacture of steroid drugs. Investigators were able to produce mutants biochemically blocked from totally degrading the steroid nucleus without the necessity of substrate modification or chemical inhibitors. The first process used UV-induced mutants of Mycobacterium sp. to produce 17-ketosteroids (Marsheck 28

et al, 1972) and was quickly followed by many others. To compete with the available technology, the substrate concentration needs to be significantly higher than 1 g/L together with high yields. At present, the Upjohn Co. is conceded to possess the best mutants (mainly M. fortuitwn ) of which some are capable of converting 10 g/L of P-sitosterol to 4-AD andl,4-ADD in approximatly 300 hours with high yields (Shoemer & Martin, 1980). Other mutants (eg: M. fortuitum NRRL B8119 by Upjohn and Corynebacterium sp. DSM1444 by Henkel) accumulate 20-carboxyl pregnenes, ie: these strains are blocked in both ring degradation and complete side-chain removal. These compounds are considered useful intermediates for alternative C21-steroid synthetic methods. Mutants which accumulate 9a-hydroxy-4-AD compounds are also of particular interest. These derivatives can be chemically dehydrated to a 9(1 l)-ene derivative from which a 9a-halogen or an 11-oxygen function can be introduced, thus replacing 11-hydroxylation processes (Kieslich, 1985). Ring A-degraded tricyclic intermediates, namely the hydroindan derivatives, have been isolated from mutants blocked further down the pathway. These are used as starting materials in the synthesis of retrosteroids which cannot be produced by biological means because of their reversed stereochemistry (Martin, 1984).

1.9. BILE ACIDS AS AN ALTERNATIVE SUBSTRATE.

The use of gall bladder bile of slaughtered cattie and sheep as a starting material has been proposed by Park (1981) as an altemative to phytosterols and cholesterol. The bile comprises about 11% solids of which approximatiy 60% is the glycine or taurine conjugate of cholic, deoxycholic or chenodeoxycholic acids. The major constiments are the conjugants of cholic and deoxycholic acid (3:1 ratio respectively). Park (1981) estimated that approximatly 25 tonnes of these conjugants could be extracted per million cattle slaughtered and this steroid 29 resource has a potential use in microbial bioconversions to useful steroid intermediates.

The cholic acids are the trivial name of cholane derivatives of which the terminal side-chain carbon is changed from -CH3 to -COOH. The position of the hydroxy groups denotes the common trivial name, for example 3a,7a,12a- trihydroxy-5|3-cholan-24-oic acid (or 3a,7al2a-trihydroxy-5p-cholanic acid) is commonly called cholic acid. Cholic acid and some of its analogues which are referred to in this thesis are shown in Figure 7. All cholanic acid derivatives in this work are of the 5|3-series, thus the 5p configuration will subsequently be omitted in future names. Where possible the trivial names will be used.

As previously mentioned, a process using deoxycholic acid for cortisone production was developed by Merck and Co., but was considered inefficient and discontinued. The process was later simplied from 37 to 11 steps although exact details are not known. Currendy the only known manufacturer using a bile acid process is Rochelle of France (R.J. Park, personal comm.). The cholic acid constituent is converted to deoxycholic acid. The 12a-hydroxy group is removed and an 11-keto group introduced. Partial degradation of the side-chain and oxidation of the A-ring results in cortisone. All the above reactions are chemical, the only microbiological step is the biotransformation of cortisone to prednisone with A. simplex . The only other known medical use for bile acids is the treatment of patients with bile disorders.

1.10. MICROBIAL DEGRADATION OF BILE ACIDS.

Microbial transformations of the bile acids were considered by Hayakawa (1973) to have no relevance for commercial exploitation. Hayakawa 30

Figure 7. The Bile Acids and Analogues, Their Trivial and Systematic Names and Structures.

COOH COOH OH

HO OH H

5p-cholanic acid Cholic acid, 5p-cholan-24-oic acid 3a,7a,12a-trihydroxy-5p- cholanic acid

Deoxycholic acid Chenodeoxycholic acid 3a,12a-dlhydroxy-5p-cholanic acid 3a,7a- dihydroxy-5p-cholanic acid

COOH COOH ,CCr HO HO H OH

Hyodeoxycholic acid Lithocholic acid 3a,6a-dihydroxy-5p-cholanic acid 3a- hydroxy-5p-cholanic acid

COOH COOH

23,24-bisnorcholanic acid Etiocholanic acid 5p-pregnane-20-carboxylic acid 5p-androstane- l7p-carboxylic acid 31 also reviewed in detail the microbial degradation of bile acids (Hayakawa, 1973; 1982). Studies by his group over a number of years, Severina et al, (1969), Bilton^rfl/., (1981) and Leppik (1981) over a range of microorganisms resulted in common metabolites being isolated. The results indicated that bile acids were degraded by one of two pathways depending on the organism used. The main features of the 2 pathways are shown in the simplified diagram in Figure 8. It is interesting to note that the 3-oxo-4-cholenic acid and the hexahydroindan intermediates are similar to the degradation intermediates of cholesterol and other sterols (Figure 6). It is probable that all microbial attacks on steroids follow the same pattern. Obviously both bile acid pathways are incomplete due to either the failure to isolate or identify some intermediates.

Both pathways differ from each other and the previously postulated pathway for cholesterol catabolism in specific areas. As observed with other sterols, Hayakawa postulated that the initial degradation reactions were the oxidation of the 3-hydroxy group and dehydrogenation at C4. In pathway A, side-chain shortening precedes ring fission, noticably after the 3-oxo-4-ene steroid formation, resulting in 17-keto steroids. These androstenes still have the 6a, 7a and 12a-hydroxy groups relevent to the parent molecule. Some strains (5. gelaticus, M. mucosum ) have C22 and C24 metabolites in equilibrium with corresponding 12-oxo derivatives (not shown in Figure 8). These and other non-degradative isolation products have for many years caused confusion in proposed pathways. Another unusual feature often observed with bile acid-utilizing micro-organisms is that not all cholic acid metabolites from a particular organism can be utilized by tiiat organism. The main differences in patiiway B witii respect to patiiway A are the dehydroxylation of C6 and/or C7 is immediately after 4-dehydrogenation and tiie side-chain shortening occurs weU after A-ring oxidation and fission. In botii pathways, ring fission is believed to 32

Figure 9. proposed Microbial Degradation Pathway of Bile Acids ^^^

Cholic acid.

I COOH OH

•OH I COOH

Pathway A •OH Pathway B

Corynebacterium equi Arthrobacter simplex Streptomyces gelaticus Streptomyces rubescens ^ ^ f Pseudomonas spp. Mycobacterium mucosum

OH """

I OH 0

•••OH

I 'COOH 0

0 = HOOC> HOOC

Hexahydroindan Perhydroindan Propionic acid. valeric acid. I ^ ^OH Further Degradation. HOOC ^^

Dicarboxylic acid. (a) From Hayakawa (1973; 1982) 33

occur via 9a-hy(iroxylation although pathways from here to the hydroindan derivatives and the dicarboxylic acid common metabolite are strain-dependent. The perhydroindan valeric acid found in pathway B was transformed by S. rebescens to the hexahydroindan propionic acid via the formation of nitrogen- containing analogues whereas A. simplex used another pathway (Hayakawa, 1973).

It is possible an industrial application may akeady exist regarding the perhydroindan valeric acid metabolite. Schoemer and Martin (1980) state that "hexahydroindan dicarboxylic acids" with structures similar to the perhydroindan and others with various chain lengths, can be synthesized from bile acids and homologous structures using Corynebacterium simplex mutants. These substances have a clinical use in the regulation of serum cholesterol levels in man.

Mason's group, located at Liverpool Polytechnic, UK, have studied the relationship between bacterial bile acid degradation intermediates and colon cancer. Strains of E. coli and Bacteraides spp., isolated from human faecal material, degraded cholic acid under strict anaerobic conditions (Owen et al, 1977; Tenneson et al., 1977a). The products isolated were bisnorcholenic acid and androstene derivatives, suggesting a degradation pathway similar to pathway A outlined in Figure 8. The bulk of their work has centred on bile acid and sterol degradation by Pseudomonas sp. N.C.I.B. 10590, an isolate from animal faeces. This pseudomonad has a wide steroid substrate range, in contrast to strains isolated by Hayakawa (1973). It has been reported to degrade lithocholic acid (Tenneson et al., 1978a), cholic acid (Tenneson et al, 1979c) and its tauro- and glyco-conjugants (Tenneson et al, 1979b), deoxycholic acid (Bilton et a/., 1981), chenodeoxycholic acid (Tenneson et al., 1979a), hyodeoxycholic acid (Tenneson et al, 1979b), cholesterol (Owen et al, 1983b) and P-sitosterol (Owen etal. 34

1985) under aerobic conditions. Resting cells of the strain degraded lithocholic acid (Owen and Bilton, 1984), cholic acid (Owen and Bilton, 1983b), deoxycholic acid (Barnes eM/., 1976; Owen a/., 1984), chenodeoxycholic acid (Owen fl/., 1983a) and hyodeoxycholic acid (Owen and Bilton, 1983a). The authors claim the conditions to be anaerobic although microaerophilic conditions may have applied.

In most bile acid degradations using this organism, similar compounds have been isolated with the only differences being the position of the 6a-, 7a- or 12a-hydroxy group commensurate with the parent substrate. The major products isolated are always 1,4-ADD derivatives, no intermediates believed to be formed further down the pathway from ADD compounds have been isolated by this group in any aerobic fermentation of a bile acid. The 12a-hydroxy group of cholic and deoxycholic acid is untouched to the 12a-hydroxylated-l,4-ADD metabolite and is then epimerized to the 12P-hydroxy isomer. This was the first report of a bacteria capable of 12a-hydroxy epimerization. The existance of intestinal microorganisms with such a capability had previously been inferred by Ali et al, (1966) and Eneroth et al., (1966). The suggestion by Tenneson et al, (1979) and Hayakawa (1982) that the epimerization is necessary before side-chain oxidation of the bisnor cholenic acid metabolites would seem incorrect considering various 12a-hydroxy androstene derivatives have been produced by Pseudomonas NCIB 10590. A more probable reason would be the 9a-hydroxylase enzyme is sterically hindered by the 12a-hydroxy group, hence the requirement for epimerization. As discussed later, all products of 12a-hydroxylated bile acid degradations at the 9a-hydroxylase step and beyond have a 12|3-configuration.

For conmiercial steroid production from bile acids, it would be necessary to be able to compete witii tiie current technology, ie: a process would 35 need low substrate extraction costs, a rapid fermentation with high substrate concentrations and product yield, cheap additional nutrients and few side-products. To facilitate this, the high solubility of bile acids may be seen as an advantage. With these objectives in mind, the C.S.LR.O. Meat Research Division, based in Brisbane, Queensland under the group leadership of Dr. R. J. Park, isolated 78 microbial strains from stale bile or soil located near a bile dehydration vat and studied their growth properties in bile acid media (Leppik et al, 1982). The isolates were divided into groups of 48 typical and 22 atypical Pseudomonas strains and 8 Gram-positive Actinomycetes. Actinomycetes studied by Hayakawa (1973) had shown only acidic intermediates from cholic acid degradation whereas pseudomonads gave rise to both acidic and neutral products (Bilton et al, 1981). All of the C.S.LR.O. isolates were obligate aerobes. Strains other than Pseudomonas generally had a poor steroid substrate range. Most could grow only on one or two of the bile acids. Tht Pseudomonas strains with the best growth rates in various bile acids were chosen for further study.

Two strams PS5-1 and PS6-1 (work numbers MR105 and MR108 respectively) have been lodged with the American Type Culture Collection and have been designated the numbers ATCC 31752 and ATCC 31753 respectively. Two other strains found to rapidly degrade cholic and deoxycholic acid which were not lodged with the ATCC were PS7-1 and PS8-1 (work no. MRl and MR 119 respectively). The strain PS5-1 was characterised by biochemical tests as P. putida biotype B (Smith & Park, 1984). The other three strains were not classified beyond the Pseudomonas group.

Aerobic batch fermentation of 40 g/L bile solids with PS5-1 could be achieved in the absence of additional carbon nutrients with good yields of hydroxylated androstenes. Yields of 12P-hydroxy-l,4-androstadiene-3,17-dione 36

(12|30H-ADD) hydroxylated derivatives were improved with restricted aeration during a 54 hr. 10 litre batch fermentation of 6.7 g/L bile solids with PS5-1 (Park, 1981). Use of this strain with selective fermentation techniques in the degradation of cattie bile solids resulted in high yields of hydroxylated androstenes and approximately 10% relative yield of 9(10)-secophenolic steroids (Park, 1984). With respect to time, media complexity, substrate concentration and by-product formation, PS5-1 held significant advantages over known commercially-used microorganisms. Similar fermentation results were obtained from PS6-1 (Smith & Park, 1984; Leppik, 1980). Their results were in contrast to those of Mason's group whose strain accumulated a broad mixture of intermediates, although 12-hydroxylated androstene derivatives were usually the major product. Leppik (1980; 1981; 1982; 1983) isolated and identified the intermediates of a deoxycholic acid (2 g/L) fermentation by PS6-1. Apart from the metabolites mentioned above, steroids with a complete or partiy degraded side-chain were isolated with the A-ring in various states of oxidation. These compounds led Leppik (1980; 1983) to propose a pathway as outlined in Figure 9. Two compounds, not previously isolated from bile acid degradations, contained unsaturated side-chains with a 3-oxo-l,4-diene A ring. One compound proved to be trans- substituted. The ap-unsaturated carboxylic acids offer evidence that side-chain shortening of bile acids is similar to the fatty acid p-oxidation mechanism, as previously postulated in tiie degradation of other sterols. Derivatives of the 17(20)-ene compound had previously been reported as degradation intermediates of cholesterol, stigmasterol and campesterol by other micro-organisms and were the subject of patent applications (Kieslich, 1980b).

The major difference oftiiis patiiwa y totiiose postulate d by Hayakawa (1982) and Bilton et al. (1981) centres on when the side-chain degradation begins. These authors proposed side-chain degradation began after the A-ring 37

Figure 9. Proposed Deoxycholic Acid Degradation Pathway by Pseudomonas spp. ^^^

XOOH OH" /h Deoxycholic acid.

OH 0 f COOH OH F I 2C I OH 0 .COOH L ff^

HO I I COOH I

OH 0 rVS 3C HO OH OH 0 I

0 0 = OH 0 HOOC-

Further Degradation

(a) From Leppik (1983)

Shadowed letters are for compound identification (see text) 38 was oxidised to a 3-oxo-4-cholenic acid derivative and that l-dehydrogenation occurred at any stage thereafter. The isolation of 3a-hydroxy- and 3-oxo-bisnorcholanic acid led Leppik (1983) to propose that the A-ring and side-chain oxidations are independent of each other and can occur simultaneously. Hayakawa (1982) contends that the above two compounds are the reduction products of other metabolites further down the pathway which have been reduced during the isolation and identification process rather than true intermediates. The possibility of some of the isolated compounds being non-degradative side-products also cannot be discounted. The failure by other research groups to isolate similar compounds could either support Hayakawa's contention or provide further evidence that strains of the same genera may have variations in their catabolic pathways. That Pseudomonas sp. NCIB 10590 differs from the CSIRO strains is beyond doubt considering the former can degrade P-sitosterol and cholesterol whereas the CSIRO strains cannot degrade steroids with a 5-6 double bond or a side-chain longer than C24 (R.J. Park, personal communication). The former strain has also been reported capable of reducing 17-keto androstene derivatives to 17p-hydroxy steroids (Bilton et a/., 1981; Owen et al, 1984).

Whether l-dehydrogenation occurs prior to or after complete removal of the side-chain, or the timing is not obligatory, is unclear but may rely on strain variation. Leppik (1981) isolated only hydroxylated-l,4-androstadienes using PS6-1 whereas Park (1981) and Smith & Park (1984) isolated hydroxylated 4-androstenes and 1,4-androstadienes from PS5-1 degradation broths. The isolated secophenol steroid indicates ring fission follows the 9a-hydroxylation step previously mentioned. The secophenol is most likely 4-hydroxylated to a seco-catechol followed by meta fission. Leppik (1981) isolated an indane-lactone which was assumed from previous publications to be a by-product of the postulated perhydroindan propionic acid previously isolated from other bile 39

acid-degradation systems.

As intermediates from Figure 9 will frequently be reported in the remainder of this thesis, each structure has been designated a letter. The compound 12a-hydroxy-3-oxo-4-cholenic acid may also be referred to as CE. The first letter coming from the A-ring structure and the second from the side-chain.

The structural similarity of the carboxylated pregnenes (CG and DG) and the 1,4-androstadienes (I and J) to steroid precursors akeady used by steroid manufacturers raised the possibility of the commercial use of these bacteria together with the previously under-utilised Australian bile acid market. Fermentation variations were limited in their ability to increase the yield of most of the metabolites. Increased yields and the accumulation of certain metabolites could be possible by the genetic manipulation of the bacteria as ahready seen with the mutation of cholesterol- and P-sitosterol-degrading Mycobacterium spp. With this purpose in mind, work reported in this thesis was undertaken with the object of generating blocked mutants capable of accumulating steroid intermediates in high yields together with a genetic characterisation of the bile acid catabolic pathway.

Most patents regarding steroid-accumulating mutants refer to Gram-positive microorganisms or yeasts which degrade phytosterols. Such processes have inherant problems such as low substrate solubility and expensive complex media, together with slow growth and transformation rates. Two patent applications specifically mention deoxycholic acid (DCA) fermentations with mutants. The mutant strain Corynehacterium sp. DSM 1444 converted 100 g DCA in 5 litres of medium in 96 hours to a mixture of 12-oxo and 12a-hydroxy mono- and di-unsaturated pregnane carboxylic acids in good yields (Bahn et al. 40

1982). The product mixture was not considered commercially useful. Tsuji et al, (1983) claimed a microbial process for the production of 12 hydroxy-ADD. If 12p-hydroxy-ADD was required, the wild-type Pseudomonas putida D4014 (isolated from soil) was used in a culture medium containing DCA. If the 12a-isomer was required, the NTG-derived mutant P. putida D4014-A1099 was used. At 5 g/L of DCA, both in 100 ml shake flasks and 4.5 L fermentations, the mutant gave yields of approximately 90% of theoretical yields in 28 hours. At the higher concentration of 20 g/L of DCA with shaking over 6 days, the yield dropped to about 29% of theoretical, indicating revertants had been selected and degraded the product. In the latter process, the claimants did not indicate the yield after 28 hours incubation. The use of the wild-type would seem to be inefficient as in 100 ml cultures of 10 g/L of DCA after 48 hours incubation, yields of about 40% of theoretical were obtained. In all cases the authors claimed the product to be 95% pure 12-hydroxy-ADD even though the growth conditions were aerobic (Tsuji et al., 1983). In contrast, Leppik (1980) using the organism PS6-1 with well-aerated fermentations at 2 g/L of DCA, found low yields of mixed products. Similar results were obtained with Pseudomonas sp. NCIB 10590 at 1 g/L of DCA (Bilton et al, 1981). Much higher purity and yields of 12P-hydroxy ADD were claimed by Smith & Park (1984) when using PS5-1 with reduced aeration.

1.11. GENERAL PROPERTIES OF TRANSPOSONS.

Transposon-associated determinants are mainly antibiotic resistance although some encode for enterotoxins, sugar utilization or arginine biosynthesis or other functions (Kleckner, 1981). There are 3 general classes of transposons, composite transposons, the Tn3 transposon family and a mixed group of unclassified transposons (Freifelder, 1987). Composite transposons have long (800-1500 bp) inverted or direct terminal repeats which are IS or IS-like elements 41

bracketing a central segment in which are encoded the determinants and possibly other functions. Composite transposons such as Tn5 and TnlO encode a functional transposase and its repressor gene within one of their terminal repeats whereas the other terminal repeat unit has slighdy diverged leaving only a residual transposase activity (Calos & Miller, 1980). The Tn3 family only have terminal inverted repeats of 30-40 bp. The uncategorized transposons either have short direct or inverted terminal repeats of varying lengths. Within the Tn3 family and presumably the unclassified transposons, the transposase enzyme/s are encoded in the central part of the transposon (Calos & Miller, 1980). Some authors also class the IS elements as transposons. IS elements are short (750-1500bp) and only encode their own transposition. Transposons carry associated determinants as well as their transposition genes and as such are longer than IS elements. All IS elements have terminal inverted repeats (10-40bp). Transposons move precisely and it is commonly believed the short terminal inverted repeats are recognition sequences for the transposing enzyme transposase as the ends of transposons must be intact for transposition to occur (Watson et al, 1987).

Most transposons can insert randomly into structural genes of phage or bacterial genomes causing recognizable insertion mutations. The affected gene may be completely inactivated and transposition into an operon will exert strong polar effects on the expression of downstream genes (Kleckner, 1981). Many transposons and IS elements have multiple stop codons in all three reading frames in both orientations which could explain their strong polar effects (Freifelder, 1987). Often transposon insertions generate chromosomal deletions upstream or downstream of the insertion site (Kleckner et aU 1977). The specificity of insertions for composite transposons seems to be totally random within the E. coli chromosome although certain regions within specific genes are preferred for insertion (Berg & Berg, 1983). The insertion sites for other transposons are not 42 so randomly distributed. Tnl, Tn3 and Tn9 preferentially integrate into certain regions of the E. coli genome, yet have many integration sites within these regions (Calos & Miller, 1980). Upon integration, a short segment of the target DNA (usually 5 or 9 bp) is duplicated and the transposon is inserted within the target and the duplicate. The length of the target sequence is constant for each transposon. DNA sequence analyses have found no extensive homology between the transposon and insertion sites.

The mechanisms that limit transposition frequencies within a host organism are not fully understood and conflicting theories have been proposed. One school of thought suggests that after integration, transposons such as Tn5 are reported to continue transposing with future generations. With transposons inserted in multiple sites, resultant homologous cross-overs can create deletions, inversions and other genomic rearrangements which often can be deleterious to the cell (Berg & Berg, 1983). Usually within surviving cells, the rate of transposition decreases and the culture acquires a stable number of transposons per cell (usually one) depending on the transposon and perhaps the organism (Freifelder, 1987). In contrast to the above, there is evidence to suggest that there is far more control. Host proteins necessary for transposition can be regulated by the host itself as well as mechanisms within the transposon. In Tn3, the multi-functional protein resolvase is capable of repressing the synthesis of both the transposase and itself (Chou et a/., 1979). With Tn5 and TnlO, the transposase promotor is very weak and subject to dam methylation controls (Roberts et al, 1985; Krebs & Reznikoff, 1986). Tn5 also seems to have a post translational inhibitor protein called p2, encoded in the IS50 element which acts against Tn5 transposition (Yin & Reznikoff, 1988).

Transposition does not require the rec gene products needed for 43

classical recombinations, instead transposons encode a transposase protein which is necessary for transposition. Two models for transposition have been proposed, conservative and replicative (Watson et a/., 1987). In the conservative model, the transposon is clipped from the donor DNA by double strand cleavages at each end of the transposon and inserted directiy into staggered cuts within the target DNA. DNA polymerase then completes the target sequence duplication. The donor (minus transposon) is lost but replication of another copy restores the loss. The replicative model again involves single strand breaks within the target sequence, but each transposon end is cut only once. Strand seperation between nicks and the joining of non-homologous strands results in two replication forks being formed. The result is a cointegrate, containing the donor flanked by two copies of the transposon and the target sequence, within the target DNA. Resolution, possibly by recombination between the transposon copies, yields the donor and recipient each with a copy of the transposon flanked by a direct repeat of the target sequence, the length most likely being the number of base pairs between the staggered cuts. Neither model explains how the target sequence and the transposon are linked although host cellular factors have been implicated in transposition of Mu (Craigie et al, 1985) and Tn5. In the case of Tn5 transposition, the transposase uses the outer ends of the compound transposon as substrate. When IS50 transposes, the outer end and the inner end of IS50 are the substrates. The sequence of Tn5 which constitutes the minimal functional outer end for transposition has been defined and was found to be homologous to a repeated sequence in the E. coli origin of replication. Purified dm A protein will bind to both the outer end of Tn5 and these repeated sequences and has been shown in vivo to be necessary for full transposition efficency (Yin & Reznikoff, 1987). The replicative model is the most favoured for the majority of transposons, especially those of the Tn3 family which have a revolvase gene within the central part of the element. Berg & Berg (1983) argue that Tn5 and Mu insertion after 44 infection are always conservative. The choice between the two models could depend on whether the second set of cuts at the transposon ends (ie:from singl e strand to double strand cleavage) occurs before the onset of replication (Biel & Berg, 1984).

Even though the biochemical and molecular details were not fully understood, Kleckner et al., (1977) proposed advantages in the antibiotic- resistance properties of transposons for some genetic manipulations. These elements offered an alternative and often easier method for the transfer of unselectable markers and cryptic plasmids, mapping and replicon fusion and the isolation of mutants and cloning of genes which lack a readily selectable phenotype.

1.12. TRANSPOSONS AS MUTAGENIC AGENTS AND VECTORS FOR THEIR INTRODUCTION INTO RECIPIENTS.

Transposons have been used in many bacterial species as mutagenic agents. In general mutations are found to be quite stable with reversion frequencies in the range of 10"^ and below. Reversion to the wild type is usually concommitant with the loss of the associated antibiotic-resistance determinant indicatingreversion i s by loss of the transposon (ie: excision) rather than a shift to a new site. Such revertants also have only one copy of the target sequence (Berg & Berg, 1983). Mutations inactivating the Tn5 transposase gene did not affect the frequency of reversion indicating that transposition and excision are seperate functions (Berg & Berg, 1983). They have proposed excision is a result of the failure to copy Tn5 and the inner core of the dupUcated target sequence rather than the "cutting out" of the transposon and a copy of the target sequence. They suggest that a loop formation due to the annealing of the ISregions woul dresult i n 45

DNA copying errors during synthesis.

Transposons can be introduced into bacteria by the use of "suicide" phage or plasmid vectors. Phage vectors are either defective in an essential integration gene or carry temperature-sensitive mutations which prevent lysogeny (Kleckner et al, 1977). Often due to packaging size restrictions, non-essential phage genes must be deleted to allow for the introduction of the transposon into the vector, Transmissable plasmid vehicles, if available, are the preferred choice. Maintenance of the plasmid vector can be prevented by many means. An RP4 mutant, temperature-sensitive for maintenance, was used to introduce Tnl into E. coli (Hayarama et al., 1981). A temperature-sensitive F ' plasmid affected in replication was used to introduce TnlO into a Salmonella strain while other researchers have used incompatability to prevent maintenance of the donor (cited in Kleckner et al, 1977). For non-enteric recipients, constructed hybrid vectors are the most conunonly used. PI group R plasmids can infect most Gram-negative bacteria. Beringer et al., (1978) used the property that the insertion of the lysogenic phage Mu into R plasmids reduced the transfer frequency and those that did transfer were highly unstable. They reasoned that such a plasmid loaded with a transposon would be an efficient system for the transfer of transposons from coli into Rhizobium spp. Other constructed vectors bear R plasmid transfer genes and the Col El origin of replication. These constructs readily transfer into, but cannot replicate in non-enteric hosts. Such vectors have been used to carry Tn7 into Pseudomonas syringae (Sato et al, 1981) and a range of transposons into Caulobacter, Actinobacter, and Rhizobium strains (Ely, 1985). Vectors with the tra region of the N group plasmid pCUl fused to pl5A repUcons (eg: pACYC184) have been successful for insertion mutagenesis of/?, melioti with Tnl, Tn5 and Tn9 (Selvaraj & Iver, 1983). Simon et al, (1983) adopted a different strategy. The RP4 mobilizing region (Mob) was inserted into cloning 46 vectors with pl5A replicons, the recombinant plasmid was then loaded with a transposon. It is possible the origin of transfer replication is located on the Mob site. These derivatives could be transferred by mobilizing Rec A" strains of E. coli SMIO which had an RP4 derivative integrated in the chromosome. The RP4- derived Mob locus on the vector is presumed to act as a substrate for the integrated RP4 plasmid transfer functions.

1.13. USE OF TRANSPOSONS IN GENE CLONING.

In addition to mutagenesis, Kleckner et al., (1977) suggested transposons could be useful in the cloning of genes where one may not have a readily selectable marker. The suggested protocol was to insert a transposon in or near the gene of interest, then to clone the whole of the transposon plus the required gene from the mutant selecting for the antibiotic-resistance marker of the transposon. Selection for loss of the transposon, ideally by precise excision, would result in a clone of the intact gene. The major difficulty could be the isolation of revertants considering some transposons are reported to revert at frequencies approaching 10"^. One may have to resort to penicillin-enrichment at this stage, or a screening of transposons for a high frequency of reversion, thus limiting the choice of transposons and cloning vectors. A modification to this protocol was used by Scott et al, (1982). The cloned inactivated gene with the transposon were used as a radioactive probe to screen a gene library of the wild-type strain. Providing the transposon and the probe vector have no homology with the DNA of the wild-type or the vector into which the wild-type library has been cloned into, then only those clones which contain wild-type genes corresponding to those on the probe should be observed. Such provisos could place limitations on the choice of transposon to be used and/or the vectors. Furuichi et al, (1985) constructed a transposon derivative of Tn5, called TnV, to 47 circumvent the limitations of homology with the probe vector. The construct was basically Tn5 with the origin of replication of pSClOl cloned between the kanamycin resistance gene and the IS50R element. TnV was introduced into the Myxococcusxanthus genomebyPI::TnV infection. Chromosomal DNA isolated from the TnV- mutagenized strain of interest was digested with restriction enzymes which did not cleave TnV. Self-ligation and transformation of the restricted DNA fragments into an E. coli host, selecting for Km^ transformants, enabled cloning of the inactivated gene in one step. The resultant clones contained only mutant chromosomal DNA and TnV. The authors claimed that clones constructed from TnV-mutagenized strains could be used directly as probes to screen a gene library of the wild-type as no vector or X phage sequences were present in the clones.

