\. CYANIDE DEGRADATION BY Bacillus pumilus Cl: CELLULAR AND MOLECULAR CHARACTERIZATION by Paul Robert Meyers A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy m the Department of Microbiology, Faculty of Science, University of Cape Town, South Africa. University of Cape Town Cape Town August 1993 The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town CERTIFICATION OF SUPERVISOR In termsof paragraph GP 8 of "General Rules for the degree of Doctor of Philosophy (PhD)" I, as supervisor of the candidate Paul R. Meyers, certify that I approve of the incorporationin this thesis of material that has already been published or submitted for publication. Pro essor D.R. Woods Deputy Vice Chancellor (Research) Uruversityof Cape Town Director of Microbial Research Units ACKNOWLEDGEMENTS I wish to record my grateful thanks to my supervisors Professor Dave Woods and Professor Doug Rawlings for supervising this research. Thank you for your enthusiasm and expert guidance in directing me to this goal. Special thanks are due to Dr George Lindsey of the Department of Biochemistry who was always available for discussion and who enthusiastically supervised a large section of this work. Thanks to Pravin Gokool of National Chemical Products for his assistance in establishing this project at the University of Cape Town and for his helpful advice and discussions. I am grateful for the assistance rendered by Stuart Glaun in constructing the first genomic library of B. pumilus Cl DNA in the vector, pEcoR251. I thank Professor Wolf Brandt of the Department of Biochemistry for amino-terminal protein sequencing, and Dr Linda Stannard of the Medical School for electron microscopy. I am grateful to National Chemical Products for funding this research, and gratefully acknowledge personal financial assistance from. the Foundation for Research Development, and the University of Cape Town. Finally, to Ann for always being there for me, and to all those with whom I have worked and from whom I have learned, thank you. May I pass on to others the knowledge I have gained. I TABLE OF CONTENTS ABSTRACT II ABBREVIATIONS V CHAPTERl GENERAL INTRODUCTION 1 CHAPTER2 ISOLATION AND CHARACTERIZATION OF B. pumilus Cl 69 CHAPTER3 THEENZYMOLOGY OF CYANIDE DEGRADATION BY B. pumilus Cl 116 CHAPTER4 STUDIES ON THE CLONING IN E. coli OF THE GENES FOR CYANIDE DIHYDRATASE 167 CHAPTERS GENERAL DISCUSSION 189 APPENDIX A MEDIA, BUFFERS, AND SOLUTIONS 191 APPENDIXB STANDARD METHODS 210 APPENDIXC CLON1NG VECTORS 216 LITERATURE CITED 217 II CYANIDE DEGRADATION BY Bacillus pumilus C1: CELLULAR AND MOLECULAR CHARACTERIZATION by Paul Robert Meyers August 1993 Department of Microbiology, University of Cape Town, Private Bag, Rondebosch, 7700, South Africa ABSTRACT A cyanide-degrading, Gram-positive, aerobic, endospore-forming bacterium was isolated from a cyanide wastewater dam by an enrichment technique and was identified as a strain of Bacillus pumilus. The bacterium was routinely cultured in Oxoid nutrient broth and rapidly degraded 100 mg/1 of free cyanide in the absence of added inorganic and organic substances. The ability to degrade cyanide was linked to the growth phase and was not exhibited before late­ exponential/ early-stationary phase. Cyanide-degrading activity could not be induced in early exponential phase by the addition of cyanide or acetonitrile to 20 mg/1. Production of the cyanide-degrading activity required at least 0.01 mg Mn2+ /1 in the growth medium (lower concentrations prevented the development of strong cyanide-degrading activity and also resulted in poor growth of the organism). No induction of cyanide-degrading activity occurred when Mn2+ ions were added to late-exponential-phase cells (or a cell-free extract from these cells) which had been grown with a low endogenous concentration of Mn2+. However, Mn2+ ions could be added to cultures growing in low-Mn2+ broth as late as the mid-exponential phase of growth with no apparent reduction of the cyanide­ degrading activity exhibited in stationary phase. Culturing the organism in Difeo nutrient broth resulted in poor growth and very low levels of cyanide-degrading activity; addition of Mn2+ to this medium did not significantly increase the levels of activity. Production of the cyanide-degrading activity required de novo transcription and translation. Cyanide-degrading activity was located III intracellulariy and cell-free extracts rapidly degraded cyanide (0.27 ± 0.08 µmole cyanide/min/mg protein at 30 °C in pH 7.4 phosphate buffer). Eight strains of cyanide-utilizing fluorescent pseudomonads were also isolated