.v 3.. .. 1.92.). y, t} :L .. ‘ v. .‘........,. wave nus ITY LIBRARIES lllllllllllllllllllll Hill ill 3 129300 This is to certify that the dissertation entitled CLONING, SEQUENCE , AND CHARACTERIZATION OF THE KLEBSIELLA AEROGENES UREASE OPERON presented by Scot t B . Mulrooney has been accepted towards fulfillment of the requirements for _Bh4_ degree in Jimhemianry flaw/534% Major professor Date 9/11190 MS U i: an Affirmative Action/Equal Opportunity Institution 0- 12771 h F mmr "Milena State ‘ Univerlity \. fi fi ~———_- PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or betore date due. DATE DUE DATE DUE DATE DUE _l __ :__l| ‘ j =‘= _J J ‘ _____Jl______ ______ MSU Is An Afflrmdive Action/Equal Opportunity Institution encircmma-pJ CLONING, SEQUENCE, AND CHARACTERIZATION OF THE KLEBSIELLA AEROGENES UREASE OPERON 3? Scott B. Mulrooney A DISSERIAIION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1990 ABSTRACT Microbial ureases play a significant role in agricultural nitrogen metabolism and the pathogenesis of several human diseases. The best-studied bacterial urease is from Klebsiella aerogenes: its regulation has been partially characterized and the heteropolymeric enzyme has been purified and shown to contain four nickel ions per native molecule. Cloning of the Klebsiella aerogenes urease genes was undertaken in order to obtain greater quantities of enzyme for study and to elucidate the regulation, genetic organization, and sequence of the urease genes. Preliminary studies were carried out using the previously cloned urease genes from Providencia stuartii. Urease was purified and characterized from recombinant Escherichia coli, and urea—induced regulation of expression was demonstrated. Urease properties were identical to the parent organism when expressed in the heterologous host. The Klebsiella aerogenes urease genes were cloned by selecting a urease-positive colony from a cosmid library and subsequently subcloned to a 5.7 kb fragment. When the recombinant plasmid was tested in several enteric hosts, urease was expressed during growth under nitrogen-limited conditions and repressed in nitrogen-rich media. Klebsiella aerogenes containing the recombinant plasmid expressed high levels of urease. These cells were used for imunogold electron microscopy to localize the enzyme to the cytoplasm. In vivo incorporation of the nickel center into urease was examined in recombinant cells overexpressing urease in a nickel-free medium with several metabolic inhibitors. Addition of nickel restored activity when a protein synthesis inhibitor was used but not when energy-utilization inhibitors were present or in sonicated cells. These results indicate that nickel ions are incorporated into pre-formed apo-urease in an energy dependent process. Sequence analysis of the urease genes revealed an operon consisting of six open reading frames: three encoding the urease subunits (ureA, ureB, and ureC) and three with unspecified functions (ureE, ureF, and urea). Deletion of the ureE, ureF, and ureG from the operon resulted in the synthesis of inactive spa-urease, however, these genes could act in trans to restore activity. This demonstrates that one or more of the UreE, UreF, and UreC gene products facilitate nickel incorporation into urease. To my family and my parents iv ACKNOWLEDGEMENTS First of all, I would like to thank.Bob Hausinger for being a source of guidance, encouragement, friendship, and for being an exceptional boss . I want to thank other members of the lab have given me invaluable assistance over the years: Mathew Todd for his companionship, expertise in kinetics and computer wizardry; Mann-Hyung Lee and Yves Markowicz for helpful technical advice, scientific discussion and occasional comic relief; Steve Anderson, Ayse Cetin, Julie Breitenbach, Lisa Gloss, and Jackie Wood for assistance and help. I am also indebted to the members of my guidance committee who have given beneficial advice and direction: Jerry Dodgson, Arnold Revzin, Larry Snyder, and John Wang. I also thank Ken Nadler, Frank Dazzo, and other members of the Nitrogen Availability Program for their support and stimulating discussions. My appreciation also goes to Chang Kao for his expert advice . TABLE OF CONTENTS List of Tables List of Figures . Chapter 1 Chapter 2 Literature Review Medical significance Other roles for microbial ureases Plant ureases Regulation of urease expression Cellular localization Urease purification Structure and kinetic properties Cloning of urease genes Sequence analysis References Purification and Characterization of Recombinant Providencia stuartii Urease Expressed by Escherichia coli. . . . . Abstract Introduction Materials and Methods Bacterial strains and growth conditions Regulation of urease expression . Assays Polyacrylamide gel electrophoresis Large-scale growth and urease purification Urease characterization . Results vi xi 10 13 22 23 24 25 25 25 26 26 27 27 28 Optimization of cloned urease expression Purification of urease Analysis of urease by gel electrophoresis Kinetic parameters Native molecular weight . Nickel analysis Discussion References Chapter 3 Regulation of Gene Expression and Cellular Localization of Cloned Klebsiella aerogenes (K. pneumoniae) Urease . . . . . Abstract Introduction Materials and.Methods Gene cloning Assays Nickel dependence studies Immunological methods Immunogold electron microscopy Results and Discussion Gene cloning Urease regulation . Characterization of the urease made by strains containing pKAUl9 Effects of nickel concentration on urease gene expression and urease activity - Immunogold localization . References Chapter 4 In.Vivo Reconstitution of Klebsiella aerogenes vii 28 28 29 34 34 34 34 37 4O 41 42 42 42 43 44 45 45 47 49 49 52 55 Chapter 5 urease apo-enzyme Abstract Introduction Materials and Methods Bacterial strains and growth conditions Assays In vivo activation of apourease Results Discussion References Sequence of the Klebsiella aerogenes Urease Genes and Evidence for Accessory Proteins Facilitating Nickel Incorporation . . . . . . . . . . Abstract Introduction Materials and Methods Bacterial strains and growth conditions DNA sequencing Construction of plasmids pKAU60l and pKAU506 Assays SDS-Polyacrylamide gel electrophoresis Purification and characterization of pKADGOl- derived urease . . . . . . . . . . . . Results and Discussion Sequence analysis of the urease operon Homology comparisons Complementation analysis of the urease operon genes Purification and characterization of pKAUGOl- viii 58 59 60 61 61 61 61 62 65 67 69 7O 71 72 72 72 73 73 75 75 76 76 83 '84 Chapter 6 Conclusions and Future Prospects Appendix derived urease References ix 88 91 95 99 Chapter 2 Chapter 3 Chapter 5 LIST OF TABLES . Optimization of recombinant P. stuartii urease expression by E. coli(pMID201) . Purification of recombinant P. stuartii urease from E. coli . Expression of recombinant Klebsiella aerogenes ure as e . Urease specific activities of recombinant E. coli cultures 31 33 48 85 Chapter 2 Chapter 3 Chapter 4 Chapter 5 LIST OF FIGURES . Phenyl-Superose FPLC chromatography of recombinant P. stuartii urease expressed in E. coli . SDS polyacrylamide gradient gel of purified recombinant P. stuartii urease expressed in E. coli . Restriction map and summary of cloned K. aerogenes urease gene fragments . Immunoblot analysis of recombinant urease expressed in E. coli DHl and S. typhimurium . . Effect of nickel concentration on recombinant urease expression and activity . Immunogold localization of recombinant K. aerogenes urease . In vivo reconstitution of urease apo-protein . Structure of the urease operon and two subclones . Nucleotide sequence of the urease genes . Polyacrylamide gel analysis of UreE, UreF, and UreG xi 30 32 46 50 51 53 63 74 77 86 CBAPTERl Literature Review 2 Urea (HzN-CO-NHZ) is very stable in solution: its half-life for spontaneous degradation is 3.6 years at 38'C and non-catalyzed hydrolysis has never been observed (4). Most urea is therefore broken down enzymatically by urease (urea amidohydrolase, EC 3.5.1.5) which catalyzes the hydrolysis of urea to yield amonia and carbamate. The carbamate spontaneously hydrolyzes to give additional ammonia and carbonic acid. In aqueous solution, the carbonic acid deprotonates and the ammonia becomes protonated resulting in a net increase of pH. Urease occurs in organisms from many different taxonomic classifications, including over 200 species of bacteria, and several plants, yeast, algae, and invertebrates (73). Much of the early urease work was done on the enzyme purified from jack bean (Canavalia ensiformis) (1,109): indeed, in 1926 it was the first enzyme to be crystallized (104). It was not until nearly 50 years later that urease was demonstrated to be a nickel-containing enzyme (22). More recent studies have focused on the role of urease in particular human pathogenic conditions and in agricultural nitrogen economy. Molecular biological and genetic tools have been employed to give more information on enzyme structure, composition, nickel incorporation, and regulation. The objective of this chapter is to present a brief overview of the significance and enzymology of ureases, and give an account of recent advances in the regulation and genetics of this enzyme. The main focus will be on bacterial ureases, although examples from plant and other sources will be mentioned where appropriate. Medical significance. Microbial urease activity has been shown to be a contributing factor in pathogenesis of several diseases. Hepatic encephalopathy (100), hyperammonemia (103), and hepatic coma (98) can occur from the effects of toxic nitrogenous compounds which have not been metabolized by the liver. Ammonia released from urea