A different strategy in the use of transposons as a cloning tool was devised by Grinter (1983). A broad-host-range cloning vector system using two compatible plasmids was based on Tn7 (Sm^, Tp^) transposition into the recipient chromosome. The first plasmid was an unstable RPl derivative, lost without Tc^ selection pressure, containing an inserted Tn7. The transposition functions were replaced with the gene of interest. A HindJR deletion in the transposition locus was filled by subcloning a functional gene of interest. The second plasmid, an RSOOB derivative, provided the missing transposition function. Both plasmids were introduced into the recipient and strains were selected in which the Tn7 derivative had transposed into the chromosome (Grinter, 1983).

Mutational blocks in steroid catabolic pathways could be generated using transposon mutagenesis. A major part of this thesis deals with the use of transposons to generate strains able to accumulate steroid intermediates. The transposons used in this project were Tn5 and Tnl. The suicide vectors used to transfer Tn5 were pJB4n and pSUPlOll and for Tnl transfer, the vector pAS8 48 was used.

The vector pJB4JI, constructed by Beringer et al, (1978) can transfer into but is highly unstable in most Gram-negative strains due to the presence of integrated Mu. A physical map was elucidated by Hirsch & Beringer (1984). The replicon is closely related to the Inc P group plasmid R751 (Figure 10). A substitution of a 2.4kb region of R751 with a section of 4.8kb from R1033 resulted in the plasmid pPHlJI (54.7kb) which conferred Gm^, Cm^, Sm^ and Sp^, the trimethoprim resistance capability of R751 was presumably lost during construction. pJB4JI is about 97.2 kb and was derived from pPHl JI. The site of Mu integration as reported by Hirsch & Beringer is shown in Figure 10. pJB4JI carries Tn5 inserted into the prophage region coding for tail proteins and was reported by Beringer et al, (1978) to confer immunity in E. coli to subsequent infection with Mu, but was unable to produce viable Mu phage. The host strain is E.coli 1830 Pro" Met". The second vector used, pSUP 1011, is carried in a mobilizing donor strain, E. coli SMIO Rec A", Thi", Thr" Leu" which has functional RP4 transfer genes integrated into its chromosome. As illustrated in Figure 10, tiie plasmid (12.5kb) is the replicon pACYC184 with an Inc P-type Mob site cloned into tiie Tc-resistance gene and Tn5 inserted in the Mob site (Simon era/., 1983). As tiie replicon has a narrow host-range, by analogy the vector should be unable to replicate in a non-enteric host.

Pseudomonads are active recipients of RP4 or IncPl group plasmids, but usually cannot maintain Col El-like plasmids. Witii tiiesepropertie s in mind, Sakayanera/., (1978) engineered a hybrid RP4-ColEl derivative fused at the EcdRl site with effective ColEl-dependent replication and maintenance functions. The transposon Tn7 (Sm^Tp^) was mserted into tiie gene for RP4-mediated replication (Sato effl/.,1981). The plasmid pAS8 Tc^ rep-l::Tn7 (pAS8 for 49

Figure 10. The Transposon-carrying Vectors pJB4JI and pSUP1011

pSUP1011, 12.5kb (Simonef a/. ,1983)

Replicon: pACYC184

Mob region H

Tn5

Mob region

Total Mob: about 2.6 kb.

pJB4JI, 97.2 kb. (Hirsch & Beringer, 1984)

P E

R751

7.0 3.3 6.2 3.2 18.0 3.1 H H H E EH Tail-end of Mu C-end of Mu

Map distances between adjacent enzyme cleavage sites given in kb.

E = EcoR1, H = Hindlll, P = Pst1, S = Sal1, B = Bam HI

NB: Not all enzyme cleavage sites are listed. 50

brevity) should transfer into pseudomonads but not be able to replicate. As RP4 contains Tnl (Cb^), pAS8 was considered a "suicide" vector containing two transposons, Tnl and Tn7. The host strain was E. coli AB2463 RecA", Thr", Leu", Thi".

1.14. AIMS OF THIS THESIS.

The aims of this thesis was a preliminary genetic characterization of the bile acid catabolising pseudomonads supplied by the CSIRO Meat Research Division and the derivation of mutants with commercial potential. Chemical and (mainly) transposon mutation methods were used to block the bile acid catabolic pathway to obtain a number of mutants capable of accumulating steroid intermediates.

Another major aim of this thesis was to apply genetic techniques to the generated mutants to study aspects of the mechanism of transposon action. The transposon encoded marker allowed the cloning of inactivated genes which could facilitate analysis of the mutations and the cloning and isolation of wild-type genes involved in steroid degradation. 51

2. MATERIALS AND METHODS

2.1. BACTERIAL STRAINS AND PLASMIDS.

These are listed in Table 3.

2.2. GENERAL PROCEDURES AND CHEMICALS.

2.2.1. Water and sterilization.

The water used in media and large-volume buffers was purified by reverse osmosis by a Millipore central lab supply system. Water was further treated by a Milli-Q deionization unit for ultra-pure buffers involving isolated DNA -eg: ligation and endonuclease restriction buffers. Unless specified, sterilization was achieved by autoclaving at 15psi for 20 minutes. All media was sterilized prior to use.

2.2.2. Growth conditions.

The Pseudomonas and temperature-sensitive E. coli strains were grown at 30°C with shaking in an orbital incubator at 200rpm, other E. coli strains were similarily grown at 37°C.

2.2.3. Chemicals and reagents.

All chemicals were of an analytical reagent grade. The media constituents Tryptone, Yeast Extract, Nutrient Broth and Nutrient Agar were obtained from Oxoid Ltd. (London). Kobe agar, used in minimal media plates. 52

Table 3. Bacterial Strains and Plasmids.

Bacteria.

Bacterial strain Relevant characteristics Source/ reference

Escherichia coli

1830 From E. coli K12 strain J53, pro Beringer a/., (1978) met AB2463 thr leu thi recA ^diioetal, (1981) ED8654 met thi recA , Murray et al, (1977) RRl ¥iom E. coli K12, pro leu thi Bolivar & Backman (1979) SmR HBlOl asRRl, rgcA, Boyer & RouUand-Dussoix (1969) SK1592 thi Kushner(1978) SMIO thi thr leu Simon era/., (1983) (contains a chromosomally integrated RP4-2(Tc::Mu) (ATnl)) V517 Macrina€ra/.,(1978)

MC4100.5F::Mu(cts) ^^ Casadaban (1976) Donated by K. Williams

continued.... 53

Tables (continued) Pseudomonas strains

Strain Work Relevant characteristics SourceAeference no. no. PArl-6 P, arvilla met (TOL) (Austen & Dunn, 1977)

PPl-8 P.putida met Met" mutant of PPl-2 (Wong & Dunn, 1976) PPl-25 P. putida trp (CAM) Trp- mutant of PPl-2 (Hewetson et al, 1978) PP7-2 P. putida trp (pND50) Trp- mutant of PP7-1 (Hewetson et al, 1978) PS5-1 MR105 P.putida biotyp&B, ATCC 31752, Smith & Park, (1984) wild-type CA"*" ATCC 31753, Leppiker a/., (1982) PS6-1 MR108 wild-type CA+ CSIRO collection PS7-1 MRl wild-type CA+ CSIRO collection PS8-1 MRl 19 wild-type CA+ NTG Trp- mutant of PS 5-1 PS5-4 105-16 trp CA+ " Leu- II II II PS5-6 105-17 leu CA+ PS5-7 105-16 Y trp CA± NTG mutant of PS5-4. Gives slow Accumulates phenol and growth on CA with yellowing of catechol secosteroids. medium. Lost large plasmid.

PS5-10 105-16 trp CA" SDS mutant of PS 5-4. Lost large plasmid. Continued 54

Tables (continued) Pseudomonas strains Strain Work Relevant characteristics Source/reference no. no. PS5-16 105(4) Km^ CA± ADD- Tn5-induced mutant of PS5-1 accumulates 12aOH-ADD PS5-18 105(6) Km^ CA± ADD" Tn5-induced mutant of PS5-1 accumulates 12|30H-ADD

PS5-25 105-16(10) Km^CA^ ADD" Tn5-induced mutant of PS5-7 accumulates phenol secosteroid PS6-3 108-8 his CA+ NTG His" mutant of PS6-1. PS6-6 108-12 phe CA+ " Phe" ti II ti PS7-4 1-4 his CA+ " His- II 11 II PS8-2 119-9 arg CA+ " Arg- " " PS8-1 PS8-10 119(2) Km^CA^ Tn5-induced mutant of PS8-1 accumulates OPDC PS8-22 119(107) Km^ Cb^CA^^ADD" Tnl-inducedmutantof PS8-10 accumulates OPDC Plasmids. Plasmid Size(kb) Relevant characteristics Derivation/reference

pACYC184 4.0 Cm^Tc^ Chang & Cohen (1978)

pAS8TcSre!/? -l::Tn7 RP4-ColEl, rep -1rp4, Tc^, Sakanyaner a/., (1978) 75 KmR Cb^CTnl), SmR(Tn7), &Sato^ra/., (1981) TpR (Tn7) Continued.... 55

Tables (continued) Plasmids.

Plasmid Size (kb) Relevant characteristics Derivation/reference pBEElO Tc^ (TnlO), Km^ pRK2013::Tnl0 Ely (1985) pBR322 4.3 ColEl ApR Tc^ BoUvar etal,{\911) pBR329 4.2 Ap^ Tc^ Cm^ Covarrubias & Bolivar (1982) pJB4JI 97.2 KmR (Tn5), Sm^ (Tn5), Gm^, pPHl::Mu::Tn5; Inc PI Beringer et al, (1978) pKan2 7.8 Ap^, Km^ (//mdin fragment ofTn5) Scott era/., (1982) pKT230 11.9 SmR, Km^ Franklin ef a/., (1981) pND200 28.6 KmÂp^Tc^ This thesis *PS5-16 pND201 42.5 KmÂpRXcR This thesis *PS5-16 pND202 14.0 KmÂpRXc^ This thesis *PS5-16 pND203 8.0 KmÂp^TcS This thesis *PS5-16 pND204 24.2 KmÂp^TcR This thesis *105-17(2) pND205 8.0 KmRAp^TcS This thesis *105-17(2) pND206 8.0 KmÂp^TcS This thesis * 105(3) pND207 8.0 KmÂp^TcS This thesis *PS5-18 Continued.... 56

Tables (continued) Plasmids.

Plasmid Size (kb) Relevant characteristics Derivation/reference pND208 8.0 KmÂpRXcS This thesis *105-17(10) pND209 17.2 KmÂpRTc^ This thesis *PS8-10 pND210 8.0 KmÂpRTcS This thesis *1-4(1) pND211 33 KmÂpRTc^ This thesis * 105(5) pND212 24 Km^ Ap^ Tc^ This thesis *105(205) pRK290 20 TcR Dittaerfl/., (1980) pRK2013 48 ColEl with RK2 Tra, Km^ Figurski & Helinski (1979) pSal52 15.5 Cm^Km^Sp^ Tait era/., (1983) pSUP202 6.2 ApR Tc^ Cm^ pBR325-Mob Simon etaL, (1983) pSUPlOl 1 12.3 Km^ (Tn5), Sm^ (Tn5), Cm^, pACYC184::Mob::Tn5; Simon era/., (1983)

RSa 38 Cm^ Km^ Sp^ Ward &Grinsted (1982)

R68-45 59 Km^ Ap^ Tc^ Haas &Holloway (1976)

* denotes Km^ fragment from the strain listed cloned into pBR322. 57 was distributed by HJ. Landgen & Co. Pty. Ltd. (Sydney). Dextran sulphate was supplied by Pharmacia AB. Sigma Chemical Co. (USA) supplied the following chemicals: amino acids and vitamins, cholic and deoxycholic acid, Tris-HCl, Trisma base, EDTA-sodium salt, mitomycin C, Lauryl sulfate (SDS), polyvinyl pyrrolidone (PVP360), Ficoll 400, bovive serum albumin (BSA), ethidium bromide and all antibiotics except the following: Carbenicillin (Carbapen, CSL, Melbourne), Spectinomycin (Trobicin, Upjohn, USA) and Piperacillin (Lederle).

Seakem agarose LE was supplied by Edwards Instruments Co (Sydney) and caesium chloride distributed by Novachem Pty. Ltd. (Melbourne). Restriction endonuclease enzymes were from Boehringer Mannheim or New England Labs Inc. Deoxynucleotide triphosphates, DNase 1, T4-DNA ligase, DNA polymerase 1 and dithiothreitol were supplied by Boehringer Mannheim. The ADD compounds, 7a,12a-dihydroxy ADD and 7a,12p-dihydroxy ADD were isolated and purified at the CSIRO Meat Research Laboratory in Brisbane from the degradation of cholic acid by PS5-1.

2.3 BUFFERS AND SOLUTIONS.

2.3.1. Amino Acids and Nucleotide Growth Factors.

Solutions were made up to 4 mg/ml water, filter-sterilized and stored at 4°C. Amino acids that were difficult to dissolve were heated to 68°C and solubilised prior to filtration. Growth factors were added to media at concentrations recommended by Davis et al, (1980). 58

2.3.2. 25% Glucose.

D-glucose was dissolved in sterile water to 25% (^/v) concentration, filter-sterilized and stored at 4°C over chloroform.

2.3.3. Antibiotics.

Antibiotic stock solutions were aseptically weighed and dissolved in sterile water (or ethanol) at lOO-times concentrations, filter-sterilized and stored at -20OC.

2.3.4. PAS Salts Concentrate.

The minimal salts solution used in PAS liquid and solid media was a lOO-times salt concentrate comprising of MgS04.7H20 (3.75%^/v), MnS04.H20 (0.5%), FeS04.7H20 (0.5%), CaCl2 (0.03%) and ascorbic acid (0.1%) dissolved in sequence at approximately 50°C in water. The solution was filter-sterilized and stored over chloroform at 4°C.

2.3.5. Vogel-Bonner Salts Concentrate.

The 50-times concentrate used in VB media comprised of MgS04.7H20 (1% w/v), 10% citric acid, 5% K2HPO4 and 17% NaNH4HP04.4H20 dissolved in sequence and prepared as per the PAS salts.

2.3.6. Saline plus 10% Nutrient Broth (SNB).

A general diluent for cells being a sterile solution of 0.85% (^/v) NaCl 59 plus 0.25% (W/v) Nutrient Broth.

2.3.7. Citrate Buffer.

Di-scxiium citrate was dissolved to a concentration of lOOmM, the pH was adjusted to 6.0 and sterilized. The solution was stored at 4°C.

2.3.8. Tris-EDTA (TE).

The DNA storage buffer comprised lOmM Tris-HCl (pH 8.0), O.lmM EDTA. The prepared buffer was sterilized at lOpsi for 15 minutes and stored at 40c.

2.3.9. Tris-Acetate-EDTA (TAE).

An electrophoresis buffer consisting of 40mM Trisma-base, 2mM EDTA, 20mM acetic acid, pH 8.1 with glacial acetic acid (Davis et al, 1980). The buffer was prepared at 50-times concentration, sterilized at lOpsi for 15 minutes and stored at 4°C.

2.3.10. Saline Sodium Citrate (SSC)

One times SSC contained 0.15M NaCl and 0.015M trisodium-citrate, pH 7.2. A 20-times concentrate was prepared, sterilized at lOpsi for 15 minutes and stored at 4°C. 60

2.3.11. Denhardt's Reagent (Denhardt, 1966).

A mixture of 0.02% C^/y) each of Ficoll 400, polyvinylpyrroUdone 360 and BSA (fraction V). The reagent was made as a 100-times stock solution using sterile water and stored at -20°C.

2.3.12. Pre-Hybridization Buffer.

The buffer was prepared as below with deionized water and stored at 40c. 5x SSC 20mM Tris-HCl, pH 7.4 5x Denhardt's reagent 200|Xg/ml sonicated denamred Calf Thymus DNA 0.5% SDS

2.3.13. Hybridization Buffer.

Prepared as above with the addition of 50% formamide C/v). The formamide was deionized by gende stirring with 1.5% (^/v) Bio-Rad Mixed Resin AG 501-X8 (D). This buffer worked well when blotting purified plasmids but as the quantity of DNA bound to the nitrocellulose would be lower when Southern blotting genomic DNA or with colony blotting, an improved method was used. Wahl et al., (1979) reported increases in the rate of hybridization up to 100-fold by the addition of 10% dextran sulphate to hybridization solutions, presumably by exclusion of the DNA probe from the volume occupied by the polymer -ie: by concentration of the probe DNA. The buffers used for these experiments were: 61

Component. Pre-hvbrid. buffer. Hybridization buffer.

SSC 5x 5x

Denhardt's reagent 5x 5x

Sodium phosphate (pH 6.8) 50mM 20mM

SDS 0.1% 0.1%

Deionized formamide 50%

Calf thymus DNA 200 |ig/ml. 100 ^ig/ml.

Dextran sulphate, Na salt 10%

2.3.14. Deoxynucleotide Triphosphates.

The dNTP was dissolved in water to a concentration of 20mM and the

pH adjusted to 7.0 using lOOmM Trizma base. Actual concentrations were

determined with a spectrophotometer using extinction co-efficients recommended

by Schleif and Wensink (1981).

2.3.15. Nick Translation Buffer (Schleif & Wensink, 1981).

The buffer (50mM Tris-HCl, pH 7.8,9mM MgCl2, lOmM DTT and 50

|j.g/ml BSA) was prepared as a 10-times concentration (NT xlO) and stored at

2.3.16. Nick Stop Buffer (Schleif & Wensink, 1981).

The buffer (lOmM Tris-HCl, pH 7.4, lOmM EDTA, 0.5% SDS and

200mM NaCl) was used to stop nick translation reactions, the addition of salt allowed for DNA precipitation. 62

2.3.17. Restriction Buffer.

The buffer (20iiiM Tris-HCl, pH 7.8, lOmM MgCl2, 5mM DTT and 100 M-g/ml BSA) was prepared as a 10-times concentrate and stored at -20°C.

2.3.18. Ligation Buffer.

The buffer (40inM Tris-HCl, pH 7.8, lOmM MgCl2, lOmM DTT and 50 |Xg/ml BSA) was prepared as a 10-times concentrate and stored at -20°C. ATP (lOmM, pH 7.0) was prepared separately by dissolving in water and adjusting the pH with O.IN NaOH.

2.3.19. Phenol.

Phenol (Wako) was melted and extracted with an equal volume of 0.5M Tris-HCl (pH 8.0) followed by two extractions with lOOmM Tris-HCl (pH 8.0). The antioxidant 8-hydroxyquinoline was added to the equilabrated phenol at 0.1% (^/v) and the prepared phenol stored at -20°C until required.

2.3.20. Chloroform.

Isoamyl alcohol was added to chloroform (1: 24) prior to use. 63

2.4. MEDIA.

2.4.1. Nutrient Broth (NB).

This generalrich mediu m for the growth of bacteria was prepared by the addition of 2.5% Nutrient Broth and 0.2% Yeast Extract to water and sterilized.

2.4.2. Nutrient Agar (NA).

The solid rich medium was prepared by the addition of 2.8% Nutrient Agar to water prior to sterilization.

2.4.3. Luria Broth (LB).

E. coli was grown in this media consisting of 1% NaCl, 1% Tryptone and 0.5% Yeast Extract The pH was adjusted to 7.4 prior to sterilization.

2.4.4. PAS Minimal Media. (Chakrabaty, 1972).

From IM non-sterile stock solutions, 2.5% (^/v) of K2HPO4,1.25% of KH2PO4 and 4% of NH4CI were added to 91 ml. water per 100 ml. of required media. Autoclavable growth substrates such as Na-cholate were added and the pH adjusted to 7.0. For a solid media, Kobe agar (1.2%) was added. After sterilization, the solution was allowed to cool to about 60°C before the addition of 1% (^/v) PAS salts concentrate plus any antibiotics or growth factors to their required concentrations. At tiiis stage, non-autoclavable growth substrates (eg: Na-benzoate) were added. 64

2.4.5. VB Minimal Media (Vogel & Bonner, 1956).

Kobe agar (1.3%) and water were sterilized and cooled to about 60°C before the addition of 1% C/w) of 25% Glucose and 2% C/\) VB salts concentrate.

2.5. METHODS

2.5.1. Curing of Plasmids.

The procedures are outlined by Negaro et al., (1980) and Rheinwald et al., (1973). The curing agent mitomycin C was added at growth-inhibitory levels of 5 M-g/ml to NB freshly inoculated to about cells/ml. The cultures were grown for 24 hours at 30°C, diluted in SNB and spread onto PAS + lOmM Na-succinate at approximately 100 colonies/plate. Surviving cells (1000) were replica-plated onto PAS plates containing 0.5% cholic acid in order to score and select clones unable to utilize the steroid. Isolated clones were analysed for the presenceAoss of resident plasmids. SDS curing was performed as such: auxotrophic cultures were grown through 3 cycles of growth in NB + 2% SDS. Cultures were diluted in SNB and 2000 survivors spread onto PAS + lOmM Na-succinate + appropriate amino acid at approximately 100 colonies/plate. The plates were replicated onto PAS + 0.5% CA + growth requirement. In both procedures, colonies that failed to grow or grew poorly on the CA media were retained for further testing.

2.5.2. NTG Mutagenesis.

The basic procedure for the derivation of bacterial mutants, using the 65

chemical mutagen N-methyl-N'nitro-N-nitrosoguanidine (NTG) was described previously (Fargie & Holloway, 1965). As NTG induces multiple mutations in linked clusters at the growing fork of replicating DNA, cultures were induced into non-syncronized growth prior to the addition of NTG. This was achieved by adding an inoculum (10^ cells) of overnight culture to 10ml NB every 10 minutes for 0.5 hour. Cells were grown to early exponential phase (90 minutes), cooled on ice and washed thrice in equal volumes of 100 mM citrate buffer. NTG (freshly dissolved in citrate buffer) was added to a final concentration of 100 |Xg/ml and the cells incubated stationary for 30 minutes. The cells were centrifuged and washed twice in sterile saline, transferred to fresh NB and grown for 4 hours. Survivors were spread onto NA at the dilution which yielded about 200 colonies/plate and after growth, replicated onto the selection medium (VB for auxotrophs, PAS + cholic acid for CA" mutants). Reversion frequencies were tested and the mutants screened for growth requirements (auxotrophs) or intermediate accumulation (CA" mutants). Growth requirements were identified by the method of White (1980), -ie: by placing 8 filter paper discs impregnated with growth factor pools around a dried lawn of a mutant spread on VB. Accumulated intermediates were identified after growth in DCA at the CSIRO Meat Research Laboratory, Brisbane.

2.5.3. Filter Mate Conjugation.

Log phase cultures of donor and recipient cells were mixed in a sterile test tube at a ratio as specified in the text. Aliquots of the mixture (1.5ml each) were collected onto two membrane filters (Gehnan, 0.45M,m) with a syringe, the filters then being placed, colonies up, on NA and incubated for 6 hours at 30°C. The trapped cells were resuspended in 3 ml. SNB cooled to 40C. Dilutions (0.15ml) were then spread onto the selection medium. 66

2.5.4. Transposon Mutagenesis.

Transposons were introduced into recipient strains by filter mate conjugation. Dilutions from 10® to lO"^ were spread on the selection minimal medium, PAS + lOmM Na-succinate + relevant antibiotics and amino acids and incubated at 30°C. The dilution tubes were stored at 4°C until the transconjugants grew. The dilution that resulted in 150 colonies/plate was spread on a further 20-50 plates. After growth, the transposon-containing recipients were individually streaked onto PAS plates containing CA, DCA and 12aOH-ADD as the sole carbon source and the growth characteristics compared to the parent strain recorded Those recipients which exhibited impaired growth relative to the parent were scored a further 3 times on the selection medium and retested. Where possible, strains were also tested for the antibiotic resistance marker carried on the suicide vector. The strains were then tested at CSIRO Meat Research Labs to identify and determine the degree of intermediate accumulation.

2.5.5. TLC Identification of Steroid Products.

The method is basically as described by Leppik (1981). Each strain was grown to late log phase in shake flasks containing 25 ml of 20mM Na-acetate media and necessary amino acids. Bile acid salts were then added to 2 g/L and their catabolism followed as such. Aiquots (5ml) were removed 24, 48 and 72 hours after the addition of tiie steroid. The aliquots were acidified witii 0.25ml of 2M HCl and extracted with an equal volume of ethyl acetate. Following evaporation of the ethyl acetate layer, the extract was dissolved in 0.3ml of ethyl acetate-metiianol (4:1) and tiie steroid components separated by applying lO^il to Merck 0.2mm Silica Gel 60 F254 TLC plates. Sheets were developed with benzene-dioxane-acetic acid (70:30:2) solvent, air dried and tiien visuaHzed with 67

anisaldehyde reagent. The migration rate and the colour of each spot was compared with standards to identify the products. Strains which accumulated products were retained by the CSIRO for further testing in fermentors and HPLC analysis.

2.5.6. Small Scale Isolation of Plasmid DNA.

A variety of extraction methods have been described for the isolation of plasmids from pseudomonads (Kado & Liu, 1981; Bimboim & Doly, 1979; Marko et al, 1982; Ranhard, 1982; Wheatcroft & Williams, 1981). AU of these methods were found to be either cumbersome or left large amounts of sheared chromosome. Plasmids hosted in E. coli were extracted by a 10-times scale-up of the method of Holmes & Quigley (1981) from 3.5ml of an overnight culture with a boiling lysis step for 2 minutes. Plasmids from Pseudomonas strains were isolated by a 20-times scale-up of the method described by Crosa & Falcow (1981) from a 15ml overnight culture with the modification that the lysis solution was pH 12.62.

2.5.7. Large Scale Isolation of DNA.

The methods described by Maniatis et al, (1982), Scott et al, (1981) and Marko et al, (1982) either resulted in poor yields of Pseudomonas DNA or were cumbersome and time-consuming. The simpler method was a 20-times scale-up of the Crosa & Falcow (1981) procedure per 1(X) ml late log-phase culture with the lysis buffer at pH 12.60. If necessary, the DNA solution was cleaned by phenol/chloroform extraction or centrifuged to equilibrium in a caesium chloride/ ethidium bromide gradient (p=1.55 g/ml.) as per Maniatis et al, (1982). Ethidium bromide was removed by repeated extractions with water-saturated butanol. The 68

DNA was desalted either by dialysis against TE (pH 8.0) or ethanol precipitation in the presence of 0.1% (^/v) sarkosyl.

2.5.8. Isolation of Genomic DNA.

Total DNA was isolated by the method described above (section 2.5.7) except the pH of the lysis solution was 12.40. The DNA was cleaned by repeated phenol/chloroform extractions rather than the equilibrium gradient step.

2.5.9. Isolation of Mu Phage DNA.

Mu is a temperate bacteriophage which during the lytic cycle enters into an uncontrolled replicative transposition phase about the host genome resulting in up to 100 phage particles per burst cell (Toussaint & Resibois, 1983). The lysogen, E. coli MC4100.5F::Mu 52) was seeded into 6 flasks containing 200 ml LB + lOmM MgSO^ and grown at 30°C to an OD^JQ^J^ of 0.6. Cultures were thermo-induced at 43°C for 50 minutes, then incubated for a further 3.5 hours at 37°C to allow packaging and lysis. Chloroform (5ml) was added to each flask and cultures incubated for an additional 30 minutes. Purification was as per Yamamoto et al, (1970) with some modifications. The precipitation with 10% (W/v) PEG 6(X)0 was stood overnight at APC rather than 1 hour while the caesium chloride step-gradient was omitted and replaced by equilibrium centrifugation (p=1.45 g/ml.) at 100,000g for 24 hours at 6°C. The purified bacteriophage was titred against E. coli Hfr H (» 10^^ pfu/ml). The bacteriophage was de-proteinised as per Maniatis et al., (1982) and the DNA stored at -20OC suspended in TE. The whole procedure resulted in 3 mg of pure Mu DNA. Prior to enzymatic digestion or nick-translation, l|Lig of DNA was incubated at 37°C in the presence of lOO^ig/ml of pre-treated Pronase E followed by phenol/chloroform 69 extractions. This was found necessary as Mu DNA is reportedly resistant to many restriction enzymes, presumably due to a modification of the Mu DNA tiiatoccur s upon induction (Toussaint & Resibois, 1983).

2.5.10. Restriction Endonuclease Digests.

DNA was digested in 1-times restriction buffer plus NaCl added to the concentration as recommended by the manufacturer. Five units of enzyme/|Lig DNA was added and the mixture incubated at the required temperature for 3-6 hours. For genomic digests, usually 20-25 units/|xg were added. Reactions were stopped either by heating at 68°C for 5 minutes or by the addition of EDTA to 20mM.

2.5.11. Dephosphorylation of Spliced DNA.

The terminal 5' phosphate of the vector was removed, to prevent self-ligation, using Calf Intestinal Alkaline Phosphatase (Boehringer Mannheim, molecular biology quality). After digestion with restriction enzymes, restriction mixtures were doubled in volume by the addition of 50mM Tris-HCl pH 8.0, 0.2mM EDTA, 0.05% SDS and incubated witii 20 units of phosphatase at for 2-4 hours. Reactions were stopped by phenol/chloroform/diethyl ether extractions followed by ethanol precipitation of the DNA.