from activated sewage sludge by an enrichment technique and tentatively identified as strains of P. fluorescens and P. putida. These isolates could not degrade cyanide rapidly. A cyanide-degrading enzyme was purified from B. pumilus Cl by column chromatography and sucrose-gradient centrifugation, and characterized with respect to physico-chemical and kinetic properties. The enzyme consisted of three polypeptides of 41.2, 44.6 and 45.6 kDa; the molecular mass of the active enzyme by gel filtration was 417 kDa. Electron microscopy revealed a multimeric, rod-shaped protein of approximately 9 x 50 nm in addition to many shorter structures. The enzyme rapidly degraded cyanide to formate and ammonia thereby exhibiting nitrilase-like activity. Activity was reduced under anaerobic conditions. Enzyme activity was optimal at 37 °C and pH 7.8 - 8.0. Activity was enhanced by azide, thiocyanate, formate, and acetate. Enhancement by azide was reversible, concentration dependent (with maximal enhancement at 4.5 mM azide), and increased with time. The enhancing effect of azide was not manifested under anaerobic conditions. Enzymatic activity was also enhanced by Sc3+, Cr3+, Fe3+ and 'fb3+; enhancement was independent of metal-ion concentration above 5 µM. Radiolabelling experiments with 51Cr3+ failed to demonstrate the binding of metal to the active enzyme. No activity was recorded with the cyanide substrate analogues CNO-, SCN-, CH3CN and N3- and the possible degradation intermediate, HCONH2• Kinetic studies indicated a Km of 2.56 ± 0.48 rnM for cyanide and a Vmax of 88.03 ± 4.67 mmoles cyanide/min/mg/I. The Km increased approximately two-fold in the presence of 10 µM Cr3+ to 5.28 ± 0.38 mM for cyanide and the Vmax to 197.11 ± 8.51 mmoles cyanide/min/mg/I. The enzyme was tentatively named cyanide dihydratase. A genomic library of B. pumilus Cl chromosomal DNA in the vector, pEcoR251, was screened in E. coli strain LK111 by a colony-Western-blot technique using antibodies to cyanide dihydratase. Failure to isolate a cyanide-degrading E. coli clone prompted the construction of a second genomic library of B. pumilus Cl DNA in the E. coli-Bacillus subtilis shuttle vector, pEB1. The pEB1 genomic library was screened in E. coli strain LK111 by two different procedures. The first involved a differential-growth method in which the fastest-growing E. coli genomic-library transformants, under cyanide-containing sloppy-agar overlays, were selected and assayed for cyanide-degrading activity. The second screening IV procedure explored the hypothesis that cyanide-degrading E.coli clones would be able to reduce 2,3,5-triphenyltetrazolium chloride (ITC) in the presence of cyanide. This latter method involved plating transformants on a cyanide­ containing indicator medium containing TIC. Neither of these screening procedures produced cyanide-degrading E. coli clones, suggesting that the cyanide dihydratase gene, or genes, are not expressed in E.coli, or that the genes are not linked on the B. pumilus Cl chromosome and could not be cloned as a unit by the cloning strategy employed. A Southern-hybridization technique was attempted using a synthetic oligomeric DNA probe corresponding to the predicted DNA coding region for the NHrterminal amino-acid sequence of cyanide dihydratase. Initial experiments, however, showed that the oligonucleotide probe was not sufficiently specific to B. pumilus Cl chromosomal DNA to warrant screening the pEB1 genomic library by a colony-Southem­ hybridization procedure. V ABBREVIATIONS A adenine ABS. absorbance Ala L-alanine (A) ampicillin 1tcc American Type Culture Collection atrn atmospheric pressure (101 325 Pa) ATP adenosine 5'-triphosphate BCW 5-bromo-4-chloro-3-indolyl phosphate BSA bovine serum albumin C cyt~sine Ci Cune Cm chloramphenicol CPM counts per minute CsCl cesium chloride Cys L-cysteine (C) Da dalton DMSO dimethY.l sulphoxide DNA deoxyribonucleic acid DTPA diethylenetriaminepenta-acetic acid EDTA ethylenediaminetetra-acetic acid ESR electron spin resonance FAD flavin adenine dinucleotide standard gravitational acceleration (9.81 m.s-2) guanine h hour(s) HPLC high-performance liquid chromatography I inosine I ionic strength (moles/I) Ile L-isoleucine (I) k kilo Michaelis constant fg1 kilobase pairs kDa kilodaltons Leu L-leucine (L) VI Lys L-lysine (K) min minute(s) Mn+ metal cation MgATP magnesium ATP MIC minimum inhibitory concentration Mr relative molecular mass mR"I\JA messenger RN A NAD(P) nicotinamide adenine dinucleotide(phosphate); oxidized NAD(P)H nicotinamideadenine dinucleotide (phosphate); reduced NBT nitro