hydrolysis contributes to these conditions (101). Furthermore, considerable recent research has focused on the role of urease in development of urinary stones and peptic ulcerations. Hman urine consists of 0.4 to 0.5 M urea (35) and presents a favorable environment for many microorganisms which can infect the urinary tract. One major consequence is infection-induced stone formation, which account for 20$ to 40% of all urinary stones (34,36,94). Infection stones are formed when polyvalent struvite and apatite salts crystallize due to alkalinization from urea hydrolysis (68,69,73). Ureolytic bacteria have also been implicated in pyelonephritis, which occurs when urea hydrolysis and its associated increase in pH results in acute kidney inflammation and tubule necrosis (96). In addition, ureolytic microbes have been shown to be important in promoting incrustation and obstruction of urinary catheters (74,112). Proteus mirabilis is the major urease-producing uropathogen in humans (96) . Experiments with animal models. have shown urease to be a significant virulence factor in infections of the kidney epithilium (9,26,29,33,58). Treatment with urease inhibitors helps reduce these effects (2,59,82) . Furthermore, recombinant DNA techniques have been used to construct urease-negative mutants of Proteus mirabilis (45) and Staphylococcus saprophyticus (30). In both cases, the mutants had significantly lower virulence. Recently, the ureolytic bacterium Helicobacter pylori (formerly Campylobacter pylori) has been implicated in promoting peptic ulcerations (8,32,38,63,72,76) . It only grows in a relatively neutral pH range and is very acid sensitive (32). A model has been proposed whereby the Helicobacter pylori, living in the stomach mucosa, survives by creating a 4 zone of favorable pH with the ammonia produced by urea hydrolysis (15). The Helicobacter urease, which has a relatively low R, value of 0.2 to 0.8 mM (41,72), is able to utilize serum urea (1.7-3.4 mM) to modulate the pH of its local environment. Consequently, gastrointestinal inflamation and lesions develop, either by interfering with diffusion of acid from the gastric glands or by tissue damage directly from the localized high amonia concentration (15,38) . Other roles for microbial ureases. Ureolytic microbes play a critical role in nitrogen cycling in ruminants: urea that has been generated from digestion and metabolism can be recycled through saliva and the bloodstream and returned to the rumen. where it is subsequently hydrolyzed to give ammonia-the major source of nitrogen of the rumen microbial population (13,42,73). Urease activity is also found in soils, either in living organisms or as free enzyme (12,55,81). Indeed, application of urea fertilizers to areas with high soil urease activities can result in plant damage due to ammonia toxicity and elevated soil pH as well as nitrogen loss from volatilization of amonia (81,99). Several studies have used urease inhibitors to reduce soil urea hydrolysis rates (ll,l4,54,64,89,90). Plant ureases. Although many members of the Leguminosae are known to possess urease activity, jack bean (Canavalia ensiformis) and soybean (Glycine max) are the best studied plant sources. Jack bean urease was the first enzyme to be crystallized (104) the first demonstrated to contain nickel (22), and its mechanism has been probed by kinetic and spectroscopic methods (1,4,23). The amino acid sequence of the jack bean urease was determined by using classical protein methods (62,105) . Soybean plants possess two immunologically and enzymologically distinguishable 5 types of urease: the embryo-specific (seed), and the ubiquitous (leaf) forms (84.85.86.87) . Molecular biological strategies have been employed to examine the tissue-specific and temporal expression of the two isozymes (39) . It was demonstrated that certain mutations would lead to the synthesis of inactive urease isozymes, indicating that some urease maturation factors may be required (71). A section of the soybean seed urease gene was cloned and sequenced (51). Regulation of urease expression. Bacteria have three modes of regulating urease expression. Organisms such as Helicobacter pylori and Morganella morganii have ureases which are expressed constitutively and are not significantly affected by components in the growth medium (72,95) . Another class, which possess urease genes that are inducible by urea, includes Providencia stuartii (Chapter 2; 75,79) and Proteus mirabilis (46). In the third group, urease expression is controlled by the global nitrogen (Ntr) system, and is best exemplified by Klebsiella aerogenes (Chapter 3; 28,60,80). Urease, like other Ntr-regulated genes, is expressed when the organism is exposed to nitrogen-limited conditions and repressed in the presence of nitrogen-rich constituents. Low nitrogen conditions initiate a complex regulatory cascade (60) which results in production.the ntrA.gene product (a.specific sigma.factor), which combines ‘with core RNA.polymerase and'begins transcription of Ntr-regulated genes. There has been a recent proposal that transcription of a subset of Klebsiella aerogenes Ntr-regulated genes, including urease, is mediated by a newly discovered nac gene (nitrogen assflmilation control; 3,61). Cellular localization. Most reports of localization of urease in cells demonstrate that the enzyme is found mainly in the cytoplasm. Cell fractionation studies of various bacteria (43,44), Providencia stuartii 6 (75), Proteus mirabilis, (46), ureaplasma urealyticum, (7,21,65, 92,97,110), and Klebsiella aerogenes (28), established that urease was in the cytoplasm. Immunogold electron microscopic localization studies of recombinant Klebsiella aerogenes urease confirmed earlier fractionation studies (Chapter 3; 80). Immunological methods were also used to localize jack bean urease in the cytoplasm (25). An alternate electron.microscopic strategy was used for Ureaplasma urealyticum where electron-dense MnOz was precipitated by the alkaline pH from urea hydrolysis: urease was concluded to be in the cytoplasm (110). Contrasting results were reported by McLean et al. (66,67) , who used a cytochemical electron microscopic method with Staphylococcus species and Proteus nfirabilis. In their strategy the bacteria were incubated with tetraphenylboron, which forms a precipitate with ammonia produced by urea hydrolysis. The tetraphenylboron-ammonia complex was then reacted to exchange the ammonium for silver ions which were then viewed by electron microscopy. For both Staphylococcus species urease was found to be membrane bound (66) , and for Proteus mirabilis it was found in the periplasm and outer membrane (67). These anomalous results are probably due to the inability of tetraphenylboron to freely enter the cell; i.e., the reagent is reacting with the ammonia product diffusing,out of the cell rather than the actual enzyme (73). Urease purification. Sumner used a very simple procedure for obtaining crystalline jack bean urease (104) which involved extraction of jack bean meal with aqueous acetone and allowing crude crystals to form. This was eventually modified to include a DEAE-cellulose step (91). The first purification of a bacterial urease was from Bacillus pasteurii and included ammonium sulfate, calcium phosphate, and acetone fractionation 7 steps (52). Modern methods have relied on a series of ion exchange, gel filtration, and.hydrophobic chromatographies to purify many ureases (73). Immuno-affinity chromatography has been used for Ureaplasma urealyticum (88,106). In addition, chromatography on immobilized substrate analogs have been used (20,70,83,ll3). Structure and kinetic properties. Native ureases have molecular weights ranging from 125,000 to 590,000 and are composed of one or more subunit types. In contrast to the homohexameric jack bean enzyme (subunit M,-90,770; 62), bacteria possess heteromeric ureases. The ureases of Klebsiella aerogenes (107), Proteus mirabilis (10,46), Providencia stuartii (Chapter 2; 79), Selenomonas rumantium (37,107), Morganella morganii (40), Ureaplasma ureolyticum (106), Lactobacillus reutri (49), and Lactobacillus fermentum (48) consist of one large (Mr-60,000 to 75,000) and two distinct small subunits (8,000 to 11,000). Many of the earlier accounts of purification of bacterial ureases reported that the enzyme consisted of multimers of a single large subunit (reviewed by Mobley and Hausinger, 73), however these studies did not look for subunits in the 8,000 to 11,000 range. Such subunits could easily be missed in SDS- polyacrylamide gel analysis unless proper precautions are taken (107). Helicobacter pylori urease is unusual in that it consists of one large (Mr-66,000) and one medium subunit (M,-29,500) (41). Whereas most ureases exhibit maximum activity near neutral pH values, the recently purified enzymes from two Lactobacillus species were unusual in that they are most active at 65'C at pH 2 (48,49). Jack bean urease possesses two nickel ions per subunit and most microbial ureases seem to contain 2 nickel ions per large subunit (22,37,48,79,107). Results reported for Brevibacterium 'amoniagenes and 8 Bacillus pasteurii (16,83) are only half this amount. Active site titration studies on jack bean (22) and on Klebsiella aerogenes (108) ureases have shown that there are two nickel ions per active site. K, values for ureases can range from 0.1 to >100 mM (73). Ureases of ureopathogenic microbes are saturated with substrate, since urine is 0.4 to 0.5 M urea (35). Specific activity for jack bean urease is approximately 3,500 umol urea min'1 mg'1 (1) , while many bacterial enzymes range from 1,000 to 5,500 (73) . Other ureases have lower reported specific activities, but this may be due to incomplete