2.5.12. Ligations.

In an ideal ligation reaction, DNA is added to achieve a vector : insert ends molar ratio of 1:2 (Dugaiczyk et a/., 1975). In practice with spUced genomic DNA, it is impossible to calculate the end molar concentration. To circumvent tiie 70 problem, digested genomic DNA (0.5-l^g) was ligated to dephosphorylated vector in a number of reaction tubes with varying quantities of vector ranging from 60-250 ng. Ligation reactions were carried out in ligation buffer plus ImM ATP in the presence of 0.2 Weiss units of T4 DNA ligase (Boehringer Mannheim). Reactions were incubated at 14°C overnight.

2.5.13. Agarose Gel Electrophoresis.

Separation of DNA fragments was achieved by electrophoresis through an agarose gel. The gel was buffered with TAE buffer. Electrophoresis was performed with gels barely submerged in TAE buffer in horizontal tanks, basically as described in Maniatis et aL, (1982). Prior to loading, DNA samples were mixed with a lOx tracking buffer (0.4% bromophenol blue, 0.4% xylene cyanol, 20% Ficoll 400 in water). Electrophoresis was carried out at 3 V/cm for CCC plasmid preparations and at 1 V/cm in the case of linear DNA (usually overnight). The gel was stained in 1 |ig/ml ethidium bromide for 30 minutes and visualized with a UV transilluminator.

2.5.14. Electro-elution of DNA Fragments from Agarose Gels.

A number of published methods to recover DNA from agarose gels (Dretzen et al, 1981; Finkelstein & Rownd, 1978; Weislander, 1979) were attempted and found to result in low yields for DNA above lOkb in size. In addition, reactions with the eluted DNA (nick-translation, Ugation) at times gave poor results, presumeably due to sulphate moities in the agarose which elute with the DNA. The method of choice resulted in high recovery yields with Uttle or no effect on enzymic activity. Agarose gel slices (IBI or Sea Plaque Low-melting agarose) containing the required bands were placed in a dialysis tube (M Wt. 71

cut-off of 20,000 D) and the tube fiUed with V4 x TAE. Electrophoresis was performed at 150 V at A^C with the tubing partly immersed in that buffer. The DNA movement could be followed by UV visualization at 310nm. Electro-elution was continued until the DNA could be seen packed on the positive side of the tubing. After gentle squeezing of the bag (not the gel), the buffer was transferred to an Eppendorf tube. The bag was washed twice with a small amount of buffer which was added to the Eppendorf tube. If yield was critical, the gel was removed, fragmented in an Eppendorf tube and centrifuged at 12,000g for 10 minutes at 4®C with any liquid being transferred to the above Eppendorf. All tubes containing eluted DNA were heated at 65°C for 5-10 minutes, quenched in ice, then centrifuged at 12,000g for 10 minutes at 4°C and the supernatant transferred to new tubes. The DNA was precipitated with ethanol and resuspended in TE.

2.5.15. Transformation of Plasmid DNA.

The method most commonly used where the recipient strain is E. coli is the CaCl2 procedure first described by Lederberg & Cohen (1974). All transformations in this project were by the CaCl2-Tris method outlined in Maniatis et fl/., (1982) with the modification that 0.4 ml of overnight culture was used to inoculate 30 ml of LB + lOmM MgS04. The Mg^"*" addition has been reported to increase the efficiency of transformation fifteen-fold(Hanahan , 1983).

2.5.16. Labelling DNA by Nick Translation.

The basic protocol was first described by Rigby et al, (1977) and modified by Schleif & Wensink (1981) to yield labeUed DNA of 5 x lo'^ cpm/p,g DNA. Purified DNA (0.5-1.0 ^ig) was mixed with 50 ^Ci of [a-^^P] - deoxynucleotide triphosphate (Amersham, 3000Ci/mmol.), 4 nmole of each 72

unlabelled NTP and 5 ^il NT xlO buffer. DNase 1 (Boehringer Mannheim, 3000

U /mg) was diluted in NT buffer such that 0.5 m Units was added to the reaction.

DNA polymerase 1 (2.5 Units) was added together with water to bring the final volume to 50 |xl. The mixture was incubated for 2 hours at IW and the reaction stopped by the addition of 150 Nick Stop buffer. The use of Sephadex G-75 columns was time-consuming, so unincorporated triphosphates were separated from the labelled DNA simply by precipitation of the DNA with isopropanol followed by a wash in 95% ethanol. The pellet was resuspended in 1 ml TE and boiled for 10 minutes to denature the DNA. A 5|il sample was measured for

Cerenkov counts, with adjustments to cpm, using a LKB 1210 Ultrobeta Liquid

Scintillation Counter for total radioactivity measurements.

2.5.17. Southern Transfer of DNA and Hybridization.

2.5.17.1. Transfer of DNA from Agarose Gels.

The method of Southern (1975) is reported to be non-conductive to the transfer of large DNA fragments (Wahl et al, 1979), so an alternative procedure was used. The protocol is basically that of Smith & Summers (1980) with some modifications, all procedures are at room temperature. After electrophoresis, the

DNA was acid depurinated to facilitate the transfer of large DNA fragments by soaking the gel twice in 0.25M HQ for 5 minutes then rinsed in water. DNA was denatured by soaking the gel twice in approximately 400 ml 0.5M NaOH, 1.5M

NaCl for 20 minutes. After a further rinse with water, tiie gel was saturated in the transfer solution of IM NH4-acetate, 0.02M NaOH for 30 minutes. Transfer of the DNA was as per Soutiiem (1975), but using tiie acetate transfer solution.

Solution is drawn tiirough tiie gel by the capillary action of paper towels placed on top of the gel and nitrocellulose filter (Schleicher & SchuU BA85, 0.45^m pore 73

size), thus carrying the DNA which is trapped onto the filter. At the completion of transfer (generally overnight), the filter was gently rinsed in 2x SSC for 30 seconds then allowed to dry on blotting paper, with the DNA up. The DNA was irreversibly fixed on the filter by baking at 8(PC for 2 hours in a vacuum oven.

2.5.17.2. Colony Blotting.

The method is an adaptation of that described in Maniatis et a/., (1982). Cells were evenly spread onto selection medium so that 10(X) colonies grew on an 82 mm plate or 3000 colonies on a 142 mm plate. The plates were incubated until very small colonies appeared, this prevented overlapping of the colonies. A nitrocellulose filter disc was wetted on a second agar plate containing the selection medium for 5-10 minutes, then removed and the side not put in contact with the agar carefully placed on top of the colonies. Small holes were pierced, around the periphery, through the filter and into the agar with an 18-gauge needle which had been dipped in Indian ink. The colonies with the filter still on top were incubated for 1 hour then the filter removed and placed, colony side up, onto the medium previously used for wetting. Both this plate and the original containing the colonies were further incubated and removed when distinct colonies could be seen on the filter or the agar. The original plate was stored at 4°C until required for identification of "light-ups". The filter disc was laid colony side up for 3 minutes onto 2-3 sheets of blotting paper sattirated with 10% SDS. The filter was transferred onto paper samrated with 0.5M NaOH, 0.5M NaCl (cell lysis and DNA denattiration) and left for 15 minutes at 37°C, then placed onto paper soaked in the neutralizing solution (0.5M Tris-HCl, pH 7.0, 2M NaCl) for 10 minutes. Filters were washed by placing onto blotting paper wetted with 2x SSC for a further 10 minutes, then allowed to dry on paper before baking in vacuo as previously described. 74

2.5.17.3. Hybridization.

The hybridization method used was dependent on the nature of the experiment. The first method was used when DNA was transferred from a gel onto nitrocellulose filters and the second for colony blotting. Both methods use 50% formamide as DNA: DNA association proceeds at lower temperatures in the presence of formamide (M^Conaughy et al., 1969) thus saving "wear and tear" on nitrocellulose filters. In all cases, the DNA being used as the probe or the plasmid from which it was isolated was blotted onto the gel to ensure the integrity of the probe. Most gels had X-Hin din standards which were subsequently blotted onto the filter. By adding radioactive X to the hybridization reactions, size determinations with autoradiographs were more easily readable. In colony blotting, the clone containing the plasmid being used as the probe, the mutant from which the clone was derived and the wild-type strain were also colony-blotted.

2.5.17.3.1. Hybridization with Soutiiem Blots.

The nitrocellulose filter was placed in a heat sealed plastic bag and incubated in 13 ml of pre-hybridization buffer overnight at 37°C. The pre-hybridization buffer was removed and replaced with 13 ml hybridization buffer and at least 5x10^ cpm of ^^P-labelled DNA probe. The buffer used depended on whether plasmid or chromosomal DNA was bound on the filter (see section 2.3.11 and 2.3.12 ). Incubation was at 42^0 for 36-48 hours with the sealed bag submerged attached in an orbital water batii shaking at 50 rpm. The filter was removed and washed twice in 2x SSC, 0.1% SDS for 20 minutes at with gentie rocking followed by a further wash in 0.2x SSC for 15 minutes. The filters were dried on blotting paper, covered in glad-wrap and exposed in an exposure cassette at -20°C to Kodak XRP-1 x-ray film with an intensifying screen (Dupont 75

Cronex Hi-Plus) for 2-24 hours depending on the requirements for a clear autoradiograph.

2.5.17.3.2. Hybridization with Colony Blots.

Up to 10 circular filters were stacked on top of one another and incubated overnight at 37°C with gende rocking in a crystallizing dish containing 40-100 ml pre-hybridization buffer (section 2.3.12), ensuring the filter discs were well submerged. The buffer was replaced with an equal volume of hybridization buffer and the radioactive probe added so that its concentration was 10^ cpm/ml. of buffer. Incubation was at 42°C for 2-4 days depending on the volume of the reaction. Filters were removed, washed and autoradiographed as described above. Prior to autoradiography, filters were taped onto a sheet of blotting paper and to assist with alignment, a few 1 }il drops of probe were randomly placed around the rim of the blotting paper, covered with tape and marked with ink. Autoradiographs which exhibited "light-ups" were aligned to the filter which in tum was orientated against the master plate to determine the colony which caused the "light-up". In most cases, the exact colony could not be determined due to distortion of the filters. In such cases, an agar plug containing the colonies aligned about the "light-up" was vortexed in SNB and various dilutions thrice spread onto the selection medium. The 82 mm plates which resulted in approximately 200 colonies were screened a second time by hybridization. Positive colonies were isolated, plasmids extracted and re-tested by Southern blotting and hybridization. 76

3. RESULTS.

3.1. PRELIMINARY CHARACTERIZATION OF BILE-UTILIZING PSEUDOMONADS.

3.1.1. Plasmid Profile.

The 4 wild-type Pseudomonas strains, which catabolized bile acids, were grown to late-log phase in NB and the DNA extracted to determine whether they harboured any plasmids. Agarose gel electrophoresis was performed together with standards of covalently closed circular (CCC) plasmid DNA of known molecular weight (Figure 11). The strain PS5-1 harbours 3 plasmids of approximately 40kb, 10.6kb and 4.1kb whereas PS6-1 has 2 plasmids of about 75 and 40kb. In contrast the remaining 2 strains, PS7-1 and PS8-1 appear to have no resident plasmids. The plasmid patterns were the same for all the strains when grown in PAS + 0.25% CA + 0.01% Yeast Extract (data not shown).

3.1.2. Catabolic Properties.

As previously stated, PS5-1 was classified by Smith & Park (1984) as Pseudomonas putida biotype B with the other strains being classified as Pseudomonas spp. unknown. The 4 wild-types were growth-tested on PAS plates containing carbon sources known to be degraded by various Pseudomonas strains (Table 4) as well as various steroid substrates. On rich media such as NA, large colonies were apparent after overnight growth at 30°C whereas 40 hours incubation was needed for the same sized colonies when grown on PAS + CA plates. For these experiments, the test plates were incubated over 4 days and growth patterns noted every 24 hours. Except for p- and m-cresol, the strains 77

Figure 11. Plasmid Profile of tine Wild-type Bile

Acid-utilizing Strains, PS5-1, PS6-1,

PS7-1 and PS8-1.

Agarose Gel Electrophoresis. From Right.

Lane Strain/plasmid Plasmld glzes (Kb).

1 PS7-1

2 PS8-1

3 PS5-1 40, 10.6, 4.1

4 PS6-1 75, 40

5 V517 (a) (57), (7.7) 5.9, 5.4, 4.2, 3.2,

2.9, 2.2

6 pKan2 7.8

7 pKT230 11.9

8 pSA152 15.5

9 pRK290 20 (appears as dimer = 40kb)

10 RSa 38

11 pAS8 75

12 pRK2013 48

13 R68-45 59

14 pJB4JI 97

(a) Plasmids in brackets not seen on gel. 14 13 12 11 10 9 8 7 6 5 4 3 2 1

kb. kb.

97.2 75 U75 40 •40

—Chromosome 11.9- -10.6

7.8-

5.9- 5.4-

4.2. -4.1 3.Z. 2.^

2.2 78

TAgLE 4. Growth Responses of the Wild-type Pseudomonas Strains on Various Carbon Sources.

Carbon Source. Strain

PS5-1 PS6-1 PS7-1 PS8-1

dl-camphor - - - _

p-toluate - - - -

p-hydroxybenzoate + + + +

benzoate + + + +

protocatechuate + + + +

p-cresol - + + +

m-cresol - - + +

starch + + + +

hemicellulose + + NT NT

cholic acid + , + + +

deoxycholic acid + + + +

lithocholic acid + + + +

hyodeoxycholic acid + + + +

chenodeoxycholic acid + + + +

androsterone + + + +

testosterone + + + + progesterone 3p-hydroxycholesterol p-sitosterol + denotes growth within 4 days. - denotes no growth NT denotes not tested (a) Results supplied by R.J. Park. 79

exhibited similar growth patterns on all carbon sources tested. The strains grew on all of the cholic acid analogues as well as androsterone and testosterone, but did not grow on progesterone, p-sitosterol or 3p-hydroxycholesterol.

3.1.3. Antibiotic-resistance Properties.

The resistance of the 4 wild-types to a range of antibiotics, and to mercuric ions, were determined by spreading approximately 10^ cells onto NA plates containing varying concentrations of the inhibitors. Sensitivity was determined in this way because of the intention of introducing plasmids that encode antibiotic resistance in later parts of this work. The lowest concentration at which no colonies were observed after 2 days incubation was considered the inhibitory concentration (IC) for that strain. Only 3 antibiotics (Km, Gm and Tc) inhibited growth of all the strains at reasonably low concentrations (Table 5) whereas relativly high concentrations were needed for the remaining antibiotics to affect growth.

3.1.4. Isolation and Identification of Auxotrophic Mutants.

To facilitate genetic studies, auxotrophs of each strain were isolated following NTG mutagenesis. The isolated mutants were characterized and tested for stability. Those with reversion frequencies of less than 10"^ were retained. In all, 15 auxotrophs were isolated fromPS5-1,7 fromPS6-l, 6 from PS7-1 and? from PS 8-1. The distribution of auxotrophic markers appeared to be fairly random. The particular auxotrophs used in the course of this project are listed in Table 3. 80

Tabi^ 5. Antibiotic and Mercury Resistance of the Pseudomonas Strains PS5-1, PS6-1, PS7-1 and PS8-1.

Inhibitorv Concentration (\C). Antibiotic ) (^ig/ml) (2) strain. Km. Cb. Sm. HqClg Gm. PS5-1 35 >2500 50 >50 7.5 PS6-1 35 2000 >500 >50 7.5 PS7-1 35 >2500 >500 >50 7.5 PS8-1 35 1000 >500 >50 7.5

Strain TQ. Cm, Pip. SCL Tm.

PS5-1 30 >100 200 > 400 >1000 PS6-1 30 >100 200 400 >1000 PS7-1 30 >100 50 100 >1000 PS8-1 30 >100 20 > 400 >1000

(1) See Abbreviations (2) Concentrations designated "greater than" (ie: Cb > 2500) denote individual colonies still appear at that concentration 81

3.1.5. Introduction of Catabolic Plasmids by Conjugation.

To determine if plasmids could be transferred into and maintained in tiie steroid-utilizing strains, plasmids of different incompatability groups were introduced into the auxotrophs, PS5-6 Leu", PS6-3 His" and the prototrophs PS7-1 and PS8-1. The CAM catabolic plasmid is in the incompatability group Inc P2, TOL and CRE (pND50) are in the Inc P9 group. All tiiese plasmids are self-transmissable and each catabolic plasmid was transferred by conjugation into the steroid strains. The selection medium was PAS minimum media + the carbon compound relevant to the catabolic plasmid + any amino acid required by the recipient

All of the catabolic plasmids transferred into the steroid-utilizing Pseudomonads tested at a relativly high frequency (Table 6). Sixteen transconjugants from each cross were purified by single colony isolation on the selection medium used, then plate-tested on PAS + CA + required amino acids. All tested transconjugants retained the ability to utilize cholic acid.

3.1.6. Curing of tiie Resident Plasmids.

It was possible that one or more of the native plasmids of PS5-1 and PS6-1 encoded genes involved in steroid utilization. To determine if this was the case, attempts were made to eliminate plasmids by curing procedures. If essential steroid degradation genes were located on a plasmid, then strains cured of that plasmid could be selected as CA" mutants.

Strains PS5-1 and PS6-1 were incubated in NB in the presence of mitomycin C and 1000 survivors of each strain growth-tested on PAS + CA. No 82

Table 6, Transfer Frequencies of Catabolic Plasmids into

Steroid Utilizing Strains.

Donor Recipient

Transconjugants per Donor Cell.

PS5-6 Leu" PS6-3 His" PS7-1 PS8-1

PP1-25 (CAM) 10- 3 10- 3 10- 6 10-2

PAr1-6 (TOL) 10- 3 10- 4 10- 4 10"^

PP1-8 (pND 50) 10- 3 NT NT NT

NT denotes not tested. 83

varients were isolated that were completely CA", however approximately 99% of clones of PS5-1 and 50% for PS6-1 grew slowly on cholic acid. This slow growth was unstable as revertants that grew normally were obtained at a frequency of 10-6.

The auxotrophs PS5-4 Trp' and PS6-3 His" were grown in NB in the presence of 2% (^/v) SDS and 2000 survivors of each strain growth tested on PAS + CA + the relevant amino acid. No CA" strains were detected for PS6-3, however one CA" varient was obtained from PS5-4. A plasmid extraction of this varient (designated PS5-10) showed that it had lost the 40kb resident plasmid. PS5-10 reverted to CA^ at a frequency of 5 x 10"^, a plasmid profile of one such revertant showed the 40kb plasmid was still absent (data not shown).

PS5-10 was assessed in a shake flask at the CSIRO Meat Research Laboratories for identification of any accumulated products. No breakdown of the deoxycholic acid substrate nor any intermediate accumulation was observed. 84

3.2. ISOLATION OF MUTANTS BLOCKED IN STEROID

BIOCONVERSIONS.

Park (1981) and Smith & Park (1984) concluded from fermentation studies with PS5-1 that the only bile acid intermediates of the catabolic pathway to accumulate to a significant extent were the 12p hydroxy-androsterones. This effect was enhanced to about 60% of theoretical yield, by the use of low aeration rates (Smith & Park, 1984), presumably because the next enzyme in the pathway, the 9a-hydroxylase system, would require molecular oxygen. In order to improve the yield of this metabolite and to produce strains able to accumulate other intermediates, a mutation programme was undertaken with a view to isolate mutants blocked in each enzyme of the pathway. Furthermore, mutations deleting specific enzymes could help to answer biochemical questions with regard to the proposed pathway.

The choice of cholic acid, deoxycholic acid and 12|30H-ADD as model screening substrates was based on the rationale that these compounds would be suitable for screening for blocks in the upper part of the pathway. Mutants with such blocks would, depending on the mutation locus, either not grow at all on the first 2 compounds or produce minute colonies, with the carbon source being the acetate or propionate residues resulting from the side-chain shortening. Providing there are no polar effects from the mutation, these mutants should grow well on the

12pOH-ADD intermediate. Mutants blocked in tiie 9a-hydroxyase gene or beyond would not grow on the 12pOH-ADD. The choice of other steroid intermediates for use in mutant selection programmes was, in fact, limited because many potentially useful intermediates were unavailable. 85

3.2.1. Isolation of NTG-induced Mutants Affected in Steroid Utilization.

The NTG-derived auxotrophs PS5-4 Tip" and PS6-3 His" were mutated using NTG and approximately 20,000 survivors from each parent were screened, by replica plating, for unusual growth responses on choKc acid medium relative to the parent. Five mutants were derived from PS5-4 and 8 from PS6-3. Plasmid profiles were performed on all of the mutants to determine the fate of the resident plasmids. The reversion frequency was also tested for each mutant by spreading 0.1ml. of overnight culture onto PAS + CA + trp/his and incubating for 8 days. All of the strains were forwarded to CSIRO Meat Research Laboratories for assessment of steroid product accumulation. The strains isolated, their phenotype on solid CA medium and the reversion frequencies are listed in Table 7. The fate of theresident plasmid s are also listed in Table 7 together with any products found accumulated after 48 hours incubation in the presence of 2 g/L DCA.

All of the mutants isolated were CA" on plates except for PS5-7 which grew poorly on cholic acid (CA-) with the growth medium turning yellow after 24 hours incubation. This strain failed to grow on 12pOH-ADD. Two strains (105-16-N3 and 108-8-N8) exhibited reversion frequencies at about 10"^ per cell plated. Revertants were not detected for the remaining mutants. Two other strains, PS5-7 and 105-16-Nl, had lost the large resident plasmid, however the remaining 3 mutants derived from PS5-4 and all of the mutants derived from PS6-3 hadretained al l of the parentialresident plasmid s (data not shown).

All mutants were initially tested by the shake flask/TLC identification procedure outlined in Section 2.5.5. Problems were encountered with growth in Uquid cultures of the mutants of PS6-3. Presumably this was because 86

TabI? 7. NTG-lnduced Mutants of PS5-4 and PS6-3 Affected in Bile Steroid Degradation

Strain Phenotype Reversion Fate of Resident Accumulated Frequency to CA"^ Plasmids Products (a) (b)

PS5-4 Parent CA+Trp- - 3 plasmids NA PS5-7 CA=^YeUow NT Lost large plasmid PS,CS+X 105-16-Nl CA- BD Lost large plasmid None 105-16-N2 CA" BD Retained all plasmids II 105-16-N3 CA- 10-9 •1 II II II 105-16-N4 CA- BD II 11 II II PS6-3 Parent CA+His- - 2 plasmids NA 108-8-Nl CA" BD retained both plasmids1 None 108-8-N2 CA- BD II n It 11 108-8-N3 CA" BD n II II II 108-8-N4 CA" BD It II II II 108-8-N5 CA- BD II II II 11 108-8-N6 CA- BD II II II II 108-8-N7 CA- BD II II II It 108-8-N8 CA- 10-9 • I M II It (a) Approximate frequency after 8 days incubation on solid CA medium NT denotes not tested BD denotes below detection levels (b) Intennediates accumulated after 48 hrs. incubation in presence of 2 g/L DCA NA denotes not applicable PS denotes 3,12P-dihydroxy phenolic secosteroid (see Abbreviations) CS denotes 3,4,12P-trihydroxy catecholic secosteroid (see Abbreviations) X denotes unknown compound None denotes DCA not utilized and no products detected 87 the parental auxotrophic strain had been affected in certain growth properties which could have arisen from the NTG affecting functions other than the auxotrophy. When cells were scraped off solid media and suspended in salts + DCA + histidine, the steroid substrate was neither utilized nor transformed to any intermediate. Of the 5 mutants of PS5-4, only one (PS5-7) accumulated any steroid intermediates. The remaining 4 mutants failed to accumulate any intermediate and, as with the PS6-3 mutants, left the steroid substrate untouched.

PS5-7 accumulated a mixture of phenolic and catecholic secosteroid compounds upon incubation with the sodium salts of cholic, deoxycholic, chenodeoxycholic, hyodeoxycholic, lithocholic, taurocholic or glycocholic acid. The steroid substrate was completely consumed within 48 hours after addition. As previously stated, the slow growth in steroid media was presumably due to utilization of the acetic and propionic acids released during side-chain cleavage.

The production of these compounds was further studied in a 4 litre fermentor by R.J. Park at the CSIRO Meat Reasearch Laboratories. The catabolism of cholic acid was followed by HPLC procedures. The mutant was grown in salts + tryptophan + 2g/L glycerol as an initial carbon source and sodium cholate added to 2 g/L after 23 hours. All of the CA was consumed 26 hours after addition and a total product yield of 72% of theoretical was obtained. Three compounds were isolated and identified as 3,7a,12P-trihydroxy phenolic secosteroid (28% of total product), 3,4,7a, 12p- tetrahydroxy catecholic secosteroid (54%) and a third unidentified compound (18%) presumed to be a product of further catabolism of the catecholic secosteroid (R.J. Park, personal communication). The first 2 compounds are represented in Figure 9 as the intermediates L and M- 88

3.2.2. Transposon Mutagenesis with Tn5 to Isolate Steroid Catabolic Mutants.

As previously stated, transposons have been widely used as mutagenic agents over a range of organisms. The two transposons most commonly used are Tn5 (Km^) and TnlO (Tc^). The structure, mechanism of transposition and application of each transposon respectively have been reviewed by Kleckner (1981) and Berg & Berg (1983). Although NTG did produce one useful mutant, the number of mutants foundrelative t o the number screened was exceedingly low. One method of increasing the ratio is the use of transposons which carry a selectable marker. Only recipients which inherit the drug resistance (ie: the transposon) need to be tested on the steroid screening plates. As the bile acid-utilizing strains were naturally sensitive to low kanamycin levels (50)J,g/ml), Tn5 was the initial transposon of choice. Two supposed suicide vectors, pJB4JI and pSUPlOll, were used as donors of Tn5 to the 4 steroid-utilizing strains. In addition transposition of PS5-7 was also studied. Since PS5-7 was able to grow slowly on cholic and deoxycholic acid, an additional mutation such as in a side-chain degrading enzyme or 1-dehydrogenase may result in the production of exotic steroids not previously found in fermentation broths. The transposon mutagenesis programme initially involved filter-mate conjugation with either E. coli 1830 (pJB4JI) or SM10(pSUP1011) as donors. The recipients used were PS5-1, PS5-6 (Leu"), PS5-7 (Trp" CA±), PS6-1, PS6-6 (Phe'), PS7-1, PS7-4 (His'), PS8-1 and PS8-2 (Arg"). The donor to recipient ratios used were 1:10 for E.coli 1830 (pJB4JI) and 1:5 for E.co/i SMIO (pSUPlOll). Filters containing the mixed donors and recipients were incubated for 6 hours on NA prior to dilution and the spreading of conjugation mixtures onto the selection plates. The selection medium for transconjugants was PAS + 5mM glucose + 10 mM Na-succinate + 150 M-g/ml kanamycin + any relevant amino acids. Each transconjugant was 89 patched onto PAS salts + steroid + kanamycin media and growth compared over 4 days with the parent grown on the same media without kanamycin. The transconjugants were also streaked ontofresh selectio n medium. Transconjugants with an unusual growth response were purified by single colony isolation and retained for further study.

In a series of conjugation and plate screening experiments, over 10,000 Km^ clones were isolated and tested for alterations in their growth response on steroid substrates (Table 8). The conjugationfrequencies fo r each cross are listed in Table 8 together with the number of transconjugants tested on the steroid substrates and the number with unusual growth responses on one or all of those substrates. When conjugation occurred, the plasmid pJB4JI yielded transconjugants at frequencies in the range of 10"^ - 10"^ per donor cell. The transfer frequency into PS5-1 and its derivatives was the highest with the result that most of the transconjugants tested were from these strains. No transconjugants were isolated from PS6-6, PS7-1 or PS7-4. In contrast, pSUPlOll did not yield transconjugants in PS5-1 or its derivatives whereas transconjugants were isolated from all the remaining recipients at transfer frequencies of between 10"^ and 10"^.

In the first E. coli 1830 (pJB4JI) x PS5-1 cross in the table, all of the transconjugants were growth-tested by patching each clone on three PAS + kanamycin plates containing either CA, DCA or 12pOH-ADD. Transconjugants with unusual growth responses were purified by single colony isolation on the original selection medium, then retested on the steroid media. In subsequent experiments, each Km^ transconjugant was first tested on PAS + CA + Km plates with those affected in their growth response being purified by single colony isolation and letested on each of the above steroids. InaU, 116 Km^ strains were 90

lahlS^ Frequency of Km^ (Tn5) Transconjugants, No. of Colonies Tested and No. of Transconjugants Affected in Steroid Substrate Utilization.

No. Transconjugants No. Affected Transconjugants Tested on Steroid in Substrate Donor Recipient per Donor Substrates Utilization

E. coli 1830 (b) PS5-1 10-4 3928 47 (pJB4J1) (b) PS5-1 10-4 1012 8

PS6-1 10-® 109 - PS7-1 no transconjugants PS8-1 10-5 552 2 PS5-6 10-4 1240 10 PS5-7 10-4 900 21 PS6-6 no transconjugants PS7-4 no transconjugants PS8-2 10-6 118 6

E. coli Smo PS5-1 no transconjugants (PSUP1011) PS6-1 10-5 782 5 PS7-1 10-6 230 1 PS8-1 10-5 795 9 PS5-6 no transconjugants PS6-6 10-4 131 3 PS7-4 10-5 146 2 PS8-2 10-4 141 2

(a) Estimation only as degree of strain interaction and sibling replication during incubation on filters is unknown.

(b) Results of two different experimental procedures (see text) 91

isolated which were affected in their growth response on at least one of the steroid growth substrates. In general the percentage of transconjugants selected as having an unusual growth response was in the order of 0.8 - 1.4% of those tested from each cross. All of the strains were tested by the shake flask procedure for the accumulation of intermediates after the addition of 2g/L DCA. The catabolism of the steroid substrate and the accumulation of steroid components were followed by TLC. Strains which accumulated products were retained by the CSIRO for further testing in fermentors.

The selected strains are listed in Table 9 by their work numbers together with their plate growth responses on the steroid substrates and their sensitivity to the antibiotic marker carried on the vector. The growth pattem of each strain in 2 g/L DCA was also recorded together with accumulated products detected by TLC analysis. The work numbers of the parent recipients are listed as such: PS5-1 is MR105, PS5-6 is 105-17, PS5-7 is 105-16 CA± PS6-1 is MR108, PS6-6 is 108-12, PS7-lisMRl, PS7-4is 1-4, PS8-1 is MR119 andPS8-2 is 119-9. All transposon-induced mutants are numbered in brackets following the parent's work number. Thus 105(4) is the fourth Km^ isolate from the parent MR 105 and 105-17(2) is the second Kn^^ isolate derived from the parent 105-17 which is itself the NTG-induced Leu" mutant of MR 105.

The plate growth responses of the Km^ isolates on the various steroid substrates were often inconsistant with other isolates which were subsequently found to accumulate similar compounds in the DCA shake flask experiments. For example, the mutants 105(205) and 105(213) both accumulated the same mixture of compounds (3KA), yet the former did not grow on CA or DCA solid media whereas the latter did. 92

Tabig 9, Growth Properties of Tn5-lnduced Mutants of Bile Acid-Utilizing Pseudomonas Strains and the Compounds Accumulated after Incubation in 2 g/L Deoxycholic Acid.

Plate Growth Growth Response on and Major Mutant Tn5 12pOH Vector Stability Product No. Vector CA DCA -ADD Marker in DCA Accumulated (a) (b) (c) (d)

1(1) pSUPIOII P - - R D

II :i) P - - S PA SKA II [2) Y Y - S B

(1) pJB4J1 - - - S D

(2) - - - S AT2 12P0H-ADD

(3) - - - S AT1 12P0H-ADD

(4) P P - S A 12aOH-ADD

(5) - P - S AT2 12aOH-ADD

(6) P P - S A 12pOH-ADD

(7) - - P S AT1 12aOH-ADD

(8) - + + S DP

(9) - - - S P (201) P + P S D (202) P P + s AT1 3KEA (203) + + P s D

(204) - P + s D

(205) - - P s AT1 3KA (208) + P P s D (213) + + P s AT1 3KA (214) P P P R D (215) P P P s D (216) P P P R D (217) P P P s D 93

Table 9 (Continued) Plate Growth Growth Response on and Major Mutant Tn5 12pOH Vector Stability Product No. Vector CA DCA -ADD Marker in DCA Accumulated (a) (b) (c) (d)

(218) pJB4JI + P P S D (219) + + P S D (220) + P P R D (221) + P P S D (222) P P P R ATI 3KEA (223) P P P S ATI 3KEA (224) + + P S D (225) + P P S D (226) + P P S D (227) + + P S D

(228) + + - S AT3 12P0H-ADD (229) + . + P S D

(232) + + - R AT2 12P0H-ADD (233) + + P S D (234) + P P R D (239) P P P S D (240) + + P S D (241) + P P S AT2 12P0H-ADD (242) + P P S D (243) P P P S D (244) + P P S D (245) + + P S D (246) + + P S D (247) + + P S AT2 PS (248) + + P S D (249) + P P S D

(251) - - - S B

(301) - P - S A PS (302) P + + S D (303) P + + s Continued. 94

Table 9 (Continued) Plate Growth Growth Response on and Major Mutant Tn5 12pOH Vector Stability Product No. Vector CA DCA -ADD Marker in DCA Accumulated (a) (b) (c) (d)

105(304) pJB4JI + + P S D

(305) P + - S AT3 12pOH-ADD (306) + P + s D (307) P + + s D (308) P + + s D

105-16(1) P P - R A PS

-16(2) P P - R A PS

-16(3) P P - R A PS

-16(4) " + P - S AT2 PS

-16(5) P P - R A PS

-16(6) + - - S A PS

-16(7) P P - R B

-16(8) P P - R B

-16(9) P P - S A PS

-16(10) " P P - S A PS

-16(11) " + P - S A PS + CS

-16(12) " + P - S A PS + CS

-16(13) " P P - S B

-16(14) " + - - S A PS

-16(15) " + P - R A PS

-16(16) " - - - S A PS + CS

-16(17) " P - - R A PS + CS

-16(18) " P + - S A PS

-16(19) " + - - S A PS -16(20) " P P - S A PS

- - PS -16(21) " - S A

105-17(1) + + P S D A 3KEA -17(2) " - P + S Continued.... 95

Table 9 (Continued) Plate Growth Growth Response on and Major Mutant Tn5 12pOH Vector Stability Product No. Vector CA DCA -ADD Marker in DCA Accumulated (a) (b) (c) (d)

105-17(3) pJB4JI P P S A 3KEA + -17(4) " P S D -17(5) " + + s D -17(6) " P s B -17(7) " P s P -17(8) s AT2 3KEA -17(9) " + s D -17(10) " s A 3KEA

108(1) pSUPIOII Y S D (2) " + S D (3) s D (4) s D (5) R D 108-12(1) S AP 3KEA -12(2) " P S D -12(3) " + S D

119(1) pSUPIOII S D (2) S AT2 OPDC (3) s D (4) " P s D (5) " Y R AP SKA (6) S D (7) S AP 3KA (8) " P R D (9) S D (10) pJB4JI + P P R AP 3KEA (11) " P + P R D

Continued.... 96

Tables (Continued) Plate Growth Growth Response on and Major Mutant Tn5 12pOH Vector Stability Product No. Vector CA DCA -ADD Marker in DCA Accumulated (a) (b) (c) (d)

119-9(1) pSUPIOII - - + S B II -9(2) - - + R B

-9(3) pJB4JI P P - R B fi -9(4) P - - R B II -9(5) - P P R B -9(6) 11 + P + S B -9(7) II P + P R P -9(8) II P P + R D

(a) Growth response after 48 hours incubation relative to parent strain on PAS + steroid substrate plate.

CA denotes cholic acid, DCA = deoxycholic acid. + = normal growth^ - = no growth P = slow or poor growth compared to parent Y = slow growth, yellow colouration about colony after 24-40 hours

(b) S sensitive R resistant

(c) A = accumulates intermediate D = degrades, no accumulation P = poor growth in liquid medium B = no modification of DCA substrate Continued. 97

Table 9. (Continued)

AT1 = product accumulates for 24 hours, then is rapidly degraded AT2= product accumulates for 48 hours, then is rapidly degraded. AT3= product accumulates for 72 hours, then is rapidly degraded.

(d) See Abbreviations for names and structures. 98

The purified clones were tested to determine the fate of the vector plasniid. In the event of true transposition, the suicide vector should have been lost. Transconjugants derived from pJB4JI were tested on NA + Gm (7.5 M-g/ml) and those from pSUPlOl 1 tested on NA + Cm (200 M-g/ml). Many Km^ isolates derived from pJB4JI were also Gm^, indicating inheritence of more than just Tn5 from pJB4JI. Some of the Gm^ strains were seen to accumulate intermediates. In fact, all of the pJB4JI-derived mutants of the parent strain MR 119 and its auxotroph 119-9 were Gm^, yet one of these, 119(10) accumulated 3KEA. The results with pSUPlOll were not so clear. All parent strains had a natural resistance to chloramphenicol with inconsistant growth responses at high levels (> 200 |ig/ml). Mutants were deemed Cm^ if the patch test resulted in a large number of colonies, however the sensitivity results were seen to be ambiguous.

In the shake flask experiments, most of the selected transconjugants degraded the DCA within 48 hours after addition. Some strains failed to grow in the acetate liquid media whereas others which grew could not utilize the DCA. However, strains were obtained which were capable of accumulating compounds. Many of these mutants were fairly unstable with some product utilization being observed 24 or 48 hours after the addition of DCA. Mutants which were blocked in the divergent upper part of the pathway generally accumulated a mixture of compounds. The designation 3KA in Table 9 indicates accumulation of a mixture of the 12a-hydroxy derivarives of 3-oxo-cholanic acid and 3-oxo- 23,24-bisnorcholanic acid (BE and BG in Figure 9) witii usually the former being the major product. Similarily, strains listed as accumulating 3KEA produced 12a-hydroxy derivatives of 3-oxo-23,24-bisnorcholanic acid, 3-oxo-4-cholenic acid and 3-oxo-23,24-bisnor-4-cholenic acid (B^ CE and CQ respectively). The ratio of these three components was strain dependant. The strain 119(2) was the 99 only mutant found to accumulate OPDC (EG) in high yields (>90%). None of the strains were found to accumulate products with a C22- or a C17(20)- double bond.

Mutants which accumulated compounds in the convergent part of the pathway generally had high yields of only one compound. The exceptions were all derived from 105-16 CA± (PS5-7) which as previously stated accumulated 3 compounds. Interestingly, of the 900 Km^ transconjugants from 105-16 CA^ that were tested, the only alteration from the parent after growth in DCA was that many of the mutants now accumulated only the phenolic secosteroid. The only other mutants obtained which accumulated one intermediate with the side-chain totally removed were generated using the vector pJB4JI and were mutants of PS5-1.

The reversion frequencies of a random sample of ten PS5-l::Tn5 mutants (all derived with pJB4JI) which accumulated products and five pSUPlOll-derived mutants of PS8-1 and PS7-4 were determined. Each mutant was grown overnight in PAS + 0.3% glucose + 0.01% yeast extract + 50 |ag/ml kanamycin (+ histidine for the PS7-4::Tn5 mutant). Cultures were washed in SNB prior to dilution, then spread on NA, PAS + CA + histidine and PAS + 12a-OH ADD + histidine plates (data not shown). For most of the mutants, small colonies appeared on the CA medium, therefore colonies that were significantly larger within 60 hours incubation were deemed to be mutants. Six of the strains had profuse growth for all dilutions down to 10'^ on both the steroid media and six others had reversion frequencies of between 10"^ to 10"^ per cell. No revertants could be isolated from 105(2), 105(4) and 105(6) thus mdicating that these mutants were extremely stable. Revertants were purified by single colony isolation on the relevant steroid medium, then tested for the loss of Tn5. Most of the revertants were Km^. 100

3.2.3. Transposon Mutagenesis with Tnl and Tn7 to Isolate Steroid Catabolic Mutants.

The derivation of stable mutants which accumulated steroidal products demonstrated that transposons, notably Tn5, were useful mutagenic agents with the bile-utilizing Pseudomonads. The use of Tn5 to generate mutants of PS5-1 and its auxotrophs resulted in strains that accumulated 5 classes of compounds (3KA, 3KEA, 12aOH- and 12pOH-ADD and PS) from deoxycholic acid catabolism. Miller et al, (1980) and other researchers have found the transposon Tn5 seemed to prefer certain regions of the E. coli chromosome, so it is plausible to expect similar results with other Gram-negative strains. To investigate this possibility, other transposons were used as mutagens. As the majority of mutants were derived from PS5-1, it was used as the recipient.

The suicide vector pAS8 hosted in E. coli AB2463 was used in a series of conjugation experiments. As outlined in Section 1.13, this RP4-ColEl hybrid derivative carries both Tnl (Cb^) and Tn7 (Sm^) and thus can be used as a source of either transposon depending on the antibiotic used in the selection media. Two donor to recipient ratios (1:1 and 1:4) were used. The mixed cells were collected on membrane filters and incubated on NA for 6 hours prior to dilution and spreading. The selection media was PAS + lOmM succinate + 5mM glucose + 2500 |lg/ml Cb (for Tnl transposition) or 100 |Xg/ml Sm for Tn7 transposition. Such high levels of Cb were necessary as PS5-1 has a natural high resistance to the antibiotic. A control conjugation, AB2463 (pASS) x PPl-2 Trp" was performed to confirm that transfer could occur within Pseudomonads using this plasmid, the selection plates being seeded with tryptophan for these crosses. Each transconjugant from PS5-1 was patched onto PAS + either CA, DCA or 12pOH-ADD + relevant antibiotic as well as fresh selection medium. Strains 101 affected in growth on the steroid substrates relevant to the parent were purified by single colony isolation and tested at the CSIRO Meat Research Laboratories for intermediate accumulation.

The conjugationfrequencies pe r transposon selected are listed in Table 10 together with the number of transconjugants tested on the steroid media and the number selected as having unusual growth responses. No transconjugants were observed with either recipient on the Sm selection media, however transfer of carbenicillin resistance into PS5-1 was about 10"^ per donor cell. The recipient PS5-1 had a high natural resistance to Cb with 2-5 spontaneous Cb^ mutants appearing on control plates. The selection plates (10"^ dilution) had 50-100 large colonies and many more small colonies on them which were deemed to be either satelite colonies or spontaneous Cb^ mutants. To eliminate the unnecessary testing of satelite colonies which had grown in the penicillin breakdown zones, only large colonies which were easily picked off the conjugal selection media were scored for growth testing on the steroid media.

A total of 1128 Cb^ transconjugants were growth-tested on each of the steroid substrates. In all, 61 clones with altered steroid-utilizing phenotypes were isolated. The Tnl-induced mutants were retested at the CSIRO by growing the potential mutants in shake flasks and testing for product accumulation by TLC against known standards. The growth responses of each clone on solid steroid media and in liquid cultues of 2 g/L DCA are listed in Table 11 together with tiie products accumulated. The work numbers are of the same pattern as the Tn5-induced mutants except the bracketed numbers begin at 401. All mutants were sensitive to Km at 80|Xg/ml indicating loss of the vector plasmid.

Of the 61 selected strains, only 9 accumulated intermediates in 102

TablelO. Frequencies of Cb'^(Tnl) and Sm^ (Tn7) Transconjugants of PS5-1 and Control Strain PP1-2 Trp", No. of Transconjugants Tested and No. Affected in Steroid Utilization.

Drug

Ratio Selection and Conjugation No.Tested No. Affected (Donor/ Concentration Frequency on Steroid in Steroid Cross Recipient) (|j.g/ml). per Donor Substrate Utilization (a)

AB2463 (pAS8) 1:1 Cb 2500 10-4 } X PS5-1 1128 61 1:4 Cb 2500 10-4 }

1:1 Sm 100 no transconjugants

1:4 Sm 100 no transconjugants

AB2463 (pASS) 1:4 Cb 2500 10 -3 X PP1-2 1:4 Sm 100 no transconjugants

(a) Estimation only as strain interaction and sibling generation

rate unknown. 103

Table 11. Growth Properties of Tn1-induced Mutants of PS5-1 and Accumulated Compounds after Incubation in 2 g/L Deoxycholic Acid

Mutant Plate Growth Responses Growth and Product Work No. CA DCA 12pOH Stability in DCA Accumulated (a) -ADD (b) (c)

105 401) S p - AT2 12p OH-ADD 402) S + + D

403) + + - AT2 12p OH-ADD

404) + + - A PS 405) + + s D 406) S s + D 408) + s + D

409) + + - A PS 410) + + s A PS 411) s s + DP 412) s + + D 413) + + s D 414) s + + D 415) s + + D 416) + p + D 417) + + p D

418) s + - D 419) s + + D 420) s s + D 421) + s + D 422) p + + D

423) s p - D 424) + + s D 425) + + s D 426) + + p ATI 12p OH-ADD 428) + + p D

429) + + - ATI 12p OH-ADD Continued. 104

Table 11 (Continued) Mutant Plate Growth Responses Growth and Product Work No. CA DCA 12pOH Stability in DC A Accumulated (a) -ADD (b) (c)

105 (430) S s s D (431) P p + D (432) + + s D (433) + + s D (434) + s s D (435) + s + D (436) + s + D (437) S + + D (438) S + + D (439) s s + D (440) + + s D

(441) - - - AP SKA (442) + + s D

(443) + - + D (444) + s + D (445) s + + D (446) + + p D (447) s + + D (448) s + + D (449) s + + D

(450) s + - AT2 3KA (451) + s + D (453) s + + D (454) s + + D (455) + + s D (456) + + s D (457) + + s D (458) + + p D (459) s + + D (460) + + s D (461) + s + D (462) + + p D Continued 105

Table 11 (Continued) Mutant Plate Growth Responses Growth and Product Work No. CA DCA 12pOH Stability in DCA Accumulated (a) -ADD (b) (c)

105 (463) + S + D (464) + S + D

(a) Growth response after 48 hours incubation relative to parent strain on PAS + steroid substrate plate.

CA = cholic acid, DCA = deoxycholic acid. + = normal growth = no growth S = slow growth compared to parent, colonies normal size 1-2 days after parent P = poor growth, minute colonies after 4 days.

(b) A = accumulates intermediate D = degrades, no accumulation P = poor growth in liquid medium ATI = Product accumulates for 24 hours, then is rapidly degraded AT2 = product accumulates for 48 hours, then is rapidly degraded.

reasonable quantities. The remainder rapidly degraded the DCA substrate with little product accumulation. No new compounds were accumulated by the presumptive Tnl-induced mutants. The only products accumulated by these mutants were SKA, 12pOH-ADD and PS. One strain, 105(441), grew poorly in the acetate liquid medium but accumulated SKA with littie loss of the product after 48 hours. The only other stable mutants with little product loss 48 hours after DCA addition all accumulated the phenolic secosteroid.

S.2.4. Transposon Mutagenesis with TnlO.

The Tc^ transposon TnlO was also used to generate mutants of PS5-1 and PS8-1. This work was carried out by an honours student in our laboratory (H. Motyka, 1986). E. coli HBlOl containing the hybrid RK2-ColEl vector pBEElO (Ely, 1985) was placed on filters with either PS5-1 or PS8-1 at a donor to recipient ratio of 1:5. Filter mates were for 24 hours on NA. The conjugation mixtures were spread on PAS + lOmM succinate + SO M-g/ml Tc. Transconjugants were patch-tested on PAS + CA + Tc and strains selected for slow or no growth on the steroid medium were purified on the original selection medium. Transconjugants were obtained at a frequency of 10'^ per donor cell. Of SOOO Tc^ transconjugants tested, 2 strains from PS5-1 and S4 strains from PS8-1 were selected on the basis of reduced growth on cholic acid. The reversion frequency to normal growth on CA was tested and found to be in the range of 10'^ to 10"^ with most strains reverting at about 10'^.

Shake flask assessments revealed that about half of the selected mutants were sensitive to sodium deoxycholate in liquid media, yet some of these still accumulated compounds, albeit slowly. Why the presence of this particular transposon should affect the strains as such is unknown. Ten of the strains 107

accumulated intermediates up to 48 hours after DCA addition. Two accumulated

3KA compounds, 2 accumulated 3KEA and 5 accumulated the OPDC product.

The remaining strain accumulated a neutral compound which was possibly

12pOH-ADD (R. J. Park, personal communication).

The vector also encoded for kanamycin resistance. Nine of the 36

selected strains were Km^ indicating loss of the vector plasmid, but the majority

were Km^, in fact 8 of the 10 intermediate-accumulating strains were Km^.

3.2.5. Summary of Transposon Mutation.

In the previous experiments, the transposons Tnl, Tn5 and TnlO were

successfully used to generate mutants using CA"*" strains as recipients.

Approximately 14,000 transconjugants were patch-tested for growth on steroid

media. A total of 213 transposon-induced clones (192 transconjugants of CA"*"

strains and 21 transconjugants of a CA~ strain) were selected as potential mutants

on the basis of impaired growth relative to growth by the parent on a test steroid

substrate. All of the above selected clones were tested for accumulation of

intermediates from the breakdown of DCA in shake flask experiments. Of the 192

clones tested which had only a single transposon-induced mutation, 3 failed to

grow or grew very poorly in liquid cultures with acetate and/or glycerol as the

carbon source. A further 128 clones degraded DCA almost as well as the parent

whilst 15 clones could not modify tiie DCA substrate at all. The remaining 46

clones accumulated various intermediates in significantiy higher yields than the parents at varying degrees of stability. The remaining 21 clones tested were

transposed derivatives of 105-16 CA± (PS5-7) which was itself a NTG-induced

mutant impaired in CA degradation. Three of tiie transposed derivatives failed to 108

modify DCA whilst 4 accumulated products similar to PS5-7. The introduction of Tn5 appeared to affect the remaining 14 strains such that only the phenolic secosteroid was accumulated. These results were tabled per transposon used and strain no. in Sections 3.2.2 to 3.2.4.

To further summarise, the results are tabulated per products accumulated and strain stability in Table 12 which is further subdivided per transposon/vector used. Six different product groups were obtained. For each product group, at least one mutant was reasonably stable in that the intermediates were not utilized 48 hours after addition of DCA. The first three product groups shown, 3KA, 3KEA and OPDC were from the upper part of the steroid catabolic pathway. Mutants generated which accumulated 3KA were derived using all three transposons, however only Tn5- and TnlO-derived mutants were generated which accumulated 3KEA and OPDC compounds. The remaining 3 product groups, formed after side-chain removal from the D-ring, were 12aOH-ADD, 12POH-ADD and the phenolic secosteroid. Only Tn5-generated mutants accumulated 12aOH-ADD whereas 12P0H-ADD was accumulated from mutants derived from all 3 transposons. The phenolic secosteroid was derived from mutants generated with Tnl and Tn5. No Tn5-derived mutant obtained using pSUPlOll as the vector accumulated a product from the convergent part of the pathway

3.2.6. Double Mutation of Bile-utilizing Strains.

From the above experiments, a range of mutants were derived which accumulated, at varying degrees of stability, intermediates from the degradation of bile acids. Even so, some intermediates such as compounds with an unsaturated side-chain or the catecholic secosteroid were not accumulated by any mutant derived by transposition. Rather than continuously generating and screening 109

Table 12. Catabolites Accumulated from Deoxycholic Acid by Transposon-induced Mutants of Bile Acid Utilizing Strains.

Product (Class) TnA/ector Stability No. of Total No. Accumulated Used of Mutants Mutants per Product (a) (b)

3KA Tn5/pJB4JI ATI 2 Tn5/pSUP1011 A 3 Tn1/pAS8 A 1 II AT2 1 TnlO/pBEElO A 1 AT2 1

3KEA Tn5/pJB4JI A 4 ATI 3 AT2 1 Tn5/pSUP1011 A 1 TnlO/pBEElO A 2 11

OPDC Tn5/pSUP1011 AT2 1 TnlO/pBEElO AT2 5

12aOH-ADD Tn5/pJB4JI A ATI AT2

12P0H-ADD Tn5/pJB4JI A 1 ATI 1 AT2 3 AT3 2 Tn1/pAS8 ATI 2 AT2 2 Continued. 110

Table 12 Continued Product (Class) TnA/ector Stability No. of Total No. Accumulated Used of Mutants Mutants per Product (a) (b)

12P0H-ADD TnlO/pBEElO AT2 12

PS Tn5/pJB4JI A 1 II AT2 1 Tn1/pAS8 A 3

PS NTG-Tn5/pJB4JI A 13 AT2 1 14

Total number of accumulating mutants 60

(a) and (b) See Table 9 and text for abbreviations. 111 mutants of the wild-type strains for these iatermediates, a further mutation of selected Tn5-induced mutants was a possible method for obtaining different products.

Three mutants accumulated products in yields approaching theoretical. These mutants 119(2), 105(4) and 105(6) grew very slowly on DCA and produced OPDC, 12aOH-ADD and 12POH-ADD respectively. The mutant 119(2) accumulated OPDC (DG) up to 48 hours after which the OPDC was rapidly depleted. Assuming that the proposed pathway is correct, then the block for this mutant would be the enzyme converting GtoH. Compounds with the 20-carboxyl pregnane side-chain (G) were deemed potentially valuable products and it was desirable to generate mutants which accumulated steroids with a partly oxidized A-ring and this side-chain. Some mutants did accumulate CG, but never as the major product, and most were subject to product depletion after 24-48 hours. If 119(2) could be further mutated in the A-ring-oxidizing enzymes, then mutants capable of accumulating high yields of these compounds may eventuate. The other two mutants 105(4) and 105(6) grew slowly on cholic acid presumeably due to carbon release during side-chain oxidation but could not grow on 12aOH-ADD. Both mutants were extremely stable. If the side-chain degradation could be blocked by the addition of another mutation, it could result in the isolation of more stable mutants which produce steroid intermediates further up the pathway.

In a previous experiment, Tnl was used to mutate PS5-1 and the donor plasmid was seen to "suicide" in all Cb^ transconjugants. The vector pAS8 was therefore used to introduce Tnl into the above Tn5-induced mutants. The donor to recipient ratio for filter mating was 1: 2 and the mating allowed to proceed for 16 hours. Cb^ transconjugants were patched onto PAS + CA + Cb media and the 112

growth response of each Cb^ isolate compared with the parent Tn5-induced mutant grown on the same medium without Cb. Strains which grew slower than their parent were purified on the selection medium and tested at the CSIRO Meat Research Laboratories for intermediate accumulation.

The conjugation frequencies (Table 13) were lower than the frequencies obtained with the wild-type PS5-1 as the recipient (Table 10) despite a longer incubation time. Antibiotic tests for the retention of the "suicide" vector in the transconjugants were not possible as both Tn5 and the vector pAS8 confer resistance to kanamycin and streptomycin, however in the experiments with PS5-1, all the Cb^ transconjugants selected as potential mutants were Km^.

Five hundred Cb^ transconjugants were growth-tested on cholic acid and deoxycholic acid media for slower growth compared to the Tn5-loaded parent strains. A total of 81 strains were selected as potentially further mutated in the cholic acid catabolic pathway by Tnl.

Of 17 presumptive Tnl-induced double mutants of 119(2) tested in shake flasks containing 2 g/L DCA, 13 were indistinguishable from 119(2) and 1 grew poorly in liquid media (Table 14), however 3 strains accumulated OPDC at high yields without the product depletion characteristic of the parent, thus the mutants were more stable than 119(2). The reversion frequency of one mutant, 119(107) was compared with 119(2). Plate tests on PAS + CA medium showed revertants of 119(107) appeared at approximately 10'^ per cell plated whereas 119(2) reverted at approximately 10'^.

Tnl mutation of 105(4) and 105(6) resulted in the isolation and subsequent screening of 38 and 28 presumptive mutants respectively (Table 14). 113

Table 13. Transfer Frequencies of Tnl Transposition into Strains 105(4), 105(6) and 119(2), No. of Transconjugants Tested and No. Affected in Steroid Utilization.

No. Transconjugants No. Affected Recipient Transconjugants Tested on in Steroid Work no. per Donor Steroid Media Utilization.

-5 105(4) 10 180 38

-5 105(6) 10 100 28

119(2) 10 -5 220 17

(a) Estimation only as strain interactions and sibling

generation rate unknown. 114

Tablg14. Growth Properties of Tnl-induced Mutants of 105(4), 105(6) and 119(2) and Accumulated Compounds after Incubation in 2g/L Deoxycholic Acid.

Parent Mutant Plate Growth Growth and Product/s Work no Response on CA Stability in DCA Accumulated (a) (b) (c)

119(2) 119 (101) S AT2 OPDC (102) P p (103) S AT2 OPDC (104) S AT2 OPDC (105) S AT2 OPDC (106) S AT2 OPDC (107) S A OPDC (108) S AT2 OPDC (109) S AT2 OPDC (110) s AT2 OPDC (111) s ATI OPDC (112) s AT2 OPDC (113) s A OPDC (114) s AT2 OPDC (115) s A OPDC (116) s AT2 OPDC (117) s AT2 OPDC

105(6) 105 (501) p AT4 12P0H-ADD (502) s ATS 12P0H-ADD (503) s ATS 12P0H-ADD (504) p B (505) s AT4 12P0H-ADD

(506) - B (507) s ATI 12P0H-ADD

(508) - B Continued. 115

Table 14 (Continued) Parent Mutant Plate Growth Growth and Product/s Work no Response on CA Stability in DCA Accumulated (a) (b) (c)

105(6) 105 (509) P PAT4 12P0H-ADD (510) S AT4 12pOH-ADD (511) S AT4 12P0H-ADD (512) S PAT34 12P0H-ADD (513) S A 12P0H-ADD (515) s AT2 12P0H-ADD (516) s A 12P0H-ADD (517) s AT2 12pOH-ADD (518) s AT2 12pOH-ADD

(519) - PATS 12P0H-ADD (520) s AT2 12P0H-ADD

(521) - B (522) p AT4 12P0H-ADD (523) s PAT4 12P0H-ADD (524) p ATS 12P0H-ADD (525) s AT2 12P0H-ADD

(526) - B (527) p AT2 12P0H-ADD (528) s AT2 12P0H-ADD (529) s AT2 12P0H-ADD

105(4) 105 (530) - B

(531) - A 12aOH-ADD (532) p A 12aOH-ADD (533) s PA 12aOH-ADD (534) s A 12aOH-ADD + X (535) s A 12aOH-ADD + X (536) s A 12aOH-ADD + X (537) s A 12aOH-ADD + X (538) s A 12aOH-ADD + X (539) s A 12aOH-ADD + X (540) s A 12aOH-ADD + X Continued. 116

Table 14 (Continued) Parent Mutant Plate Growth Growth and Product/s Work no Response on CA Stability in DCA Accumulated (a) (b) (c)

105(4) 105 (541) P A 12aOH-ADD + X (542) S A 12aOH-ADD + X (543) S A 12aOH-ADD + X (544) S A 12aOH-ADD + X (545) P A 12aOH-ADD + X (546) S A 12aOH-ADD + X (547) P A 12aOH-ADD + X (548) P A 12aOH-ADD + X (549) S A 12aOH-ADD + X (550) S A 12aOH-ADD + X (551) S A 12aOH-ADD + X (552) P A 12aOH-ADD + X (553) S A 12aOH-ADD + X (554) s A 12aOH-ADD + X (555) s A 12aOH-ADD + X (556) s A 12aOH-ADD + X (557) p A 12aOH-ADD + X (558) s A 12aOH-ADD + X (559) p PA 12aOH-ADD (560) s A 12aOH-ADD + X (561) p A 12aOH-ADD + X (562) s A 12aOH-ADD + X (563) s A 12aOH-ADD + X (564) s A 12aOH-ADD + X (565) s A 12aOH-ADD + X (566) p B (567) s PA 12aOH-ADD + X

Continued. 117

Table 14 (Continued)

(a) growth response after 72 hours incubation relative to parent strain on PAS + steroid substrate plate.

CA = cholicacid, = no growth S = slow growth compared to parent, colonies normal size 1-2 days after parent P = poor growth, minute colonies after 5 days

(b) DCA = deoxycholic acid A = accumulates intermediate P = poor growth in liquid medium B = no modification of DCA substrate ATI = product accumulates for 24 hours, then is rapidly degraded AT2 = product accumulates for 48 hours, then is rapidly degraded. ATS = product accumulates for 72 hours, then is rapidly degraded. AT4 = product accumulates for 96 hours, then is rapidly degraded.

X = Unknown product 118

With respect to mutants of 105(4), 2 strains failed to grow in liquid media and another 2 grew poorly but accumulated 12aOH-ADD, 2 were indistinguishable from 105(4) and the remainder accumulated an unknown neutral steroid compound in low yields in addition to the 12aOH-ADD, Of the mutants of 105(6), 4 failed to grow and the remainder accumulated the same compound as 105(6) with the differences between mutants being in growth rates and the retention time of the product before depletion began.

The results showed that the insertion of Tnl did affect the Tn5-induced mutants but the effects were phenotypically different for each mutant Except for the unknown compound, no mutants were isolated which accumulated a product different to the parent strain, although it must be said that the number of Cb^ transconjugants tested was low. Tnl mutation of 105(6) and 119(2) affected the "stability" of the strains insofar as deletion of the accumulated product was at times dramatically altered.

3.2.7. Fermentation Studies of Transposon-induced Mutants.

In view of the possible commercial importance of the phenolic secosteroid, 12aOH-ADD, 12pOH-ADD and the OPDC compound, the most stable mutants which accumulated these intermediates were chosen to study the intermediate production and catabolism of bile acids in more detail. This work was performed by R.J. Park at the CSIRO Meat Research Laboratories. The mutants were grown in a 4L fermentor with 2 g/L glycerol as the initial source of carbon at 25°C. Various bile acid substrates were added after substantial growth had occurred, usually about 24 hours. Catabolism of the steroid substrate and formation oftiie product s was monitored by HPLC, spectrophotometric and TLC procedures as outiined by Park (1981) and Leppik et al, (1982). The results of 119

some of these fermentation runs are shown in Table 15. The NTG-derived mutant

PS5-7 is shown for comparative purposes. The Tn5-induced mutant of PS5-7 that

accumulated the phenoUc secosteroid, 105-16(10), was designated PS5-25. The

mutant 105(4) which accumulated 12aOH-ADD was designated PS5-16 and

105(6),which accumulated 12pOH-ADD, was designated PS5-18. Two strains

which accumulated OPDC were studied, 119(2) and its Tnl-induced mutant

119(107), and were designated the strain numbers PS8-10 and PS8-22 respectively. The amount of steroid substrate added, incubation time, product and product yield are also presented in Table 15. The incubation time was the time

after the initial steroid addition up to when the fermentation was completed and the products harvested. Generally this occurred when it was determined that all of the steroid substrate was consumed.

Successful fermentations were conducted with substrate concentrations ranging from 2 g/L up to 50 g/L deoxycholic acid. The amount of bile solids are shown as cholic acid equivalents, in fact 120 g/L of mixed ovine and bovine solids were added for these fermentations.

In all cases, the final product was dependent on the structure of the substrate raw material. PS5-7 accumulated a mixture of phenolic and catecholic secosteroids and an unidentified product. In other fermentation studies, the 2 secosteroids had reached their maximum values by the time all of the substrate was consumed whereas the unidentified product slowly increased in concentration until the fermentation was concluded (data not shown). This led to the conclusion that this catabolite was probably formed subsequent to the secosteroids and that its production was due to strain instability. The transposon-induced mutant, PS5-25, appeared to be a more stable mutant as it produced only phenolic secosteroid in high yields(80%) with higher substrate concentrations and longer incubation times. 120

Table 15. Production of Steroid Intermediates from Bile Acids by Fermentation with Mutants.

Substrate Incubation Product Compound Quantity (g) time (hr) Compound Quantity (g) Yield (%) (a) (b) (c) (d) (e)

Strain PS5-7 CA 8 26 3,7a,12p-trihydroxy phenolic secosteroid (1.3g) 3,4,7 a, 12P-tetrahydroxy catecholic secosteroid (2.5g) Unidentified product (0.8g) 4.6 72 Strain PS5-25 CA 24 19 3,7,12a-trihydroxy phenolic secosteroid 15.0 81 CA 20 32 tt ti If 15.0 97 DCA 24 17 3,12P-dihydroxy phenolic secosteroid 14.6 80 CDCA 19 3,7a-dihydroxy phenolic secosteroid 5.5 91

Strain PS5-16 CA 8 24 7a,12a-dihydroxy-1,4- androstadiene-3,17-dione 5.7 97 CA 120 49 II II 78.0 88 DCA 8 21 12a-hydroxy-l,4- androstadiene-3,17-dione 5.6 97 DCA 200 69 II II 140.0 96 Bile solids 166 46 total ADD-type products 108.0 75

Continued... 121

Table 15 (Continued) Substrate Incubation Product Compound Quantity (g) time (hr) Compound Quantity (g) Yield (%) (a) (b) (c) (d) (e) Strain PS5-18 CA 24 49 7a,12p-dihydroxy-l,4- androstadiene-3,17-dione 16.9 96 DCA 8 40 12P-hydroxy-l,4- androstadiene-3,17-dione 5.5 95 Bile solids 166 42 total ADD-type products 114.0 90 CDCA 15 48 7a-hydroxy-l,4,- androstadiene-3,17-dione 9.0 78 HDCA 8 47 6a-hydroxy-l,4- androstadiene-3,17-dione 6.0 98 Strain PS8-1Q DCA 8 23 12a-hydroxy OPDC 6.8 98 DCA 8 48 12a-hydroxyOPDC 4.1 59 CA 8 21 7a, 12a-dihydroxy OPDC 6.0 86 CDCA 8 26 7a-hydroxy OPDC 5.2 72 Strain PS8-22 DCA 16 47.5 12a-hydroxy OPDC 11.3 82 DCA 40 48.5 12a-hydroxy OPDC 30.6 88 (a) CA sodium cholate DCA sodium deoxycholate CDCA chenodeoxycholic acid HDCA hyodeoxycholic acid Bile solids = amount of bile solids added as cholic acid equivalents (b) amount added into 4 litres for fermentation (c) incubation time is that between initial steroid addition and harvest at completion of the fermentation. (d) see Abbreviations (e) product yield is the sum of both the soluble and precipitated product 122

PS5-16 accumulated only ADD-type compounds with the 12-hydroxy group being in the a-position. Product yields were very high irrespective of the substrate concentration indicating the extreme stability of this mutant. No intermediates were accumulated if the substrate, such as CDCA or HDCA, did not contain a 12-hydroxy group (data not shown). This would suggest that only the

12a-isomerase gene was affected by the transposon insertion, that is there was no polar mutation which affected the production of enzymes further down the pathway. This was apparent with the results from the bile steroid fermentation of which only a 75% yield was obtained. Most likely the low yield was because some of the steroids in the bile solids did not contain a 12-hydroxy group and were thus utilized by the mutant. PS5-18 accumulated 12P-hydroxy ADD type product from CA and DCA as well as hydroxylated ADD products from chenodeoxycholic and hyodeoxycholic acid. Yields were very high over long incubation times, except for CDCA, indicating that this mutant was also very stable.

PS8-10 and PS8-22 accumulated hydroxylated OPDC products corresponding to the substrate. PS8-10 accumulated the product at 98% theoretical yield after 23 hours incubation in 2 g/L DCA and 59% yield after 48 hours. The product was rapidly depleted if the fermentation was allowed to proceed longer than 24 hours. Similar results were obtained with CA and CDCA as substrate.

The stabilized derivative, PS8-22, accumulated OPDC in yields greater than 80% with longer incubation times and substrate concentrations up to 10 g/L. PS8-22 could be used for up to 96 hours before revertants became enriched with considerable product loss. 123

3.3. MECHANISM OF TRANSPOSITION BY TN5 FROM pJB4n.

In Section 3.2.1., use of the Tn5-loaded vector pJB4JI effectively produced desired mutants of the bile-utilizing pseudomonads. As diagrammed in

Figure 10, the plasmid carries Tn5 integrated within the Mu sequence. Mu can also transpose at random and shares all of the properties characteristic of transposable elements (Toussaint, 1985). In order to obtain an understanding of the transposition events within the mutants, 5 strains derived using pJB4JI were further investigated.

The chosen mutants investigated were 105(3), PS5-16 (105(4)), PS5-18

(105(6)), 105-17(2) and 105-17(10), which accumulated from an aerobic fermentation in 2 g/L DCA, the products 12P0H-ADD, 12aOH-ADD,

12pOH-ADD, 3KEA and 3KEA respectively (Table 9). All 5 mutants were sensitive to gentamicin indicating that pJB4JI was not present. The transposon was inserted in the chromosome within each mutant as was shown in this section

(see later) when total DNA of each mutant was hybridized with Tn5 sequences.

The product class 3KEA was a mixture of partly oxidized acidic intermediates.

The mixtures comprised of the 3-oxo-12a-hydroxy-derivatives of 4-cholenic acid, bisnorcholanic and 4-bisnorcholenic acid (CE, BG and CG in Figure 9).

Fermentation using the mutant 105-17(2) in the presence of 2 g/L DCA for 48 hours resulted in the accumulation of the above products in the ratio of 5:30:60 respectively whereas 105-17(10) accumulated only the first 2 intermediates in the ratio of 10:80 with the 3rd intermediate undetected (R.J. Park, personal communication). Although 105(3) and PS5-18 both accumulated 12p hydroxy-ADD, product disappearance with the former began after 24 hours incubation whereas PS5-18 accumulated near theoretical yields with no subsequent product utilization. As previously described, PS5-16 accumulated 12a 124

hydroxy-ADD in near theoretical yields with no product utilization being observed.

To enable characterization of the region about the Tn5 insertion site, DNA fragments encoding the transposon and flanking DNA were cloned and then studied. This section reports the isolation and characterization of a number of clones from the above mutants. In addition, to confirm whether the DNA sequences within these clones were common with all or the majority of mutants derived with pJB4JI, a further 15 mutants were investigated by Southern hybridization analysis.

3.3.1. Selection for Clones Encoding Kanamycin Resistance from pJB4JI-derived Mutants.

The small cloning vector pBR322 was preferred as it does not encode Km^, the Tn5 encoded antibiotic-resistance determinant, but does encode Ap and Tc resistance. EcoRl was the enzyme of choice as there are no EcoRl restriction recognition sites within Tn5 (Jorgensen et al, 1979) and the vector contains a unique EcoKl site. Cloned DNAfragments woul d therefore carry Tn5 (5.7kb) as well as flanking DNA at least to the adjacent EcoRl sites. In order to maximize the ligation efficiency using this system, a series of reactions were performed with varying amounts of digested genomic and vector DNA.

Genomic DNA of each mutant was digested to completion with concentrated EcoRX, ligated at various concentration ratios to 0.1-0.25 |ig calf alkaline phosphatase-treated pBR322 previously cut with EcoKl, and transformed into E. coli RRl or HBIOI. Clones that inherited the transposon were selected by plating the transformation mixture onto NA + Km (30|J.g/ml). Colonies were growth-tested on NA + Ap (30^g/ml) and NA + Tc (20^g/ml) to 125

ensure they were not spontaneous Km^ mutants. Each clone was screened for plasmids which after purification were transformed into the cloning host E. coli ED8654 to confirm the antibiotic resistance properties were carried on the plasmid.

Kanamycin-resistant clones were isolated from each mutant and were numbered in groups according to their approximate molecular weight and the host strain in which they were isolated (Table 16). All of the clones encoded Ap^, but for the majority the Tc resistance determinant of the vector pBR322 was not expressed. Each clone that was Ap^Km^Tc^ harboured a plasmid of far greater molecular weight than pBR322, which when transformed into E. coli ED8654 also transferred the antibiotic resistance markers. The size of these plasmids indicated that both the transposon and a large flankingregion o f DNA was inserted in the vector. Four classes of clones were isolated from PS5-16. The 2 largest (28 and 40kb) encoded Ap^Km^Tc^ whereas the 2 smaller clones encoded only Ap^Km^ (8 and 12kb). Two classes of clones (8 and 25kb) were isolated from 105-17(2) and, as in the case with clones from PS5-16, only the largest clones encoded Tc^ in addition to Ap^Km^. In some instances, more than one class of clone was isolated off the same selection plate. One clone of each class from PS5-16 and 105-17(2) was retained for further analysis. From PS5-16, 4 clones were retained. They were (with their molecular weights) pND200 (28kb), pND201 (40kb), pND202 (12kb) and pND203 (8kb). Two clones retained from 105-17(2) were pND204 (25kb) and pND205 (8kb).

All of the clones from 105(3), PS5-18 and 105-17(10) only encoded Ap^Km^ and harboured a plasmid approximately 8kb in length. As insertion into the EcoRl site of pBR322 does not inactivate the tetracyclineresistance gen e and these clones were less than lOkb, which is the combined molecular weight of pBR322 and Tn5 (4.3 and 5.7kb respectively), there was a possibility that either 126

TABLE 16, Clones of Eco R1 -restriction Fragments of Tn5- Induced Mutants, their Approximate Size and Tetracycline Sensitivity.

E. coli No. Clones Approx. Tc Mutant No. Clone No. Host strain Isolated M. Wt Sensitivity (kb) (a)

105(3) K1-11 RR1 11 8 S

105-17(2) A1-A6 HB101 6 25 R It A7-A11 HB101 4 8 S If A10 RR1 1 8 S

PS5-16 D1-D2 RR1 2 28 R II D3-D9 HB101 7 8 S II D10-D16 RR1 7 12 S II D17 RR1 1 40 R

PS5-18 G1-6 RR1 6 8 S

105-17(10) F1-3 HB101 3 8 S

(a) R and S denote resistant and sensitive respectively. 127

a fragment of the transposon or the vector, together with most of the flanking region had been excised and deleted. For those strains from which only Ap^Km^ clones were derived, the cloning experiments were twice repeated using freshly isolated total DNA of the relevant mutant and EcoRl from different suppliers. The last step was thought necessary as the unexpected low molecular weight clones may have arisen from the activity of contaminating restriction enzymes in the EcoRl preparation. In these repeat experiments, E. coli HBlOl was used as the host strain to eliminate any possible RecA-initiated recombination. Transformants were screened on both NA + Km and NA + Km + Tc (15^g/ml) as the vector carrying a large insert could be unstable in the absence of Tc selection pressure. In all cases, similar clones carrying an 8kb plasmid were isolated off NA + Km plates but no clones were isolated off the NA + Km + Tc medium. One clone from each mutant was retained for further study. They were pND206 from 105(3), pND207 from PS5-18 andpND208 from 105-17(10).

3.3.2. Restriction Mapping and Southem Hybridization Analysis of Clones isolated from pJB4JI-derived Mutants.

Restriction enzyme mapping of the clones pND200, pND201 and pND204 were performed to determine their insert sizes and similarities. The clones were digested with the enzymes EcoRl and Hin^Sl separately and were also digested with both enzymes together, the DNA fragments were then separated on an agarose gel (Figure 12A). In addition, digests with Bam HI andX/z<9l as well as double digests ^XhBamYillEcoRl^XhollEcoRl m6.XhoHBamYi\ were performed and the DNA separated on an agarose gel (Figure 12B). The restriction digests of a clone of a pSUPlOl 1-derived mutant (pND209) are included and will be referred to in a later section. Digestion of each plasmid with EcoRl indicated that pND200 and pND204 had inserts of approximately 24.3 and 19.9 kb 128

Figure 12A. Restriction Endonuclease Digestion of the Clones pND200, pND201, pND204 and pND209 with the Enzymes EcoR^, H/ndlll and Double Digests.

Agarose Gel Electrophoresis from Right. Lane Clone and Enzyme/s used.

1 X-Hin6\\\ 2 pND209 -Eco R1 3 II -H/ndlll 4 II -Eco RI/H/ndill 5 pND204 -Eco R1 6 II -H/ndlll 7 II -Eco RMHin6\\\ 8 pND200 -Eco R1 9 II -H/7?dlll

10 II -EcoR1/H/>?dlll (Partial digest) 11 pND201 -Eco R1 (Partial digest) 12 II -Hind\\\ 13 II -Eco RI/H/ndlll

Numbers on the right are X-Hin dill weight markers in kb.

Gel Conditions: Horizontal gel, 0.8% agarose,

35 volts, 18 hours, 4°C 13 12 11 10 9 8 7 6 5 4 3 2 1

•«»»« ma- 129

Figure 12B. Restriction Endonuclease Digestion of

the Clones pND200, pND201, pND204

and pND209 with Various Enzymes.

Agarose Gel Electrophoresis from Right.

Lane Clone and Enzyme/s used. 1 pND201 'Bam H1 2 X-Eco RMHin 6\\\ 3 pND209 -Bam W 4 " -Xho 1 5 " -Bam HMEco R1 6 pND204 -Bam H1 7 " -Xho 1 8 " -Bam HMEco R1 9 " -Bam HMXho 1 10 " -XhoMEcom 11 pND200 -Bam HI 12 " -Xho 1 13 " -Bam HMEco 14 " -Xho^/EcoR^ 15 " -Bam W/Xho ^ 16 pND201 -Xho^ 17 " -Bam HMEco 18 " -BamHMXho^ 19 pND200 -XhoMEcoR^

Numbers on the right are X-Eco RMHin dill weight markers in kb.

Gel Conditions: Horizontal gel, 0.8% agarose, 40 volts, 20 hours, 4°C 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

5i5.0 130 respectively (Figure 12A) of which Tn5 contributed 5.7kb (in Figure 12A, the EcoKHHindlYL double digest of pND200 (lane 10) and the EcoRl digest of pND201 (lane 11) are partial digests. A clearer double digest pattern of pND200 is shown in Figure 24). There appeared to be only one copy of the transposon in each clone thus sizable fragments of the flanking DNA from the mutants PS5-16 and 105-17(2) had been cloned. The clone pND201 had 2 generated inserts, 24.3 and 13.9kb, which considering both pND200 and pND201 were derived from PS5-16 suggested pND201 was simply pND200 with an extra 13.9kb chromosomal fragment. It was not known if the extra fragment was contiguous with the pND200 fragment in the chromosome (ie: a partial EcoRl digestion of PS5-16 during the cloning procedure) or came from elsewhere in the chromosome and had been incorporated during ligation.

The clones had similarities in their restriction pattems in addition to those expected from the internal fragments of Tn5 and pBR322. The similarities in the digest pattems of pND204 and pND200 in Figures 12A and 12B indicated that DNA from pJB4JI may have inserted together with Tn5 in the mutants 105-17(2) and PS5-16. A comparison of the restriction map of pJB4JI (Figure 10) with the above 2 clones suggested tiiis was the case. The analogous restriction bands of pND200 and pND204 were approximately the same molecular weight as the sizes oftheMu::Tn5 fragments of pJB4JI reported by Hirsch & Beringer (1984). The vector pJB4JI is an R751-R1033 hybrid construct (pPHlJI) containing an integrated Mu with an inserted Tn5 in tiieM u DNA (Figure 10). Hybridization of tiie cloned DNA witii constituent parts of the pJB4JI would confirm whetiier this was tiie case. Neither the construct pPHlJI nor R751 could not be obtained for tiiis study, however Mu phage was propagated from a lysogenic E. coli strain MC4100.5F:: Mu cts62 ^y ^ thermal shift to tiielyti c stage and Mu DNA extracted from tiiephage . This thermoinducable prophage was also used in the construction 131 of pJB4n (Toussaint, 1985).

The digested DNA of the clones in Figures 12A and 12B was transferred onto nitrocellulose filters and hybridized to radioactively labelled Mu DNA. DNA fragments of pND200, pND201 and pND204 hybridized to the Mu probe. The fragments of pND200, pND201 and pND204 which hybridized to Mu in the EcoRl-HindTR double digests (Figure 13A) were the 2.8 and 6.0kb fragments common to each clone, the 7.7kb fragment of pND204 and the common 12.1kb fragment of pND200 and pND201 (the highest molecular weight fragment of pND200 which hybridized to Mu DNA in lane l^hould be ignored as this is a / result of a partial digest). The clone derived fom the pSUPlOl 1-generated mutant served as a negative control as there was no Mu DNA reported in pSUPlOl 1 (Simon a/., 1983). Similarly in Figure 13B, Mu hybridized to common sized fragments in each clone as well as fragments which were unique. The restriction digest and hybridization results enabled a restriction map of each clone to be deduced (Figure 14). The restriction map of pJB4JI did not indicate the exact distance between the Hin6Bl site in the left arm of Tn5 and the tail end of Mu (Figure 10). The only indication of size from that //indlQ site is T.Okb to an upstream Pstl cleavage site and 29.7kb to the next EcoRl site, both being located on the R751 replicon. Assuming the distance from the above ///ndin site to the tail end of Mu is between 6.5 to T.Okb, then the clone pND204 would contain 0.7 to 1.2kb of flanking DNA not containing Mu sequences and pND200 would have between 5.1 and 5.6kb. As pND200 hybridized to PS5-1 DNA (see Section 3.5.1) and pJB4JI did not (data not shown), it was assumed that the non-Mu DNA flanking Tn5 within this clone was of Pseudomonas origin. Similar hybridization experiments with pND204 failed, presumably due to the small size of that flanking DNA. It was therefore reasonable to presume that the transposing mutagenic agent in the mutants PS5-16 and 105-17(2) was Mu and not solely the transposon Tn5. 132

Figure 13A. Hybridization of Digested Clones in Figure 12A with Mu DNA.

Lanes as per Figure 12A.

Numbers on the right are X-Hin dill weight markers in kb. 13 12 11 10 9 8 7 6 5 4 3 2 1

kb 23.1

9.4 6.6 4.3

2.3 2.0 133

Figure 13B. Hybridization of Digested Clones in Figure 12B with Mu DNA.

Lanes as per Figure 12B.

Numbers on the right are X-Eco RMHin dill weight markers in kb. 19 18 1716 1514 13 12 11 10 9 8 7 6 5 4 3 2 1

— 2.0 1 Q 134

Figure 14. Restriction Digest Maps of the Clones pND200, pND201 and pND204.

Distances in kb between cleavage sites are indicated by the numbers.

Legend. Ap denotes Ampicillin resistance Tc II Tetracycline E II EcoR^ X II Xho^ H II Hin6\\\ B II BamW

thick lines in insert denotes Mu DNA double thick lines denotes Tn5 X H X B H X H

Mu Mu

• • • 0.7 1.6 0.3 1.5 0.7 5.5.3

—• '-li! 0 .4

pND200. M. Wt = 28.6 kb. 3.9

E X B E X H X B H X H

Mu Mu

3.1 11.4

" .. • • •

pND201. M. Wt. = 42.5 kb 3.9

X H X B H X H

Mu Mu

7.0 0.7 1.6 0.3 1.5 0.7 5.3

pND204. M. Wt. = 24.2 kb. 3.9 135

The 8kb plasmids, pND202, pND203, pND205, pND206, pND207 and pND208 were digested with EcoRl, Hin^m, BamRl, Pstl m&Xhol, All of the plasmids had the same digest pattern (data not shown), the molecular weight was 7.9 - 0. Ikb. The plasmids had one EcoRl site as only one linear band of 8kb was observed when the plasmids were digested with EcoRl, When digested with Hindlll, 2 fragments of 3.4 and 4.4kb were observed, it was not known if small fragments of below SOObp were present. Double digests with BamHl/EcoRl, Pstl/EcoRh Hindlll/EcoRl, Hindlll/Pstl.Xhol/EcoRl and 5am Hl/Pst 1 suggested that these clones were pBR322 with an insert of approximately 3.6kb located in the tetracycline resistance gene within lOObp downstream of the HindHI recognition site in pBR322. The insert appears to be the internal fragment of Tn5. Comparison of the insert's restriction map with that of Tn5 (Jorgenson et al, 1979) suggests that the ends of the insert are approximately lOObp upstream of the HinWi sites within the IS50 sequences.

3.3.3. Southern Hybridization Analysis of Tn5-induced Mutants Derived from the Vector pJB4JI.

In the previous 2 Sections, flanking DNA sequences about the transposon from two pJB4JI-derived mutants were seen to contain Mu DNA. Attempts to clone the transposon and flanking regions from 3 other pJB4JI-derived mutants were unsuccessful, presumably due to deletion/excision events. It was of interest to determine (a) if the above 3 mutants and other pJB4JI-derived mutants also contained Mu DNA, (b) the location of the transposon and Mu, (c) whether pJB4JI remained in these mutants and (d) the number of copies of Tn5 within each mutant. In addition to the 5 mutants used for the cloning experiments in Section 3.3.1, an extra 15 pJB4JI-derived mutants were studied by Southern hybridization analysis. The 20 mutants used for this work, the products accumulated by each 136

Table 17. pJB4JI-derived Mutant Strains Investigated by Southern Hybridization Analysis.

Mutant Product Accumulated Strain (a) by mutant strain (b)

105(2) 12I30H-ADD 105(3) 12P0H-ADD PS5-16 12aOH-ADD 105(5) 12aOH-ADD PS5-18 12P0H-ADD 105(7) 12aOH-ADD * 105-16(1) PS 105-16(10) PS 105-17(2) 3KEA 105-17(10) 3KEA 105(202) 3KEA 105(205) 3KA 105(213) 3KA * 105(214) No accumulation * 105(220) No accumulation * 105(222) 3KEA 105(247) PS 105(301) PS 105(305) 12pOH-ADD

* 119-9(5) No growth in DCA NB: (a) * denotes Gm^. (b) products as per Table 9. 137

strain and the response in the presence of 2g/L DCA are listed in Table 17. The sensitivity of each mutant to gentamicin is also noted. The wild type parent of 19 of the mutants was PS5-1 and the remaining mutant was a PS8-1 derivative. These particular strains were chosen for this study for they either:

a) accumulated similar products, but differed in yield or the time before product disappearance began

b) expressed the antibiotic resistance determinant of the vector c) did not degrade the steroid substrate.

The fate of the vector pJB4JI and the location of the transposon Tn5 was investigated by physical means. Total DNA from each of the selected pJB4JI-derived mutants and the parent strains was isolated and subjected to agarose gel electrophoresis together with pJB4JI. Some of the mutants appeared to have lost the 40kb resident plasmid (Figure 15 A and Figure 15B). It is doubtful if this was significant as the strain 105-17 Leu" (PS5-6) lost this plasmid over a period of time whilst stocked in glycerol at -20°C without losing its ability to grow in cholic acid media (data not shown). Thus its Tn5-derived mutants, 105-17(2) and 105-17(10), also lack this plasmid. In addition, the mutant 105(202) had lost the 10.6kb resident plasmid. All of the resident plasmids which were retained by the mutants appeared to have molecular weights unchanged from the equivalent plasmids carried by the parent strain. No new plasmids could be seen in any of the mutants except for 105-16(1) which had a plasmid corresponding in size to pJB4JI suggesting the vector had not "suicided" within this strain. This result was unexpected as 5 of the mutants, 105(214), 105(220), 105(222), 105-16(1) and 119-9(5), exhibited Gm^ suggesting that some of the Tn5 transconjugants derived fi-om pJB4JI had inherited aU or a fragment of the vector in addition to the transposon Tn5 or Mu/rn5. 138

Figure 15A. Total DNA of PS5-1 and pJB4JI-derived Mutants.

Agarose Gel electrophoresis: From Right Lane Strain/Plasmld 1 pJB4JI 2 3 PS 5-1 4 105(2) 5 105(3) 6 PS5-16 7 105(5) 8 PS5-18 9 105(7) 10 105-16(1) 11 105-16(11) 12 105-17(2) 13 105-17(10)

Gel Conditions: Horizontal Gel, 0.8% agarose, 60 volts, 8 hours, 4°C. 13 12 11 10 9 8 7 6 5 4 3 2 1

pJB4JI 40.0 Chromosome

10.6 139

Figure 15B Total DNA of PS5-1, PS8-1 and pJB4JI- derived Mutants.

Agarose Gel electrophoresis: From Right

UOfi Strain/Plagmid

1 pJB4JI 2 PS5-1 3 105(202) 4 105(205) 5 105(213) 6 105(214) 7 105(220) 8 105(222) 9 105(247) 10 105(301) 11 105(305) 12 PS8-1 13 119-9(5)

Gel Conditions: Horizontal Gel, 0.8% agarose, 60 volts, 8 hours, 4°C. 13 12 11 10 9 8 7 6 5 4 3 2 1 kb

pJB4JI

40.0 Chromosome

10.6

4.1 140

The location of the transposon within each mutant was determined by Southem hybridization analysis with Tn5 sequences. The DNA of the above 2 gels was transferred onto nitrocellulose filters and hybridized to radioactively labelled pKan2 DNA. pKan2 is a recombinant plasmid containing the 3.3kb HindSR inner fragment of Tn5 plus the first 1100 base pairs of each IS50 arm cloned into pBR322 (Scott et al., 1982) and as such served as a hybridization probe of Tn5 sequences. The transposon probe hybridized to the suspected pJB4JI plasmid of 105-16(1) as well as to the chromosome in that mutant. In all other mutants, only the chromosomal DNA hybridized to pKan2 indicating that in each mutant tested, Tn5 had inserted into the chromosome (Figures 16A and 16B). Faint hybridization was observed in the 10.6kb resident plasmid band of the wild tpye PS5-1 and its mutants in Figure 16A, however this is believed to be an artifact due to non-stringent washing of the filter as these plasmids did not hybridize in Figure 16B. It would appear that this plasmid has weak homology with pKan2.

Chromosomal DNA of 8 mutants was digested with ///ndin, separated on an agarose gel and transferred onto a nitrocellulose filter which was probed against pBR322 and pKan2. Hybridization bands were only observed with the pKan2 probe and all of the mutants showed only one hybridization band of a molecular weight of 3.3kb (data not shown). This corresponded to the ///«din fragment of Tn5 suggesting that the only hybridization between the mutant DNA and pKan2 was caused by the Tn5 sequences. To ascertain the number of copies of Tn5 within each mutant and the size of the restriction fragmentswhic h contain the transposon, hybridization studies were performed with digested DNA. Chromosomal DNA, rather than total DNA of each of the above 20 mutants was isolated, as Tn5 insertion in the chromosome was believed to affect the steroid catabolic pathway. The DNA was digested with EcoRl (as there are no EcoRl restriction recognition sites witiiinTn5) and separated on an agarose gel (Figures 141

Figure 16A. Hybridization of Total DNA ofpJB4JI-

Derived Mutants in Figure 15A with pKan2 13 12 11 10 9 8 7 6 5 4 3 2 1

pjB4n I Chromosome 142

Figure 16B. Hybridization of Total DNA ofpJB4JI- Derived Mutants in Figure 15B with pKan2 13 12 11 10 9 8 7 6 5 4 3 2 1

pjB4n m Chromosome

A •••it" ".-S'-'K; , . ••-

^ . ' = - ^ + -A

'•I ' > 143

17A and 17B). The digested DNA was transferred onto nitrocellulose filters and hybridized to labelled pKan2. The sizes of the restriction fragments which hybridized to pKan2 were estimated by comparison with the X.Hin6ni standard.

No hybridization was detected between pKan2 and the DNA of the controls PS5-1 in Figure 18A. In Figure 18B, a weak hybridization signal at approximately 9.0kb was observed in both the control and mutant DNA. This was suspected to be an artifact as such a signal was not seen in Figure 18 A. Generally all of the fragments which hybridized to pKan2 were greater than 15kb in size. This was expected as it has been previously shown (Section 3.2.2) that Mu was incorporated with Tn5 in PS5-16 and 105-17(2). The Mu/Tn5 fragments of pND200 and pND204 were about 19.2kb. As it was likely that Mu had co-transposed with Tn5 in all of the pJB4JI-derived mutants, then the expected size of EcoRl digest fragments which would hybridize to pKan2 would be at least 19.2kb plus Pseudomonas DNA to the next EcdRl site. One mutant, 105(5) had a single fragment of 17kb which hybridized to pKan2 and as such may not contain Mu DNA. PS5-16 had 2 fragments which hybridized to pKan2, one was approximately 24kb which is most probably the fragment that was cloned in pND200 and the other about 20kb. The mutant 105-17(2) had only one fragment of about 20kb which correlates with the insert size of pND204. In 12 of the mutants tested, only one fragment hybridized to pKan2 indicating that these mutants either had only one copy of Tn5 inserted within the chromosome or had more than one copy and these transposons were located quite close to each other. The remaining 8 mutants, PS5-16, PS5-18, 105(202), 105(214), 105(220), 105(222), 105(305) and 119-9(5) had 2 hybridized fragments. All had at least one fragment over 20kb which hybridized to pKan2. The size of the second fragment ranged between 15-22kb for 5 of these mutants, however for the mutants PS5-18, 105(202) and 105(214) the lower hybridized fragment was 3.4kb. 144

Finure 17A EcoR^ Digests of Chromosomal DNA of pJB4JI-derived Mutants of PS5-1.

Agarose Gel Electrophoresis: From Right

Uofi Strain

1 X-Hin dm; size standard

3 Eco R1 digest of PS 5-1 4 105(2) 5 " " " 105(3) 6 " " " PS5-16 7 " " " 105(5) 8 " " " PS5-18

9 " " " 105(7) 10 ...... 105-16(1) 11 ...... 105-16(10) 12 ...... 105-17(2) 13 " " "105-17(10)

Numbers on the right are X-Hin dlli weight markers in kb.

Gel Conditions: Horizontal gel, 0.75% agarose, 45 volts, 20 hours, 4°C 13 12 11 10 9 8 7 6 5 4 3 2 1 145

Finure 17B EcoR^ Digests of Chromosomal DNA of pJB4JI-derived Mutants of PS5-1 and PS8-1.

Agarose Gel Electrophoresis: From Right Lane Strain 1 X-Hin dm; size standard 2 3 Eco R1 Digest of PS5-1 4 105(202) 5 105(205) 6 " " " 105(213) 7 " " " 105(214) 8 " " " 105(220) 9 105(222) 10 ...... 105(247) 11 ...... 105(301) 12 ...... 105(305) 13 14 ...... 15 119-9(5) 16

Numbers on the right are X-Hin dlli weight markers In kb.

Gel Conditions: Horizontal gel, 0.75% agarose, 45 volts, 20 hours, 4°C 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 146

Figure 18A. Hybridization of DNA Digests in Figure 17A to

pKan2 Plasmid Probe.

Numbers on the right are X-Hin dill weight

markers in kb. 13 12 11 10 9 8 7 6 5 4 3 2 1

kb 23.1

9.4 6.6 4.3

I 2.3 2.0 147

Figure 18B. Hybridization of DNA Digests in Figure 17B to

pKan2 Plasmid Probe.

Numbers on the right are X-Hin dill weight

markers in kb. 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

"23.1

9.4

6.6

4.3

2.3 2.0 148

The presence of Mu DNA in the mutants was investigated by hybridization. Chromosomal DNA of each mutant was isolated, digested with EcoRl and separated on an agarose gel (Figure 19). X-Hinm. standards were not included due to space restrictions of the gel, instead the fragment sizes of pJB4JI and Mu derived from publications were used. The restriction pattem of these were confirmed by electrophoresis on an agarose gel with a X-size standard (data not shown). The EcoKl digest pattem of pJB4JI concurred with the restriction map derived by Hirsch & Beringer (1984), ie: fragments of 42.5, 18.0, 12.2, 11.4, 7.3 and 5.0kb. Viral Mu DNA extracted from phage particles consists of Mu DNA (37.8kb) with random host DNA sequences of variable length between 1.5 and S.Okb at the tail end of each molecule (Toussaint, 1985). In the main, the observed EcoKl digest pattem of the isolated Mu DNA agreed with published maps (Toussaint, 1985) except two small fragments (2.5 and 2.2kb) could not be accounted for. Two fragments, 18.2 and 5.1kb, being the central and a regions of Mu were clearly discemible. The p region containing the variable ends was seen in Figure 19 as an indiscreet band ranging between 13.5 and IT.Okb in length. The largest fragment was assumed to be undigested DNA due to the previously-noted difficulty in digesting Mu DNA with restriction enzymes. Hirsch & Beringer (1984) reported Tn5 was located in the p region of an integrated Mu within pJB4JI. They mapped the distance from the tail end of Mu to the adjacent EcdRX site within Mu to be approximately 19.2kb. This figure agrees well with the published size of the p region plus an added Tn5. In the case of pND200 and pND204, the inserts of 24.3kb and 19.9kb would therefore consist of the p region of Mu plus Tn5 plus Pseudomonas DNA to the next EcoRl site. The hybridized fragments in each strain other than the central fragment would contain DNA sequences from either pJB4JI or the Pseudomonas strain, ie:flanking DNA . The molecular weight of each fragment would vary depending on the precise integration point of the Mu DNA relative to the nearest EcoRl site within the 149

Figure 19. Eco R1 Digested Chromosomal DNA of pJB4JI-derived Mutants of PS5-1

Agarose Gel Electrophoresis: From Right

Lane Strain/Plasmid 1 Eco R1 digest of Mu DNA 2 " " " pJB4JI 3 " " " PS5-1 4 105(2) 5 105(3) 6 " " " PS5-16 7 " " " 105(5) 8 PS5-18 9 " " " 105(7) 10 105(202) 11 105(205) 12 ...... 105(213) 13 105(214) 14 ...... 105(220) 15 105(222) 16 105(247) 17 ...... 105(301) 18 105(305) 19 105-16(1) 20 " " " 105-16(10) 21 105-17(2) 22 105-17(10)

Numbers on the right are fragment sizes of EcoRI digested Mu and pJB4JI in kb. Gel Conditions: Horizontal gel, 0.8% agarose, 35 volts, 18 hours, 4°C 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

— 42 18 Variable region 150

flanking region, but would be expected to be greater than the a region of Mu (5.1kb) and the p region with the incorporated Tn5 (19.2kb).

The fcoRl-digested DNA from the pJB4JI-derived mutants was transferred onto a nitrocellulose filter and hybridized to radioactively labelled Mu DNA. The autoradiograph is shown in Figure 20. The three fragments of pJB4JI (42.5, 18.0 and 7.3kb) which hybridized to Mu DNA correlated to the Mu- containing fragments shown in the restriction map of Hirsch & Beringer (1984). No hybridization between PS5-1 DNA and Mu was observed. Almost all of the pJB4JI-derived mutants hybridized to Mu DNA. The exceptions, which therefore contained no Mu DNA, were 105(3), 105(5), 105(7), 105(205) and 105(213). The intensity of the autoradiograph signals made accurate sizing of the fragments difficult although some generalizations could be made. All of the strains which contained Mu sequences had the 18.0kb central fragment and except for 105(214) and 105(301), each mutant also had a hybridized fragment greater in size than the central fragment. Possibly for 105(214) and 105(301) there may have not been enough DNA in the gel or on the filter to obtain a strong signal. The largest sized hybridized fragment of each mutant relative to the size of the largest fragment in the other mutants was of the order PS5-18>105(202) and 105(220)>105(2), PS5-16, 105(222), 105(247), 105(305) and 105-16(11)> 105-16(1)>105-17(2) and 105-17(10). The relative pattern of these large fragments generally paralleled the largest fi^gment of that mutant which hybridized to pKan2 (Figures 18A and 18B). All of the mutants also had fragments smaller than 18.0kb which hybridized to Mu DNA. The size of the fragments differed in each strain and there was no clear pattern. Some of the mutants had more than one small fragment, for example the mutants PS5-16, 105(220), 105(222) and 105(247) had 2 small fragments and 105(305) had 4 which hybridized to Mu DNA. The reasons for the disparities are not known and were not investigated. 151

Figure 20. Hybridization of Eco R1-digested Chromosomal DNA of pJB4JI-derived Mutants of PS5-1 with Mu DNA Probe.

Numbers on the right are fragment sizes of EcoRI digested Mu and pJB4JI in kb. 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 152

3.4. MECHANISM OF TRANSPOSITION BY Tn5 FROM pSUPlOll.

In Section 3.2.1., mutants capable of accumulating steroid intermediates were derived using the vector pSUPlOll. As diagrammed in Figure 10, pSUPlOll has pACYC184 as its replicon and encodes the Cm^ determinant from that plasmid. Chloramphenicol sensitivity tests for the pSUPlOl 1-derived mutants were ambiguous because of the high resistance of the parent strains, so the presence of some or all of the vector in these mutants could not be discounted. Two of these mutants were selected for further investigation to determine the fate of the vector. As outlined in Section 3.3, to enable the characterization of tiie regions about the transposon, clones were isolated from both mutants by selecting for the Km^ determinant of Tn5. The chosen mutants PS8-10 and 1-4(1) accumulated the 12a hydroxy derivatives of OPDC and the product class 3KA respectively after 48 hours fermentation in the presence of 2 g/L DCA.

This section reports the isolation and characterization of such clones from the above mutants. In addition. Southern Hybridization analysis of the 2 mutants and a further 3 mutants derived frompSUPlO l 1 were performed.

3.4.1. Selection for Clones Encoding Kanamycin Resistance from pSUPlOl 1-derived Mutants.

As outlined in Section 3.3.1, EcoRl digested genomic DNA from each mutant was ligated into the EcoKl site of pBR322. Clones were selected by spreading the transformation mixtures onto NA + Km(30 M.g/ml). One clone was isolated from PS8-10 which harboured a plasmid approximately 18kb in lengtii and encoded Ap^Km^Tc^. This clone was designated pND209. Six clones were isolated from 1-4(1) and all harboured a plasmid approximately 8kb which 153

encoded Ap^Km^ only. One clone which was designated pND210 was retained for further study. Despite repeated experiments, as previously described, with a Rec A" host HBlOl and the addition of Tc to the selection medium, only similar clones of approximately 8kb were isolated from 1-4(1).

3.4.2. Restriction Mapping of pND209.

pND209 was digested with EcoKl, HindJS. and with both enzymes together and separated on an agarose gel (Figure 12A, Lanes 2-4). The EcoKl digest showed that the clone was a 12.9kb fragment inserted in pBR322. Digests with Hindm and EcoKllHindJH indicated that the central Hin6ni fragment of Tn5 was flanked by 2 fragments of 7.3 and 2.2kb. Further digests with BamUl, Xhol and BamUl/EcoRl (Figure 12B) together with the digest patterns in Figure 12A enabled a restriction map of the plasmid to be deduced (Figure 21). The clone did not contain any sequences homologous to Mu DNA (Figures 13A and 13B). The inserted DNA consisted of one copy of Tn5 with a flanking region located at each end. The distance fromth e Xhol site in the IS50 element to the end of Tn5 is 0.4kb, therefore the two flanking regions about Tn5 were 6.2 and l.lkb.

3.4.3. Hybridization of pND209 with Fragments of pSUPlOll.

It is possible that pND209 contained DNA sequences of the vector pSUPlOll as well as the transposon Tn5, for pSUPlOll had been found integrated in the chromosome of Zymomonas mobilis by Warr (1985). To confirm that all of the flanking regions about Tn5 within pND209 were in fact Pseudomonas DNA and did not contain vector DNA, it was necessary to hybridize fragmentso f pSUPlOll against pND209. That pND209 did contain 154

Figure 21. Restriction Enzyme Map of pND209.

X H X B H X

t t i t t i

6.6 0.7 1.6 0.3 1.5 0.7

./"•:••;

pND209. M. Wt. = 17.2 kb.

Distances in kb between cleavage sites are indicated by the numbers.

Heavy line as such. represents Tn5. 155

some Pseudomonas DNA was beyond doubt as in previous hybridization experiments (data not shown) the clone hybridized to PS8-1. Examination of the restriction map of pSUPlOll showed a 2.6kb region containing a Mob site inserted in the Tc^ gene of the replicon pACYC184, with Tn5 integrated within the 2.6kb region (Simon etal, 1983; Warr, 1985). To separate the Mob region and the replicon pACYC184 completely from the transposon in a restriction digest of pSUPlOl 1 was an onerous task and it was simpler to use plasmids containing the constituent segments. The plasmid pSUP202 has a 1.9kb fragment, which encodes the mobilizing region of RP4, inserted into the cloning vector pBR325. The source of this insert was a Sau 3A partial digest of RP4, the same source as the 2.6kb Mob region within pSUPlOll (Simon et al, 1983). Hybridization of pSUP202 and/or pACYC184 against pND209 fragments would indicate if pND209 (and by definition the mutant PS 8-10) contained any homologous regions to either of the constituent segments of pSUPlOl 1.

The pBR322 repUcon of pND209 and pBR325 from pSUPlOll have homologous sequences which would confuse the results, therefore pND209 was double digested with EcoRl and Hin^iBl and separated on an agarose gel. The 7.3 and 2.2kb fragments were separated from the pBR322 and the inner Hindlll fragment of Tn5 by electroelution, ie: the only DNA to be used as the probe were the outer arms of tiie IS50 elements and the cloned flanking regions. The plasmids pSUPlOll, pACYC184, pSUP202 and pND209 were digested with various restriction enzymes and separated on an agarose gel so that as far as possible the regions of interest were well separated. Genomic DNA of PS8-1 and PS8-10, digested with was included in the gel (Figure 22A). The separated DNA fragments were transferred onto a nitrocellulose filter and hybridized to the radioactively-labelled electroeluted fragments of pND209. 156

Figure 22A. Restriction Endonuclease Digests of the Plasmids, pACYC184, pSUP1011, pSUP202 and pND209, and Chromosomal DNA of PS8-1 and PS8-10.

Agarose Gel Electrophoresis. From Right.

Restriction Fragment Sizes (kb)

lane Plagmid/PNA Enzyme Used (from well down^

1 2 pND209 Hin dlll/Eco R1 7.4, 4.3, 3.4, 2.1 3 4 PSUP202 Hin6\\\/Ecom 4.8,3.1 5 " undigested 7.9 6 PSUP1011 H//?dlll/EcoR1 5.1,3.3,2.5,1.6 7 " H/A7dlll/Sa/1 3.6,3.0,2.5,1.9,1.5 8 PACYC184 H/ndlll/EcoR1 4.3 (partial cut), 2.8,1.5 9 X Hin6\\\/EcoR^ 21.2,5.1,5.0,4.2,3.5,2.0 1.9,1.6,1.3,1.0, 0.8, 0.6 10 11 PS8-1 Eco R1 12 PS8-10 13 PS8-1

Numbers on the right are fragment sizes of >--H/hdlll/EcoR1 in kb. Gel Conditions: Horizontal gel, 1.0% agarose, 30 volts, 23 hours, 4°C 13 12 11 10 9 8 7 6 5 4 3 2 1 157

Figure 22B. Hybridization of Digested Component

Fragments of pSUP1011, PS8-1 and

PS8-10 with Isolated Fragments of

pND209.

Lane Plasmid/Enzvme. Hybridized Fragment Sizes. m

2 pND209-Eco R1/H/ndlll 7.3,2.1

6 pSUPIOII-EcoRI/H/ndlll 5.1,2.5

7 " -Hin6\\\/Sal^ 3.0,2.5

9 X-Eco RMHin dlii per Figure 22A

11 PS8-1 - Eco R1 7.8

12 PS8-10- " 14.0

13 PS8-1 - " 7.8

Numbers on the right are fragment sizes of

X-H/ndlll/EcoRI in kb. 13 12 11 10 9 8 7 6 5 4 3 2 1

- 21.2

^7.3 ^5.0 4.2 '3.5

2iLl.9 1.6 1.3 158

There was no hybridization between either pACYC184 orpSUP202 with the probe DNA (Figure 22B). The only plasmid bands to show extensive homology with the probe were those fragmentso f pSUPlOl 1 which contained the outer 1.2kb arms of IS50 and the two fragments of pND209 which comprised the probe DNA. The digested chromosomal DNA of PS8-1 and its mutant showed weak signals on the autoradiograph. A strong signal was obtained after a longer exposure time (36 hours) although the plasmid section of the autoradiograph showed extensive background. To clarify the signals, the chromosomal section of the 36 hour autoradiograph was pasted onto the back of the shorter exposed autoradiograph. The EcoKl digested fragment from PS8-1 which hybridized to the pND209 fragmentsprob e was approximately 7.8kb. The fragment of PS8-10 which hybridized to the probe was approximately 14kb. The difference in molecular weight of the hybridized fragments of the mutant and its parent was approximately 6kb which correlates reasonably well with the M. Wt. of Tn5 (5.7kb). These results suggested that none the vector DNA was inserted in the site of the mutant from which pND209 was derived.

3.4.4. Southern Hybridization Analysis of Tn5-induced Mutants Derived from the Vector pSUPlOll.

The strain PS8-10 was the only mutant derived using Tn5 as the mutagen in this project which accumulated the compound 12a hydroxy-ODPC from the degradation of DCA in approximately 90% yields. Its double transposon-mutated derivative PS8-22 accumulated the same compound, but exhibited a longer accumulation time before product utilization. For this reason, these two mutants were investigated by Southern Hybridization analysis. The mutant 1-4(1) was also investigated and for comparison purposes, 3 other mutants derived from pSUPlOl 1 were included in tiiestudy . These mutants were 119(5), 159

119(7) and 119(8). The first two accumulated SKA products whereas 119(8)

degraded DCA with no accumulation. Two of these mutants, 119(5) and 119(8),

appeared to be more resistant to chloramphenicol than the others suggesting that

some of the mutants may have inherited all or a fragment of the vector in addition

to Tn5. As in the previous section, the fate of the vector plasmid was investigated

by physical means.

Each mutant was grown in liquid cultures containing Km (50 |ig/ml) as a

selective pressure. Two cultures of PS 8-22 were grown, one in the presence of

kanamycin only and the second in the presence of kanamycin and carbenicillin

(200 |ag/ml). Total DNA of the parent strains, PS7-1 and PS8-1, in addition to

the mutants was isolated and subjected to agarose gel electrophoresis. The

plasmids pSUPlOll and pAS8 were also run for comparison. Neither of the

parent strains had resident plasmids, so any plasmids seen in the mutants would

presumably be the vector or a derivative.

There were no plasmids observed in the mutants investigated (data not

shown) suggesting that either pSUPlOll had indeed "suicided" or it may have integrated in the chromosome. The DNA of the above gel was transferred onto a nitrocellulose filter and hybridized to radioactively labelled pKan2. No hybridization was observed between the probe and the parent controls (data not

shown). The chromosomal DNA of each mutant hybridized with pKan2 confirming that Tn5 was inserted in the chromosome. Both pSUPlOl 1 and pAS8 hybridized to the probe. As pSUPlOll contained Tn5, this resuh was expected.

It was not determined where the homologous region/s between pKan2 and pAS8 were, however it may possibly have been the Km^ genes encoded on the two plasmids 160

Chromosomal DNA of the parent strains and the mutants was digested with the restriction enzyme EcoRl and separated on an agarose gel. The digested DNA was transferred onto a nitrocellulose filter and hybridized against pKan2. Each mutant had one -digested DNA fragment which hybridized to pKan2 (data not shown). The fragments of PS8-10, PS8-22 and 119(8) which hybridized to pKan2 were all approximately 13.5kb whereas the fragments of 1-4(1), 119(5) and 119(7) were approximately 15-18kb. The size of the PS8-10 fragment was in accordance with the size of the insert of pND209. It would appear that the Tnl-derivative, PS8-22, did not have any extra DNA inserted into the EcoRl fragment of PS8-10 in which the Tn5 had transposed. 161

3.5. CLONING OF THE STEROID CATABOLIC PATHWAY.

The objective of the project was the construction of strains capable of steroid bioconversions to desired products. In a previous section, transposon mutagenesis was attempted which resulted in a number of mutants being isolated. An alternative approach is to clone the genes and operons which encode the steroid degradation pathway. Following characterization, genes encoding different pathway enzymes could be separated by sub-cloning, thus making it possible by the fusion of specific segments to construct strains which could carry out pre-determined steroid bioconversions. Using such constructs, the suspected revertants which have caused the eventual bypass of the transposon-induced mutations may be eliminated. Expression modifications such as the coupling of genes to strong promotors and the elimination of repression operators are future altematives.

The rationale to select clones encoding steroid conversion genes has been used previously to isolate Rhizobium trifoli genes involved in nitrogen fixation (Scott et al, 1982). Assuming that a mutant contains a single Tn5 which has inactivated a particular gene, the transposon and flanking regions of genomic DNA can be cloned into a plasmid. The flanking DNA would presumably encode the inactivated gene and possibly other steroid catabolic genes. The flanking regions can then be used as hybridization probes against a gene bank of the parent strain to detect clones containing the corresponding wild-type genes. If the whole operon is not on tiie clone selected against tiie "flanking region" probe then the gene bank could be further probed using a clone, derived from another transposon-induced mutant, which contained those genes missing from the original selected clone. Providing restriction endonuclease sites were available, further genes upstream or downstream could also be isolated by tiie use of chromosome 162

walking.

To enhance the probability of cloning the whole of the upper part of the bile acid pathway, restriction fragments from various mutants were cloned

(Sections 3.3.1 and 3.4.1). The mutants chosen were each reported to accumulate different products from a deoxycholic acid fermentation and so each mutant was deemed to possess a transposon inserted in a different site of the pathway. The particular inactivated enzyme for each mutant was assessed by a comparison between the accumulated products and the pathway. In some cases, the enzyme chosen could not be determined definately because of the uncertainty regarding the pathway. Of the clones derived from mutants of PS5-1, only pND200 appeared to have a sizable fragment of Pseudomonas DNA. The remaining flanking DNA was derived from Mu phage. In addition to pND200, only one clone, pND209 which was derived from the mutant PS 8-10, appeared to contain a large fragment of Pseudomonas DNA.

Although the cloning of Pseudomonas cholic acid catabolic genes into

E. coli would not provide for any positive selection procedure, E. coli was the host of choice as it is well characterized and could be easily transformed by exogenous DNA at a high frequency. The cloning vector initially used was pKT230, an 11.9kb plasmid containing unique restriction endonuclease sites within its two selectable markers, Km^ and Sm^ (Figure 23). This construct was derived by cloning the pACYC177 vector of Chang & Cohen (1978) into the Pst 1 site of RSFlOlO (Franklin et al, 1981). The replicon RSFlOlO is a small multi-copy plasmid (15-20 copies) with a broad host range and can be transformed mioE.coli md P. aeruginosa strains. The inclusion of pACYC 177 provided tiie plasmid with additional antibiotic markers and restriction endonuclease sites. In addition to transformation, pKT230 could be transferred at high frequencies by 163

Figure 23. Restriction Map of

Pstl Eco R1 Sst 1 BamHI

Hindm Xma 1

Xhol

(a) From Bagdasarian & Timmis (1981) 164

conjugative plasmids such as pRK2013 from E. coli into other Gram-negative bacteria (Bagdasarian & Timmis, 1981), thus making the transfer of selected clones or gene libraries from E, coli hosts into other strains easier. Lucas (1987) cloned the P-glucosidase gene of Pseudomonas PS2-2 and the endoglucanase gene of Xanthomonas XAl-1 into the //mdin site of pKT230. The cloned genes were expressed in both P. putida 17527 and E. coli ED8654. The promotor region was also present in both clones. Franklin et al, (1981) cloned the catechol-2,3-oxygenase gene from the TOL plasmid pWWO into both the Xhol and Sst 1 sites of pKT230, located in the Km^ and Sm^ genes respectively. It was found in both cases that expression of the cloned enzyme was governed by the constitutive promotor of the relevant antibiotic resistance gene. Measurement of the cloned enzyme activity indicated that the promotor of the Km^ gene was about 8 fold more active that that of the Sm^ gene.

This section reports the preparation and screening of gene banks of the two wild type strains, PS5-1 and PS8-1, for clones containing genes relevant to the steroid catabolic pathway. Two strategies were applied to generate and screen gene libraries of the wild type strains. The first method used a complementation screening procedure and was performed by 2 Honours students in this laboratory with research direction and technical contribution from the author. The second method using colony hybridization was performed solely by the author (see later).

3.5.1. Hybridization of DNA from PS5-1 and PS8-1 with the Clones pND200 and pND209.

To ensure that the flanking DNA of the clones used as probes for the gene bank was of Pseudomonas origin, it was first necessary to probe wild type DNA with the clones. It was also necessary to determine which enzymes could be 165

used to obtain a single fragment of wild type DNA, preferably within the size range of 10-20kb. This was achieved by digesting chromosomal DNA of wild type strains with a variety of restriction endonucleases and hybridizing the separated digested DNA with the probes of interest. Previous experiments showed that the flanking regions of pND200 and pND209 did not hybridize to each other (data not shown). Thus even if both clones contained DNA of Pseudomonas origin, the sequences differed. To maximize the probability of cloning the whole of the pathway both pND2(X) and pND209 were used as hybridization probes. As Pseudomonas DNA in pND2(X) originated from PS5-1 and that of pND209 from PS8-1 both wild-type strains were used for the construction of gene libraries.

The restriction endonucleases chosen were commonly used enzymes with a hexameric recognition site. Chromosomal DNA of PS5-1 and PS8-1 was digested with ////idlll, Pst 1, BamHl, EcoRl, Sal 1 andX/zol and separated by agarose gel electrophoresis, two gels contained digested PS5-1 DNA and two gels contained digested PS 8-1 DNA. A X-HinWl standard was also loaded onto the gels as were Hin^EcoKl double digest of pND200 (for PS5-1) and pND209 (for PS8-1) as controls (the gel for PS5-1 digested DNA is shown in Figure 24, the remaining gels are not shown). The DNA in all of the gels was transferred onto nitrocellulose filters. The 12.2kb EcdRlJHinWL digest fragment of pND2(X) and the 7.3 and 2.2kb fragments of EcoRHHindSl digested pND209 were electroeluted from an agarose gel and radioactivly labelled. One of the filters containing digested DNA of PS5-1 was hybridized to the fragment of labelled pND2(X) whereas the other filter was hybridized to the Augments of pND209. The same hybridization procedure was repeated for the gels containing digested PS 8-1 DNA. Radioactively labelled X DNA was added to the hybridization mixtures to serve as a weight marker. 166

The resulting autoradiograph of digested PS5-1 DNA hybridized with pND200 is shown in Figure 25. In all cases, the digested pND200 or pND209 hybridized to the appropriate probe ensuring the integrity of the experiment. For each restriction enzyme used, at least one fragment of digested DNA hybridized with the probe relative to the mutant of that wild-type strain. The approximate molecular weight of each hybridized fragment was calculated by comparison with the X weight markers. The fragments of PS5-1 DNA which hybridized to the pND2(X)fragment prob e and thefragments o f PS8-1 DNA which hybridized to the pND209fragments prob e are listed per enzyme used in Table 18. Digestion of PS5-1 with Pstl and PS8-1 with Pstl, Sail SindXhol resulted in more than 1 small hybridized fragment. Digestion of PS5-1 with HindHl mdXhol and PS8-1 with HindHI resulted in one fragment greater than 23kb. For both strains, the enzymes which resulted in afragment, whic h hybridized to the appropriate probe, within the preferred size range were EcoRl and BamHl, For PS8-1 DNA, an -digested fragment of approximately 7.8kb hybridized to the pND209 fragments and for PS5-1 DNA, the fragment which hybridized to the pND200 fragment was approximately lOkb. Insertion within the EcoRl site is reported to inactivate the Sm^ gene of pKT230 (Bagdasarian & Timmis, 1981) therefore £coRl was the enzyme of choice for cloning wild-type DNA into pKT230.

The EcoRl-digested fragment size of PS8-1 DNA (about 7.8kb) correlated well with the estimated size of the flanking DNA of pND209 believed to be of Pseudomonas origin. The EcoRl/Hindni fragments of pND209 used as probes in this experiment totalled 9.5kb. However approximately l.lkb of each of theflanking fragments wa s IS50 DNA (Figure 21), thus the Pseudomonas DNA within thesefragments woul d total about 7.3kb. A similar correlation could not be made with pND2(X) as the DNAflanking on e side of Tn5 in pND200 comprised solely of Mu DNA. 167

Figure 24. PS5-1 Genomic DNA digested with Various Restriction Endonucleases.

Agarose Gel Electrophoresis: From Right lane DNA Source and Restriction Enzyme Used.

1 pND200-EcoR1/H/ndlll 2 dill standards 3 PS5-1 DNA-X/70 1 4 " " -Sa/1 5 " " -EcoR1

6 " " -Bam H1 7 " " -Psf1 8 " " -H/ndlll 9 " " -H/ndlll 10

11 X-Hin6\\\ standards

Numbers on the right are X-Hin6\\\ weight markers and the pND200 fragment size in kb.

Gel Conditions: Horizontal gel, 0.8% agarose,

35 volts, 18 hours 11 10 9 8 7 168

Figure 25. Hybridization of Digested PS5-1 Genomic DNA with Flanking Regions of pND200.

Lanes as per Figure 24.

Numbers on tlie right are X-Hin dill weight markers and the pND200 fragment size in kb. 11 10 9 8 7 5 4 3 2 1

m 169

Table 18. Estimated Sizes of Restriction Fragments of

PS5-1 and PS8-1 which Hybridize to the

Clones pND200 and pND209 Respectively.

Strain Probe Restriction Endonuclease Used Hybridized Fragment Size inkb. (a)

PS5-1 pND200 HinWl 26 Pst\ 4.2,2.5, 1.6 Bamm 22 EcoRl 10 Sail 4.5 (plus possibly other larger fragments) Xhol >30

PS8-1 pND209 Hin(m >23 Pstl 5,3 BamUl 11 EcoRl 7.8 Sail 3.5,2.5, 2.0 Xhol 3.5,2.5

(a) Estimated sizes only due to width of A,-size standards of strong signals. 170

The hybridization pattern of the PS5-1 digested DNA was different when the pND209 fragments were used as the probe. The EcoKl digest had 4 fragments, approximately 4.6, 4.0, 1.9 and 1.6kb, which hybridized to the pND209 fragments. All of the enzymes used resulted in more than one DNA fragment of PS5-1 hybridizing to the pND209 fragments probe. PS8-1 did not hybridize to the pND200 fragment.

3.5.2. Derivation of Gene Libraries of PS5-1 and PS8-1 using the Vector pKT230.

Chromosomal DNA of PS5-1 and PS8-1 was digested to completion with EcoRl, The digested DNA was size-fractionated by sucrose gradient ultra- centrifugation. The collected fractions containing fragments in the 6-16kb size range were pooled and the DNA concentrated to 100}ig/ml by ethanol precipitation. pKT230 DNA was digested with EcdRl, dephosphorylated and concentrated. The fractionated Pseudomonas DNA was ligated, at various total DNA concentration ratios (8-30^g/ml), to 0.15-0.3|Xg dephosphorylated vector in 30^il reactions. Ligation mixtures were transformed into E. coli ED8654 with selection for Km^.

A control plate from the reaction containing only dephosphorylated vector plus ligase contained 35 transformants whereas most of the reactions with added fractionatedDN A generated between 50-450 transformants per plate. This suggested that most of the digested vector was dephosphorylated. Two of the ligation reactions fromeac h strain resulted in 3000-4500 transformants each. Each probable gene bank was pooled, by washing the colonies into LB + 25% glycerol, and stocked at -20°C. One hundred transformants from each pool were patched onto NA + Sm media to determine tiie degree of insertional inactivation. In each case, less than 5% of the transformants were Sm^. This suggested that either the 171

transforaiants did not contain a plasmid with an insert or the plasmids with inserts were unstable and quickly lost the insert. The whole experiment was repeated using ligation conditions considered from the previous attempts to be the optimum. One ligation reaction using PS5-1 DNA resulted in a total of 6000 transformants and one with PS8-1 DNA resulted in 4500 transformants. Tests for insertional inactivation with 100 colonies from each again showed that less than 5% were Sm^. Twenty transformants from each reaction were grown in LB + Km and screened for the presence of plasmid DNA. All bar 1 of the tested transformants contained a plasmid which co-migrated on an agarose gel with pKT230. Digests of the plasmids revealed all were similar to the vector (data not shown). The one exception from the PS8-1 gene bank, which was Sm^, appeared to contain 2 plasmids. When digested with EcoRl, there were 2 bands, one dense band which co-migrated with pKT230-£coRl and a much lighter band which was estimated to be lOkb in size.

3.5.3. Cloning of Pseudomonas DNA into the HinWi Site of pKT230.

Valentine (1986) generated gene libraries of each strain by ligating //mdm-digested chromosomal DNA to pKT230 which had been cut with HinWL and dephosphorylated. Ligation mixtures were transformed into E. coli ED8654 and the transformed cells selected by plating the transformation mixtures onto NA + Sm (20\ig/m\), For experiments in which approximately 10^ transformants per mixture were obtained, the cells were resuspended in LB + 25% glycerol and stored at -70®C. To determine the percentage of transformants with an inactivated Km^ gene, 100 transformants from each stock mixture were patched onto NA + Km(30^g/ml). The gene Hbraries with greater than 85% insertional inactivation were retained for further work. Plasmid DNA was isolated from 20 randomly 172

selected Sm^Km^ clones and digested with //mdlH to confirm the presence of insert DNA. All of the samples tested contained foreign DNA within the size range of 2.4 - 9.8kb. The screening method employed to isolate clones encoding steroid bioconversion genes was by complementation with the catabolic mutant strains PS5-7, PS5-16, PS5-18 and PS8-22. The gene banks were transferred into the mutants by conjugal transfer with the mobilizing plasmid pRK2013. Conjugation was by a 3-way filter mate. Complemented mutants were screened by replica plating the conjugation mixtures onto PAS salts + 12aOH-ADD. Eight separate screening experiments were performed with clones from gene banks of each strain. In all, approximately 15000 clones from PS 8-1 gene banks and approximately 25000 clones from PS5-1 gene banks were screened for the desired clones. Despite high transfer into the recipients and maintenance of the clones, no complemented transconjugants were isolated.

Welch (1987) also generated gene libraries of PS5-1 and PS8-1. The experimental procedure was similar to that of Valentine (1986) except that only ///ndHI-generated fragments greater than 9kb (isolated by sucrose gradient) were added to the ligation reactions. Insertional inactivation in excess of 70% was obtained in a number of gene libraries containing over 10^ transformants. Plasnoid isolation of Sm^Km^ clones showed that all had a plasmid larger than the vector and approximately 15% of these carried an insert greater than 12kb. Approximately 8000 transformants from each gene bank were mobilized using pRK2013 into PS5-18 and PS8-22. No complementing clones were isolated. Both Valentine (1986) and Welch (1987) demonstrated that pKT230 with foreign DNA inserted in the Hin6Bl site could be transferred and maintained in transposon-induced mutants of PS5-1 and PS8-L These results suggested that a more direct selection procedure in which a greater number of clones could be screened, such as colony hybridization, was required to detect the clones of interest 173

3.5.4. Screening of the Pseudomonas Gene Libraries for Clones Encoding the Steroid Catabolic Pathway using Colony Hybridization.

In a series of experiments, the author screened a total of approximately 20000 transformants from each wild type derived by Valentine and 50000 transformants of each wild type derived by Welch by colony hybridization. Welch (1987) noted that the percentage of recombinants in the pKT230-//mdin derived gene banks decreased with the time that cells were grown in liquid media. Thus for colony hybridization experiments, diluents of the glycerol-stocked clones were spread directly onto the solid medium. Transformants derived with PS5-1 DNA were screened with the electro-eluted 12.1kb EcoRHHinWl fragment of pND200 which was radioactively labelled. Transformants derived with PS8-1 DNA were hybridized with the 7.3kb EcoRllHindBl fragment of pND209. Isolation of these fragments was necessary as it had been previously observed that the Km^ gene of Tn5 located in pND200 and pND209 hybridized with the Km^ gene of pKT230 (data not shown). To further eliminate the possibility of false "light-ups" caused by the Km^ gene of pND200 and pND209, denatured and fragmented "cold" pKan2 DNA was added to hybridization reactions at a concentration of 0.05M,g/ml reaction buffer. The relevant wild-type and its mutant, the clone from which the probe was derived, HBlOl (pKan2) and SK1592 (pKT230) were also colony blotted onto 2 filters in each experiment as controls.

In all experiments, the first three controls clearly hybridized to the probe with a strong signal whereas pKan2 and pKT230 showed only background hybridization. On those filters on which a hybridization signal appeared, the corresponding colonies were isolated and plasmid DNA extracted. The plasmids were digested with //mdlll, separated on an agarose gel and retested by Southem 174

hybridization against the probe of interest. The initial signals were deemed to be artifacts as no hybridization signals were observed with the purified digested plasmids (data not shown).

The above experiments were repeated using transformants derived by

EcoRl digestion. From each cloning experiment, approximately 50000 colonies were screened in a similar manner as above. No colonies were isolated which hybridized to either probe.

3.5.5. Preliminary Cloning of PS8-1 and PS5-1 DNA into the

Cloning Vector pBR329.

It was necessary to test the reactivity of the dephosphorylated vector and to test the stability of recombinants with inserts in the EcoRX site. To achieve this,

EcoRl- digested pBR322 was ligated in the presence of 0.15|ig dephosphorylated pKT230. E. coli ED8654 was transformed with the ligation mixture and recombinants selected for Km^Ap^. Ten transformants together with controls

SK1592 (pKT230) and ED8654 (pBR322) were patched onto NA + Km + Ap +

Tc, NA + Sm + Ap and NA + Sm plates. Six of the transformants grew on all of the plates whereas 4 grew only on tiie NA + Km + Ap + Tc plates. The SK1592

(pKT230) control only grew on the NA + Sm media and the ED8654 (pBR322) control did not grow on any plates. From overnight cultures grown in NB and NB

+ Ap + Km, plasmids were extracted and digested with EcoRl. The DNA isolated from strains grown in just NB consisted of pKT230 only, whereas the DNA isolated from strains grown in the presence of the antibiotics, all contained pKT230 and an insert which co-migrated with digested pBR322 (data not shown).

This suggested that although foreign DNA could be inserted into the EcoRl site of pKT230, the recombinant could only be maintained under selective pressure. The 175

use of pKT230 as the cloning vector was discontinued.

Preliminary experiments have begun in the preparation of gene banks of PS5-1 and PS8-1 using the cloning vector pBR329, a 4.2kb derivative of pBR328 which carries resistance genes for Ap, Tc and Cm (Covarrubias & Bolivar, 1982). A unique EcoRl site is located in the Cm^ gene, which lacks its original promoter but is transcribed by a promoter located within the Tc^ gene. Ligation experiments using the size fractionated genomic DNA previously isolated (Section 3.5.2) and dephosphorylated £coRl-digested pBR329 were performed. Experimental conditions were similar to those previously described except ligation reactions were at various total DNA concentration ratios (5-15 |Xg/ml) with 0.05-0.15 |xg of vector in a 30^1 reaction and transformants were selected for Ap^.

Two ligation reactions from each strain resulted in approximately 1000 transformants each. Tests for insertional inactivation with 50 colonies from each reaction showed that 70% were Cm^. Six Ap^Cm^ transformants from each strain were grown in NB + Ap and screened for the presence of plasmids. All contained a plasmid greater in size than the vector (data not shown). Eleven of the plasmids were digested with EcoRl and separated on an agarose gel together with X-Hindm standards and Eci^Rl-digested pBR329. Six of the clones contained an insert of molecular weight between 9-17kb (Figure 26). Experiments are currendy underway to optimize the ligation reaction conditions in order to generate gene banks of each wild-type with approximately 10000 transformants. From there each gene bank will be screened for clones containing steroid catabolic genes by colony hybridization with pND200 and pND209. 176

Figure 26. Eco R1 Digests of Sample of Apf^Cm^ Transformants Containing PS5-1 and PS8-1 DNA Cloned into pBR329.

Agarose Electrophoresis: From Right

lane. Strain Origin of Insert DNA Size of Insert DNA

(Kb) 1 >.-H/V7dlll 2 51 PS5-1 3.3 3 52 II 4.5 4 53 II 9.5 5 54 II 6.8 6 55 n 6.6 7 56 n 18 8 81 PS8-1 9.5 9 82 n 6.5 10 83 n 9.8 11 84 H 20 12 85 II 10

13 pBR329 -

Gel Conditions: Horizontal gel, 0.8% agarose,

30 volts, 16 hours, 4°C 13121110 9 8 7 6 5 43 2 1

Linear Plasmid pBR329 177

4. DISCUSSION.

Most manufacturers of commercially available steroid drugs use processes which partially degrade sterol raw materials obtained from plants or animals. The manufacturing processes have incorporated microbial biotransformations to replace the more difficult chemical steps. The combination of chemical and bio-transformation processes has allowed manufacturers to expand both their product and raw material range. As an alternative process in the synthesis of the precursors, bisnorcholanic acid, 17-ketosteroid and hexahydroindan derivatives, some manufacturers have recently favoured fermentation processes using microbial mutants. The production of these compounds in one controlled fermentation offers a promising alternative to the multi-step chemical /biotransformation processes currently in use.

Bile acids are an inexpensive steroid raw material which are readily available from abattoirs. Researchers at the CSIRO Meat Research Division investigated the catabolism of bile acids by several Pseudomonas strains isolated from soil near an abattoir. A steroid degradation pathway was proposed by Leppik (1983). Some of the intermediates of this pathway were structurally similar to steroids used as precursors in current manufacturing processes. Use of these Pseudomonads in a fermentation process to produce these intermediates was deemed a potential commercial process.

The initial aim of this project was the derivation of stable mutants capable of accumulating steroid intermediates in high yields. The intermediates which were considered as potential valuable precursors were in the upper part of the catabolic pathway (Figure 9). A preliminary characterization of these strains was necessary prior to the derivation of mutants. 178

The 4 parent strains generally exhibited similar growth patterns on non-bile steroidal media (Table 4). In addition, the strains gave a similar growth response when tested on steroid media and accumulated similar metabolites from bile acid fermentations suggesting the catabolic pathways of each strain are similar, if not the same (RJ. Park, Personal communication). Positive growth on cholic, hyodeoxycholic and lithocholic acid suggested that the 6-, 7- and 12-hydroxy groups did not affect breakdown of the steroid structure, however specific groups about the ring structure did. The presence of a 3-oxygen function was necessary for growth as no strain grew on 5p-cholanic acid, however the configuration of the 3-hydroxyl group was irrelevant as growth was observed on both androsterone (3a-hydroxy) and SpOH-etiocholanolone. The presence of a 5-6 double bond or an 11-oxygen function also prevented growth (R.J. Park, Personal communication). The strains grew on both testosterone and androsterone suggesting that the oxidized state of C17 was irrelevant, but failed to grow on cholesterol, P-sitosterol and progesterone. The latter results suggest that the terminal carbon must be either a C22 or C24 for side chain removal, however whether a carboxylated terminal carbon was also necessary for degradation to commence or the oxidized C20 of progesterone prevented growth is unclear.

Resident plasmids were detected in only 2 of the 4 parent strains, PS5-1 and PS6-1. One plasmid of each strain was of a similar molecular weight (40kb), but restriction analysis with EcoRl (data not shown) showed that the plasmids of each strain had a different restriction profile. The phenotype of these plasmids was considered cryptic and there was no evidence to suggest that any of the resident plasmids play an essential role in bile acid utilization. The strains with no plasmids had the same steroid catabolic properties as those that had plasmids. Attempts to transfer the ability to utilize cholic acid by conjugation (with auxotrophic CA"*" donors) and transformation of the resident plasmids into the non-bile utilizing 179 pseudomonad PPl-8 were unsuccessful (data not shown). Curing experiments with mitomycin C and SDS only resulted in the isolation of one CA" mutant (PS5-10). This strain had lost the 40kb resident plasmid, however revertants to the CA"*" phenoptype were observed. This indicates that the plasmid is not essential for steroid utilization. The auxotrophic mutant PS5-6 Leu" had also lost the same large plasmid after a period whilst stocked in glycerol at -20°C, yet its ability to degrade CA or 12a hydroxy-ADD was unimpaired. In spite of these results, it could not be discounted that some complementary genes encoding steroid catabolic functions were located on both a plasmid and within the chromosome. The observation in Section 3.3.3 that Tn5 was inserted only in the chromosome of mutant derivatives of PS5-1 affected in the bile acid degradative pathway added further evidence that the resident plasmids may play no role in steroid breakdown.

In contrast, Tenneson et aL, (1979d) reported that plasmids in bacteria do encode for cholic acid breakdown, but the genetic evidence was weak. A number of wild-type strains capable of degrading bile acids were maintained on NA, then retested on DCA. The majority lost that degradative ability. The remainder were treated with mitomycin C and survivors replica-plated onto DCA media. The isolation of DCA" mutants by the two methods led them to state that this was evidence of a plasmid-mediated breakdown pathway. This implied role of plasmids would have been more convincing if the presence of plasmids within the wild types (and the subsequent loss of those plasmids in the DCA' mutants) had been demonstrated by plasmid extractions. Further, evidence that the re- iQtroduction of the plasmid/s into these mutants was concomittant with regaining of the CA"^ phenotype was required.

As it appeared that the genes necessary for bile acid breakdown were 180

located in the chromosome, work aimed at the manipulation of the resident plasmids was discontinued. A mutation programme was commenced primarily to produce strains capable of accumulating intermediates with high yields. It was possible that the mutants could also be used to study the pathway of the bile acid catabolism. Leppik (1983) proposed that the A-ring oxidation and side-chain degradation were independant, thus a mutant blocked at the 4-dehydrogenase gene would degrade the side-chain to at least the 17-ketosteroid resulting in an androstanedione product. In contrast, Tenneson et. al, (1979c) and Hayakawa (1982) proposed that the 4-dehydrogenation of the 3-keto steroid was a prerequisite to side-chain shortening, therefore the above mutant would accumulate only a 3-oxo cholanic acid product. From the product accumulated, the point where the degradation stopped could be determined thus indicating at which step the introduction of a 4,5-double bond was necessary for the next reaction to proceed. The possibility of accumulating the potentially valuable precursor, 9a,12-dihydroxy-4-androstene-3,17- dione with a block in the 1- dehydrogenase enzyme could not be excluded.

Auxotrophs of the bile acid-utilizing pseudomonads, PS5-1 and PS6-1 were treated with the chemical mutagen NTG and survivors screened by replica-plating onto CA media. Twelve CA" mutants failed to modify the DCA substrate in shake flask tests and were discarded. The remaining mutant, PS5-7, accumulated a mixture of phenolic and catecholic secosteroids and an unidentified compound, irrespective of the bile acid substrate used. Aerobic fermentation studies using PS5-7 in the presence of 2g/L DCA demonstrated that the catecholic secosteroid was formed subsequent to the phenolic secosteroid thus confirming the order of intermediate formation in the pathway. The isolation of a catecholic 9,10-secosteroid from the catabolism of bile acids or sterols has not previously been reported, rather the formation of these compounds was implied by the 181 isolation and identification of other intermediates (RJ. Park, Personal communication). The unknown compound tended to increase in concentration with fermentation times, with a concommitant reduction in concentration of the phenolic and catecholic secosteroids. This suggested that the unknown compound is an intermediate formed subsequent to the secosteroids. It was unclear whether the block in the pathway was immediately after the formation of the unknown compound, as seems most likely, or just after the catecholic secosteroid formation with the accumulation of the unknown compound a result of mutation instability or even a non enzymatic modification.

A total product yield of 72% of theoretical was obtained using PS5-7 in an aerobic fermentation 26 hours after the addition of 2 g/L CA. The cells had previously been grown with glycerol as the sole carbon source to mid-log phase (23 hrs). The yields of the phenolic and catecholic secosteroids were 20% and 39% of theoretical respectively. Production of the phenolic secosteroid from CA by PS5-7 had increased when compared to the parent strains. Leppik (1981) isolated a phenolic secosteroid from the aerobic fermentation broth of a PS6-1 culture using DCA as the sole carbon source. The yield was only 0.25% of theoretical. Park (1984) obtained higher yields from a variety of bile acids using PS 5-1 with limited aeration. The high yields obtained were attributed to the differing fermentor conditions rather than the different Pseudomonas strain. PS5-1 was grown for 17 hours with 2g/L CA as the sole carbon source and the yield of the relevant phenolic secosteroid was 11.7% of theoretical. The fermentation was terminated when a component had reached an absorption maximum near 280nm. At this point the concentration of the relevant androstadienedione was rapidly decreasing and the steroid substrate was absent. It was hypothesized that as a consequence of the restricted aeration, the 9a-hydroxylation of the androstadienedione intermediate and the 4-hydroxylation 182

of the phenolic secosteroid would be rate limiting.

The yields and fermentation times to reach the maximum appeared to be mainly dependent on the growth phase of the culture prior to addition of the steroid substrate. This was demonstrated by Park (1984) when PS5-1 was grown in glycerol for 50 hours to a stationary phase, then DCA (2g/L) added. The fermentation was terminated 18 hours later with a yield of phenolic secosteroid of 72% of theoretical. In contrast, PS5-1 was grown in glycerol for only 7 hours prior to the addition of 2g/L HDCA and CDCA. Fermentation times, for both substrates, in excess of 43 hours were required to reach the absorption maximum, even so the yields for both was 8% of theoretical. The results with PS5-1 suggested that relatively low yields were obtained if the steroid was added to growing cells, due to cellular growth requirements for carbon. Higher yields could be obtained, such as in the case with DCA, if the steroid was added to fully grown cells. Each of the above fermentations were conducted using different growth conditions and fermentation times,consequentl y comparisons of the yields obtained by PS5-7 and PS5-1 cannot be made. Unfortunately, no fermentations were conducted using PS5-7 with limited aeration. Even so the yield of 20% of phenolic secosteroid in an aerobic fermentation confirmed that PS5-7 was indeed a biochemically blocked mutant useful in the production of secosteroids.

Chemical mutagenesis was discontinued as replica-plating was not considered an effective screening method when looking for reduced growth patterns. Cross-feeding of mutants, affected in the bile-utilizing pathway, by surrounding unaffected survivors could not be controlled.

An altemative procedure which allows potential mutants to be isolated from non-affected cells is transposon mutagenesis. Successful transposition with 183

the suicide vectors used in this study relied on the ability of the vector to transfer into, but not be maintained in the recipients. The screening method used relied on isolating strains with reduced growth on any one of the three steroid substrates used. The unavailability of other steroid compounds limited the choice of selection substrates. The inclusion, of partly oxidized steroids as selection agents, would have clarified the screening of mutants with blocks in the upper part of the pathway. Compounds such as 3-oxo-7a,12a-dihydroxy-l,4-choladiene-24-oic and 3a,7a,12a-trihydroxy-androstan-17-one used as screening substrates would have simplified the selection of mutants blocked in the side-chain degradation or the oxidation of the A-ring. Even so, careful comparison of the growth patterns of each mutant relative to the parent on the screening media was deemed a sufficient method to isolate such mutants. It was reasonable to presume that a mutation in a regulatory gene, or a polar mutation, would cause a mutant to exhibit little or no growth on any of steroid media used. The lack of exotic steroids for use as test substrates also hindered determination of the location of the point of mutations. It was necessary to further test each presumptive mutant by the shake flask procedure for identification of any accumulated products after incubation in the presence of

DCA. It was possible that comparison of the accumulated product/s and the proposed pathway could indicate the point of mutation.

Using the vector pJB4JI, Km^ tranconjugants were obtained with all the recipients used except PS6-6, PS7-1 and PS7-2, with very low conjugation fi-equencies with PS6-1. Likewise, Km^ transconjugants were isolated with all the recipients except PS5-1 or PS5-6 using the vector pSUPlOll. The results suggest that the failure to detect Km^ transconjugants in these cases was not due to the failure of Tn5 to transpose in the recipients nor to the inability of the recipient strains to express the antibiotic resistance of Tn5. Rather it would appear that the vector could not transfer mto these recipients as in each case, use of the alternative 184

Tn5-loaded vector produced Km^ transconjugants.

Almost 10,000 Km^ transconjugants were plate-tested on steroid substrates. In all, 95 presumptive mutants of CA"^ strains blocked in the steroid catabolic pathway were selected (1.0% of transconjugants tested) on the basis of impaired growth on one or more of the steroid substrates. Forty one (0.4%) had altered growth properties in the presence of DCA compared to their parents (Table 9). This compares well with reports of Tn5 used to isolate auxotrophs. Srivastava et al, (1982) obtained mutants of Alcaligenes eutrophus at a frequency of 0.8% using E. coli 1830 (pJB4JI) and Turner et al, (1984), using E, coli SMIO (pSUPlOl 1) obtained auxotrophs of Xanthomonas campestris at 0.2%.

pJB4JI was also introduced into the NTG-derived CA- mutant PS5-7. Fourteen of the 21 presumptive mutants isolated by plate tests which appeared to be affected in steroid utilization accumulated only the phenolic secosteroid. Five of the above 14 Km^ transconjugants were also Gm^. Two of the mutants, 105-16(1), which was Gm^, and 105-16(10), Gm^, were further studied. In addition to the resident plasmids found in the wild-type, 105-16(1) harboured a plasmid which was the same molecular weight as pJB4JI. Hybridization studies with total undigested DNA demonstrated that Tn5 was located in the new plasmid of 105-16(1) and the chromosome of both 105-16(1) and 105-16(10) (Figure 16A). The results with 105-16(1) suggested that pJB4JI was stably maintained in this PS5-7 transconjugant and that a copy of Tn5 had transposed into a site within the host genome. However the results with 105-16(10), and the proportion of Gm^ strains, demonstrated that the vector in most cases was lost and that transposition had occurred.

In other Pseudomonas recipients, the vector pJB4JI often appeared to 185

be stably maintained with no loss of transfer functions. Almost all of the presumptive Km^^ mutants of PS8-1 and PS8-2 inherited the Gm^ marker of pJB4JL Even so the presence of the transposon/vector affected the recipient's steroid catabolic capabilities. Most of the mutants of 119-9 Arg" (PS8-2) could not modify the DCA substrate, even though the parent PS8-2 degraded DCA. In addition, one mutant of PS8-1 accumulated 3KEA products (119(10), Table 9). In a separate experiment, 44 Km^ transconjugants of the cross, E. coli 1830 (pJB4JI) X PS8-2 were tested for gentamicin resistance. All were Gm^ and could donate pJB4n to PPl-8 at high frequencies (data not shown). One Km^Gm^ mutant (119-9(5)) was further studied. No plasmid was observed (Figure 15B) and hybridization of total DNA revealed that Tn5 was located in the chromosome (Figure 16B). As this mutant could co-transfer both markers to PPl-8, it appeared that most, if not all, of pJB4JI was integrated in the chromosome.

Six of the 65 presumptive mutants of PS5-1 and PS5-6 were also Gm^ indicating these transconjugants had also retained some part or all of pJB4JI. None of these mutants could transfer Km^ or Gm^ to PPl-8. Two of the Gm^ strains, 105(222) and 105(232), accumulated products in shake flask experiments with DCA as carbon substrate, however product disappearance began after 1 or 2 days in a similar fashion to many other mutants which were Gm^ (Table 9). Various reports have suggested that the suicidal behaviour of pJB4JI is strain-dependent. Meade et al., (1982) found 1% of'Kvc^ Rhizobium meliloti transconjugants were also Gm^ when using this plasmid. Srivastava et al, (1982) reported that 30% of pJB4JI-derived Km^ transconjugants of A. eutrophus were Gm^, however the Gm^ was lost on non-selective media and none of the transconjugants, be they Gm^ or Gm^, could transfer Km^ back to E. coli. Bellard & Trust (1985) found that 50% of pJB4JI-derived Km^ transconjugants of Aeromonas salmonicida also inherited Gm^, all of which harboured an additional 186 plasmid which co-migrated with pJB4n. Co-inheritence of the Gm^ marker was reported with 100% of pJB4JI-derived Km^ transconjugants of X. campestris (Turner era/., 1984) and Azospirillum brasilense (Singh & Klingmuller, 1986). The Km^Gm^ transconjugants in both reports could transfer pJB4JI back to E. coli at high frequencies. No reports on the behaviour of pJB4n in pseudomonads were found, however some strains of Pseudomonas solanacearum could stably maintain RP4::Mu (Boucher et al, 1977), a plasmid genetically close to pJB4n.

Even so the isolation of a number of strains which accumulated steroid intermediates established that Tn5 could be used as a mutagen for the bile-utilizing pseudomonads. The plate comparison method of detecting potential mutants proved to be a more effective procedure that replica plating. The non-uniformity of the plate growth responses amongst mutants which were subsequently found to accumulate similar intermediates may be due to varying degrees of stability of each transposon-induced mutant. By the same token the transposon may have inserted into different sites within an operon resulting in slightly different phenotypic expression on various steroid substrates. The number of mutants detected with various stabilities and plate growth responses together with the range of intermediates accumulated suggests that Tn5 insertion was fairly random.

Of the 73 pJB4JI-derived presumptive mutants, selected for unusual growth responses on the test media, 22 accumulated products. Mutants were isolated which accumulated products from the divergent upper part of the pathway (3KA and 3KEA) or a non-acidic intermediate (12aOH-ADD, 12pOH-ADD or PS). Further studies of 18 selected mutants showed that although some had lost the 40kb resident plasmid, none harboured any new plasmid species (Figures 15 A and 15B). This was unexpected as 4 of the mutants studied were Gm^, yet unlike 105-16(1) did not contain a plasmid of the same size as pJB4JI. It is possible that 187

the plasmid was either unstable in liquid cultures and was lost in the absence of gentamicin, is not easily extracted from the mutant strains, or that at least a part of pJB4JI had integrated into the chromosome. Tn5 was located only in the chromosome of each mutant studied as shown by hybridization of the total DNA of the mutants with pKan2 (Figures 16A and 16B).

Five out of 22 pSUP 1011-derived presumptive mutants accumulated products, those products being either SKA, 3KEA or OPDC. Why no mutants were isolated which accumulated non-acidic products is unclear, but is likely because fewer pSUPlOil-derived transconjugants than pJB4JI-derived transconjugants were tested for unusual growth responses. pSUP 1011-derived Km^ transconjugants were generated with PS6-1, PS7-1 and PS8-1, but not with the P. putida strain PS5-1 or its derivatives. Morgan & Chatterjee (1985) obtained pSUPlOl 1-derived auxotrophs of Pseudomonas syringae and Zumft et al, (1985) used pSUPlOll to isolate Nos" mutants of Pseudomonas stutzeri. Both reports concluded that the mutations were by simple random insertion of Tn5. Use of the E. coli SMIO (pSUPlOl 1) system also failed with P. aeruginosa unless a complementing RP4 Mob region was cloned into the recipient prior to mobilization of pSUPlOl 1 (Schilf & Krishnapillai, 1985). It was postulated that a )d\\{Kil )/kill owcrndtikor) compensatory system of RP4 was located in the Mob region of pSUPlOl 1 and that Tn5 was inserted in the kor gene, thus resulting in host-cell killing for specific recipients. Whether the compensatory system or other reasons were responsible for the failure to isolate PS5-1 Km^ transconjugants was not investigated.

The bile acid-utilizing recipients were tested to determine if the non transposable marker in pSUPlOll (Cm^) was present, however the chloramphenicol sensitivity tests were ambiguous. No plasmids were observed in 188

total DNA extractions with a sample of 5 presumptive mutants suggesting loss or integration of the vector had occurred. Hybridization studies with these strains revealed that Tn5 was inserted within the chromosome of each. Turner et al,

(1984) obtained pSUPlOll-derived Km^ transconjugants of Xanthomonas

campestris, all of the transconjugants tested were Cm^. Simon et al, (1985) also

reported the complete loss of the vector with pSUPl Oil-derived Km^

transconjugants of Rizobia meliloti. In contrast, Warr (1985) found that

pSUPlOll had integrated into the chromosome ofZymomonas mobilis 7M6

recipients and also had co-integrated with a ZM6 resident plasmid pNSWl. In

ZM6 (pNSW2) Km^ transconjugants, the vector was stably maintained. It was

concluded that one of the IS50 elements located on pSUPlOl 1 was responsible for

co-integration. If this is the case, it would most probably be IS50R which has a

far higher transposase activity than IS50L which contains an ochre mutant allele of

transposase (Hirschel & Berg, 1982).

The vector pAS8 Tc^ rep -l::Tn7 (pAS8 for brevity) was used as a suicide vector for two transposons, Tnl (Ap^) and Tn7(Sm^). No Sm^ transconjugants were obtained with either PS5-1 or PPl-8 Met" as a recipient.

Sato et al., (1981) reported Tn7 transposed into plasmids of phytopathogenic

Pseudomonads at high frequencies with a subsequent loss of pAS8. Tnl-derived

Ap^ transconjugants were also isolated using the same vector. Krishnapillai

(1979) obtained Tra" Cb^ Sm^ mutants of the R91-5 plasmid of P. aeruginosa using an RP4::Tn7 vector. The reason why Tn7 did not transpose into PS5-1 or

PPl-8 is unclear although it does have unusual site specificities. Tn7 will insert into many plasmid sites of RP4 (Barth et al, 1978) as well as the Pseudomonas plasmid mentioned above, but has a single "hot spot" in E. coli (Lichtenstein &

Brenner, 1981) and other bacteria (Caruso & Shapiro, 1982; Ely, 1982). To be sure, the reason was not the inability of pAS8 to transfer into PS5-1 as Cb^ 189

transconjugants were generated. The vector appeared to suicide within PS5-1 as all of the presumptive mutants were Km^. Further, no plasmids were observed in PS8-22.

No new product classes were accumulated by Tnl-derived mutants, however only a small number of transconjugants were growth-tested on the 3 steroid substrates. Most of the presumptive mutants were selected on the basis of slow growth on one or more of the substrates, however subsequent tests in shake flasks revealed that the majority degraded DCA almost as quickly as the parent. The 9 mutants which did accumulate products (0.8% of transconjugants tested) generally had growth responses in DCA media similar to Tn5-induced mutants which accumulated the same compounds. There appeared to be no site specificity by the transposon as the mutants isolated accumulated a wide range of intermediates (Table 11).

The vector used to introduce TnlO into PS5-1 and PS8-1, (pBEElO), also appeared to suicide in Tc^ transconjugants. The presence of TnlO in PS8-1 resulted in two major differences to previously obtained transposon-induced mutants. Of the 10 mutants which accumulated products, 5 accumulated OPDC. Previously only one transposon-induced mutant (PS 8-10) had been isolated which accumulated this compound. The reason for this observed preference is unclear although Kleckner (1981) reported that TnlO does favour certain "hot spots". TnlO insertion specificity appears to be determined by a symmetrical 6bp consensus sequence located within the 9bp target sequence duplicated during transposition (Hailing & Kleckner, 1982). The probability that this consensus sequence is present at least once in the genes encoding for a large pathway such as the bile acid catabolic pathway cannot be discounted. The second outstanding feature regarding TnlO transposition was the extreme sensitivity, by over half of 190

the mutants, to sodium deoxycholate in liquid cultures. The cause was not investigated as this difficulty was surmounted by the slow feeding of sodium deoxycholate together with the addition of free acid (RJ. Park, Personal communication).

In all, a total of 60 single transposon-induced mutants which accumulated intermediates were isolated (Table 12). Most of the mutants accumulated a product for between 1-3 days after the addition of DC A substrate before rapid utilization of the product occurred. Eventually, both the substrate and the product was consumed. Even so, for most product classes, at least one stable mutant was isolated. It would seem unlikely that the product utilization was due to the switching on of a "shunt pathway" to avoid the mutation as: (a) the phenomenon was observed for all product classes and (b) there were large variations in the stability of mutants which accumulated the same compound. A more likely reason was reversion resulting in a partial or complete return of the parental phenotype.

The reversion frequencies of ten pJB4JI-derived mutants of PS5-1 and five pSUPlOll-derived mutants of PS8-1 and PS7-4 were examined (data not shown). For 6 mutants, the frequency could not be calculated due to either profuse growth on both the CA and 12aOH-ADD media or no revertants were observed. Such growth was presumably due to very high reversion frequency rates. The frequency of revertants that were isolated ranged from 10""^ to 10"^ per cell plated with the majority about 10"^. The reported reversion frequencies of other pJB4JI-derived mutants were similar. Beringer et al, (1978) found auxotrophic mutants of Rhizobium meliloti reverted at frequencies between 10"^ and 10"^ while Srivastava et al, (1982) fo\xnd Alcaligenes eutrophus auxotrophs reverted to prototrophy at frequencies between 10"^ to 10"^. There was no clear 191 correlation between the product accumulation time exhibited by each mutant and its reversion frequency.

The reversion to CA+ 12aOH-ADD+ was not always associated with loss of kanamycin resistance. Generally, 50-80% of revertants were Km^, thus reversion did not arise through a precise excision of Mu/Tn5 unless they excised precisely and relocated. The retention of the Km^ marker in revertants appears to be strain dependent. Beringer et al., (1978) reported the Km^ retention rate varied from 10% to 90% of revertants depending on the mutant used, all of the mutants were derived from the same parent. In addition, all of the revertants of A. eutrophus were Km^ (Srivastava et al, 1982). Possibly the reversion is a result of the re-insertion of the transposon to a new site. Analysis of the clones pND204 and pND200 suggested that Mu, and not Tn5, was the transposing agent in the mutants 105-17(2) and PS5-16. The Tn5 was inserted in the tail region of Mu and may simply have been carried by Mu into the Pseudomonas recipient's genome. Mu DNA was found in most of the pJB4JI-derived mutants that were studied, therefore for the generation of revertants, re-location of Mu would most likely be required. Spontaneous reversion of Mu-induced mutations are extremely rare in E. coli (Howe & Bade, 1975). The reversion frequencies obtained in this study would suggest that the re-location of Mu from the Pseudomonas chromosome is more frequent than from the chromosome of E. coli. Further hybridization studies on some of the revertants may have shown whether Mu had relocated to a new site, and if so whether Tn5 relocated with Mu. It was also possible that the Mu/Tn5 had excised and had dissociated, but prior to dissociation the Tn5 had reinserted into the genome at some other site. However the high rates of reversion of some mutants would make this event unlikely.

The mutants accumulated 6 classes of products (Table 12) which 192 allowed a study of the mechanism of the bile acid degradation pathway. From the divergent part of the pathway, mutants accumulated the 12aOH-derivatives of either 3KA, 3KEA or OPDC products from the degradation of DC A. No mutants were isolated that accumulated a fully oxidized A-ring with an unmodified side-chain. Further no 3a-hydroxylated compounds with a partly degraded side-chain were accumulated by a mutant. In addition the only products with a 17-keto function all contained a fully oxidized A-ring, eg: 12aOH-ADD. The accumulation of 3KA products (BE and BG in Figure 9) and the fact that no mutants were isolated that accumulated 3a-hydroxy-23,24-bisnorcholanic acids (AG) suggest (a) the degradation of the side-chain to a 23,24-bisnor acid can proceed only after the oxidation of the 3a-hydroxy group and (b) further degradation of the C22 side-chain cannot proceed until after further oxidation of the A-ring. The accumulation of 3KEA products (BG, CE and £G in Figure 9) and the fact that no 4-ene-3,17-dione compounds were isolated suggest that the degradation of the C22 side-chain could not proceed even after 4-dehydrogenation of the A-ring. The degradation of the C22 side-chain was only observed in mutants which could also oxidize the A-ring to a 3-oxo-l,4-diene structure. The above proposals conflict with the hypotheses of Leppik (1983), Tenneson etal, (1979c) and Hayakawa (1973; 1982). Leppik proposed that A-ring oxidation and side-chain degradation were separate and independent. Production of the 3KA and 3KEA products by the above mutants suggests that side-chain degradation to the bisnor acid and androsterone appears to be dependent on the oxidized state of the A-ring.

Hayakawa (1973) proposed that a 3-oxo-4-ene A-ring is a prerequisite to side-chain degradation. Cholesterol and other sterols with a 5-6 double bond are isomerized to a 4-5 double bond prior to side-chain degration (Sih & Whitlock, 1968). The isomerization reaction may be necessary to remove steric hinderence 193 caused by the 5-6 double bond rather than the requirement for the 4-5 double bond. Hayakawa (1982) contends that the A-ring saturated bisnorcholanic acid products reported by Leppik (1980; 1982) in low yields are reduction products of once-formed unsaturated bisnorcholenic acids. One of the saturated bisnorcholanic acids, (BG), was accumulated by many of the mutants which accumulated 3KA products in fair yields (R. J. Park, personal communication). The accumulation of this compound by many mutants is evidence that side-chain degradation to the 23,24-bisnorcholanic acid can proceed with only the 3-keto group being present, ie: 4-dehydrogenation is not a prerequisite. The isolation of 4-androstene derivatives as degradation products of bile acids by Barnes et al, (1976) and Bilton et al, (1981) led them to conclude that further removal of the side-chain can occur with 3-oxo-4-ene-steroids. It is possible that Pseudomonas NCIB10590 degrades bile acids by a slightly different pathway. No such compounds were isolated in this project with any mutant suggesting that for these strains, 1-dehydrogenation was necessary prior to complete removal of the side-chain.

Analysis of the products accumulated by the mutants suggests that the A-ring oxidation and side-chain removal are separate. Thus one pathway catalyses the A-ring oxidation (in order), 3-hydroxysteroid dehydrogenase, 4-dehydrogenase and 1-dehydrogenase and the other pathway catalyses the side-chain degradation from C24 to the C22 intermediate and then to the 17-keto steroid. It is proposed by the author that these two pathways are encoded on separate operons (or regulons) as, remembering that transposons cause polar mutations, it is the only model that can adequately explain the mixtures of products accumulated. By combining this model with the side-chain degradation enzymes' dependency on the A-ring structure, then those mutants which accumulate 3KA products are blocked in the 4-dehydrogenase gene, mutants which accumulate 3KEA products are blocked in the 1-dehydrogenase gene and mutants which 194

accumulate OPDC products are blocked in a gene involved in removal of the C22 side-chain. Mutants which were blocked in the 3-hydroxysteroid dehydrogenase gene would not modify a cholanic acid substrate. Mutants blocked in a gene involved in removal of the C24 side-chain would presumably accumulate a

3-oxo-l,4-choladienic acid, a compound which had previously been isolated by

Leppik (1980; 1983) as a degradation product of DCA by PS6-1. No such

mutants were isolated.

The isolation of mutants which accumulate 12aOH-ADD, 12pOH-ADD

and the phenolic secosteroid confirm the convergent part of the postulated

pathway. The above mutants would appear to be blocked in the 12a-hydroxy

isomerase gene, one of the genes effecting the 9a-hydroxylation and the

4-hydroxylation gene respectively. PS5-16 accumulates 12aOH-ADD from the

degradation of DCA and 7a,12a-dihydroxy-ADD from CA. With substrates

which have no 12 hydroxy group such as chenodeoxycholic and lithocholic acid,

no products were accumulated. It would appear that the Tn5-induced block of the

12a-hydroxy isomerase gene does not affect other genes involved in the catabolic

pathway. In order to study the production of the above compounds by mutants,

the strains PS5-16, PS5-18 and PS5-25 were grown in fermentors in the presence

of various bile acids at different concentrations. All three Tn5-induced strains

accumulated the relevant product in yields greater than 80% of theoretical, even

with high substrate concentrations (Table 15).

An attempt was made to stabilise the Tn5-induced mutant PS8-10 by a

second mutation using Tnl. It was also possible that the introduction of Tnl into

PS8-10, PS5-16 and PS5-18 could result in the accumulation of novel

compounds. 195

The conjugation frequencies (Table 13) were one order of magnitude lower than the frequencies obtained with the wild-type PS5-1 as the recipient

(Table 10) despite a longer incubation time. Whether this was due to donor-recipient interactions or, as is most likely, the presence of the Tn5 in the recipients is unclear. The insertion of Tnl affected the mutants in a different manner. PS8-10 and PS5-18 accumulated the same compound, however the growth rate and product retention times were altered (Table 14). The mutant

PS8-22 (119(107)) was particularly stable compared to its parent. Fermentation studies demonstrated that the Tnl-induced stable derivative accumulated high yields of 12a hydroxy-OPDC from DC A with longer fermentation times and higher substrate concentrations than PS8-10 (Table 15). The reasons for this phenomenon are unclear and were not investigated, however the use of transposon "double mutations" was demonstrated as a useful tool and could provide a means of reducing loss of accumulated products in mutants which currently are of little value in fermentation studies. The Tnl-induced mutants of

PS5-16 in general were not affected as above, instead the Tnl appeared to cause an additional compound to accumulate. At present too little is known of the degradative pathway, its control and the ordering of the relevant genes to surmise the basis of such an altered phenotype.

The mechanism of transposition within mutants derived with pJB4JI was studied. It appears that in most mutants the transposition event was Mu- rather than Tn5-mediated. The derivation of the clones pND204 and pND200 from the mutants 105-17(2) and PS5-16 confirmed this view. These clones were isolated by cloning DNA fragments, which encoded the transposon and flanking

DNA, from the mutants into pBR322 with selection for Km^. The clones were mapped by restriction analysis and Southern hybridization with both pKan2 and

Mu DNA. Both clones contained in addition to Tn5 the tail end fragment of Mu up 196 to an upstream EcoRl site and flanking DNA presumed to be Pseudomonas DNA to a downstream EcoKl site. This presumption appeared to be valid as digested PS5-1 DNA hybridized to pND200 (Figure 25), but not to pJB4JL These results agree with other reports which used vectors which carry one transposon inserted into another. About 25% of R. meliloti mutants derived with pJB4JI had Mu sequences contiguous with Tn5 (Meade et al, 1982). Forrai et al., (1983) also found pJB4JI-derived mutants of R. meliloti contained both Mu and Tn5. An analagous result has been reported with a RK2-ColEl::Tn7::Tn5 hybrid plasmid with Tn5 inserted in a non-essential region of Tn7. It is reported that a high number of Km^ transconjugants were due to Tn7 rather than Tn5 transposition (cited in Berg & Berg, 1983).

Attempts to clone similar Km^ fragments from 105(3), 105-17(10) and PS5-18 were unsuccessful as all of the clones isolated only consisted of all or almost aU of pBR322 plus an insert which comprised the internal fragment of Tn5 (encoding Km^) and the inner fragments of the two IS50 elements. Each clone had the same antibiotic resisitance properties, molecular weight and restriction pattern suggesting that the reason that such clones were produced was the same for each clone. The results are difficult to explain. Mu was not involved as 105(3) does not contain Mu. By the same token, it is difficult to conceive that Tn5 caused deletions that removed the outer regions of the IS50 elements. Neither Isberg & Syvanen (1981) nor Berg & Berg (1983) reported such a phenemenon with pBR322::Tn5 plasmids. The retention of the unique EcoRX site of pBR322, the loss of Tc^ and the fragmented Tn5 would suggest that it is more likely that these clones were due to a reduced specificity of EcoRl to "star activity" caused by high enzyme concentrations. Computer searches of the sequences of pBR322 and IS50 with known EcoKl"^ recognition sites (listed in Boehringer Mannheim Biochemicals catalogue, 1985) failed to find any such sites within lOOObp of the 197

Hindm site of IS50, although one EcoRl'' recognition site was near the region of pBR322 where the insert was located.

Of the 20 pJB4JI-derived mutants investigated, 15 appeared to contain a single Tn5, the remainded appeared to contain 2 copies of the transposon. Of particular significance was that PS5-16 appeared to contain 2 copies of the transposon (see later). All of the mutants, bar 105(5), had at least one EcoRl- digested fragment of 20kb or higher that contained Tn5. This was expected as previous studies with pND200 and pND204 suggested that the mutants PS5-16 and 105-17(2) would have similar fragments in excess of 19kb with most of the fragment containing DNA of Mu or Tn5 origin. Subsequent hybridization with Mu DNA determined that 14/19 mutants contained Mu DNA (Figure 20). Generally each mutant contained at least one copy of the whole of Mu. To determine if Mu and Tn5 had co-transposed, as was the case with 105-17(2) and PS5-16, it was necessary to determine whether the EcoRl-digested fragment of each mutant that hybridized to Tn5 was the same size as the fragment that hybridized to Mu. Accurate estimations of the size of the large £c^?Rl-digested fragments which hybridized to Mu were not possible due to systematic errors. However for each mutant, the relative size of the largest fragment that hybridized to Mu paralleled the relative sizes of the largest fragment that hybridized to pKan2. Although this evidence is not conclusive, it does suggest that Tn5 and Mu sequences are on one DNA fragment in common, ie: are contiguous. If this is the case then the mutation in the 14 mutants which contained Mu DNA was Mu-mediated and Tn5 was "carried" by Mu into the recipients. The 5 mutants which did not contain Mu, 105(3), 105(5), 105(7), 105(205) and 105(213) all had one £c(9R1-digested fragment, either 17kb or 20kb, which hybridized to pKan2. The use of pJB4JI with PS5-1 or derivatives therefore resulted in only approximately 26% of mutants being genuine Tn5-induced mutants. 198

Mutants derived with pSUPlOl 1 were also studied. The transposon and the flanking regions about Tn5 were cloned from PS8-10. This clone (pND209) had flanking regions of 6.2 and l.lkb in length (Figure 21) which are believed to be of Pseudomonas origin. The evidence is that the flanking regions (a) hybridized to PS8-1 DNA and (b) did not hybridize to the replicon of pSUPlOl 1 (pACYC184) nor the Mob site (pSUP201) (Figure 22B). Southern hybridization analysis of five pSUPlOl 1-derived mutants indicated that only one copy of Tn5 was inserted in the chromosome of each mutant. The fragments that hybridized to pKan2 were in the range of 13-18kb. Interestingly, the Ec^jRl-digestion fragment of PS8-22 which hybridized to pKan2 was the same molecular weight as that of PS8-10. As no plasmids were observed in PS8-22, it must be assumed that any integrated DNA is located in the chromosome. It appears that if Tnl has integrated into the chromosome, then it has done so at a reasonable distance from the Tn5 integration site and yet still affects the stability of the Tn5-induced mutation. The reasons are unclear and were not investigated.

The final section of this project was the cloning of the genes and operons of the steroid catabolic pathway. The screening of gene banks for clones encoding the above genes was by colony hybridization with the flanking regions of pND2(X) and pND209 as probes. pND200 was derived from PS5-16 which was blocked in the 12a hydroxy isomerase gene. pND209 was derived from PS8-10 which was presumed blocked in the gene/s encoding removal of the bisnor side-chain. As both clones contained Pseudomonas DNA, it was likely that these genes and others from the relevant operons were located on the above clones. The DNA of two wild type strains, PS5-1 and PS8-1, was used in the preparation of gene banks. This was considered necessary as although pND209 hybridized to both PS5-1 and PS8-1 DNA, pND200 only hybridized to PS5-1 DNA. One EcoRl digest fragment of PS8-1 DNA hybridized to pND209, however pND209 199 hybridized to 4 EcoRl digest fragments of PS5-1 (Table 18). The Pseudomonas DNA inserted in pND200 did not hybridize to pND209, Thus the Pseudomonas DNA derived from the two mutants was not homologous and probably encode for completely different genes.

Attempts by Valentine (1986) and Welch (1987) to screen gene banks by phenotypic complementation of a range of mutants were unsuccessful. The gene banks were prepared by ligation of digested genomic DNA into the Hin&lll site of pKT230. Genes cloned into the site are transcribed by the Km^ promoter (Franklin et al„ 1981) and as pKT230 has a broad-host range and can be mobilized into many Gram-negative bacteria, it was considered a useful vector for complementation screening. The probability of detecting a clone encoding steroid catabolic genes was low as hybridization of Hindlll digested PS5-1 and PS8-1 DNA with pND200 and pND209 respectively revealed that the fragments to be cloned using HindlLl were above 23kb in size (Table 18). As mobilization of the gene banks into CA' or 12aOH-ADD" mutants was restricted to using about 200 colonies/plate, the number screened by this method was generally limited to less than 20000 clones per experiment. Colony hybridization allowed a greater number to be screened, however no clones were detected that hybridized to either pND2(X) or pND209.

Use of EcoRl reduced the size of the fragments to be cloned to about lOkb. The insertion of foreign DNA into the EcoRl site of pKT230 was reported by Bagdasarian & Tinmiis (1981) to inactivate the Sm^ gene which is under the control of a p-lactamase promo tor upstream of the structural Sm^ gene (Bagdasarian et al, 1981). In eveiy ligation experiment performed, the number of Km^Sm^ transformants obtained was about 5% of total transformants, however agarose gel electrophoresis of ligation samples indicated that ligation into pKT230 200 was occurring. No clones that hybridized to either probe was detected. To obtain positive selection for an insert, pBR322 was cloned into pKT230 at the EcoRl site. It was demonstrated by growth of these clones on NA + Sm + Ap that the Sm^ gene was usually not inactivated. In addition, the clones were highly unstable in the E. coli host when grown in liquid media without the Ampicillin selection pressure. Instability of clones derived with pKT230 have been reported elsewhere. Sykes et al, (1987) cloned the structural genes for the subunits of a keto acid dehydrogenase multienzyme complex into pKT230. The gene bank was derived by ligating EcoKHSst 1 double digested genomic DNA from Pseudomonas putida with similarily digested pKT230. Recombinant plasmids, which were Km^Sm^ were transformed into P. putida mutants to achieve direct selection for the required clone by phenotypic complementation. The required clone carried a 7.8kb insert. The presence of insert DNA in pKT230 drammatically affected the stability of the vector in the host P. putida with only 3% of colonies retaining Km^ after 3 serial transfers of overnight growth. The vector without insert DNA was extremely stable in the P. putida host strain even without selection pressure.

Preliminary experiments with pBR329 as the cloning vector are more promising. EcoRl generated gene banks have been prepared with up to 70% of transformants indicating insertional inactivation. A random sample of Ap^Cm^ transformants all contained plasmid DNA with sizable inserts. Future work in this project will involve the preparation of a gene bank from each wild-type with a sufficient number of recombinant clones to constitute a full gene library. The screening procedure used will be by colony hybridization.

Two £c<9Rl-digested fragments of PS5-16 DNA hybridized to pKan2 (Figure ISA). It is therefore possible that the Pseudomonas DNA cloned into 201

pND200 does not contain genes relevant to the steroid catabolic pathway. In addition, neither of the probes being used in the colony hybridization experiments may contain genes involved in A-ring oxidation. It was previously proposed that only the mutants which accumulate SKA or 3KEA products are blocked in this part of the pathway and neither parent of pND200 or pND209 accumulated these compounds. To overcome these two possibilities, the EcoKl generated Km^ fragments from a further two mutants (105(5) and 105(205)) have recently been cloned into pBR322 (data not shown). The particular mutants were chosen as neither have Mu DNA (Figure 20) and both have only one EcoRl digest fragment which hybridizes to pKan2 (Figures 18A and 18B). Both clones are

Ap^Tc^Km^. pND211 was cloned with DNA from 105(5) which accumulates

12aOH-ADD. As with pND201, pND211 contained two £:c6>Rl-generated inserts in pBR322. The fragment sizes are 17kb, which correlates with the mutant DNA fragment size that hybridized to pKan2 (Figure 18A), and 12kb, which is most likely a fragment that ligated to the 17kb fragment prior to ligation to pBR322. It would be of interest to hybridize this clone against pND200. The second clone, designated pND212, was derived using EcoRl digested DNA of 105(205). This mutant accumulates 3KA, therefore the clone should contain genes encoding for enzymes relevant to A-ring oxidation. A preliminary restriction analysis of pND212 indicated the clone has one EcoRl digested fragment of approximately

20kb which again correlates to the JEcoRl-digested DNA fragment size, of the parent mutant, which hybridized to pKan2.

There has been very little fundamental genetic research with microorganisms involved in steroid biotransformations or degradation. Most research is based on enzyme regulation (usually induction), or the generation of blocked mutants or varients with fewer side reactions (Sedlaczek, 1988). As a result, the genetic background of steroid biotransformation and catabolism remains 202 unknown. Cloning of genes which encode for specific enzymes or whole operons involved in a steroid catabolic pathway would allow a more detailed investigation of the structural organization and regulation of such a pathway. Some reports of the cloning of genes encoding for steroid transforming enzymes have recently been published. Komel & Saurugger (1985) cloned a steroid 1,2-dehydrogenase gene from Norcadia restricta. To date, no further reports on this work have been published. A gene encoding the bile acid 7-dehydroxylase from a Eubacterium sp. has been cloned using a synthetic oligonucleotide as the screening agent (Coleman et al, 1987). In the parent strain, the enzyme is induced by bile acids with a 7-hydroxy function (CA, CDCA). Choi & Benisek (1987) cloned the structural gene for a 3-keto-5-ene steroid isomerase from Pseudomonas testosteroni. The clones were isolated using antibodies raised against purified isomerase.

In this project, one aim was to clone whole operons encoding sections of the pathway. With a more suitable vector and clones to be used as probes on hand, the future work in this project is to clone all the genes encoding the upper part of the bile acid catabolic pathway down to the formation of the indane lactone or possibly beyond. Studies and manipulation of the pathway will then be possible. It is reasonable to assume that future strain improvements in the steroid transformation area will include microorganisms generated by recombinant DNA methods. 203

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