purification or inactivation (73). Ureaplasma urealyticum produces an unusual urease in that specific activities have been reported as high as 180,000 (102). These high activities are consistent with a proposal that urease may be involved in energy transduction in Ureaplasma. Urea is an absolute requirement for growth of Ureaplasma, but very little of the ammonia or carbon is used by the cell (27) . It is thought that the cytoplasmic increase in pH resulting from urea hydrolysis leads to formation of a proton gradient which drives ATP formation (65,93). Cloning of urease genes. The screening method for all of the bacterial ureases that have been cloned, with the exception of Ureaplasma urealyticum, was to transform libraries of the urease-containing microbes into a urease-negative one; usually E. coli. Selections were made on agar .plates which were only slightly buffered and contained urea and a pH indicator that gave a color change around urease-positive colonies. Christensen urea agar (17) has been used, but works best with constitutively expressed ureases. Other variations have been described which give an indicator color change upon overnight incubation (19,24,46,50,79). The urease genes of Bacillus pasteurii (50) and Klebsiella pneumoniae (31) were isolated by ligating size-fractionated chromosomal DNA into positive selection vectors. Providencia stuartii urease genes were cloned from its large conjugative plasmid by ligating fragments into vector pBR322 (75). The urease genes of a urease-positive E. coli (19), Klebsiella aerogenes (Chapter 3; 79), Klebsiella pneumoniae (31), Morganella morganii (40), and Proteus mirabilis (46,111) were cloned by partially digesting chromosomal DNA with Sau3A and ligating into various lambda and cosmid vectors. For Proteus vulgarus (77) , the same strategy was used, except that plasmid pUCl8 served as the vector. Furthermore, a similar approach was taken for Staphylococcus saprophyticus in which chromosomal fragments were ligated into a Staphylococcus vector .and recombinant plasmids screened in urease-negative Staphylococcus carnosus (30). In each of these cases, the cloned fragments contained the necessary regulatory regions that were recognized by the E. coli transcription and translation mechanisms. For Providencia stuartii and Klebsiella aerogenes clones, expression from the recombinant multi-copy plasmid allowed exceptionally high production of urease which was advantageous for purification of large amounts of enzyme (Chapter 2, 3; 79,80). To circumvent any possible problems of heterologous expression, Helicobacter pylori chromosomal fragments were cloned into a vector containing a lac promoter which controlled synthesis of the recombinant proteins (18). The urease genes of Ureaplasma urealyticum were detected by hybridization to other cloned ureases rather than selecting for a urease-positive phenotype (6). Expression of urease activity was not 10 possible in heterologous hosts because Ureaplasma uses a UGA codon for tryptophan rather than a stop. Sequence analysis. Recent DNA sequencing of the complete operons of Proteus mirabilis (47) revealed six open reading frames, three of which encode the urease subunits. The Proteus mirabilis urease subunit genes are preceded by a ureD gene, which may be involved in regulation (46). Next, are the r, B, and a urease structural genes, designated ureA, ureB, and ureC, respectively. Between ureA and ureB is a short region with homology to a eucaryotic splice junction, and the ureB and ureC genes overlap by four base pairs. Following ureC are ureE, and ureF which have unknown functions. These results, combined with earlier transposon mutagenesis studies, lead to the conclusion that all of the genes except ureE are necessary for urease activity. The Klebsiella aerogenes operon also consists of six open reading frames (Chapter 5; 78), however, there were two significant differences. An upstream ureD was lacking and an additional open reading frame, designated ureG, was found just downstream of ureF. These findings, together with partial sequence information of the Proteus vulgarus (77), Helicobacter pylori (18), and Ureaplasma urealyticum (5) urease genes, reveal that the amino acid sequences of the multiple bacterial urease subunits are remarkably similar to the single subunit jack bean enzyme: there is about 50 to 60% identity between the sequences of the bacterial and the plant ureases. Indeed, 37% of the Klebsiella aerogenes amino. acid residues are present in all four bacterial and the jack bean enzymes (Chapter 5). Nickel incorporation into urease. Several studies have indicated that one or more gene products, in addition to the enzyme subunits, are 11 required to incorporate nickel ions into urease. For example, pleiotropic mutations have been found which result in the production of inactive ubiquitous and seed ureases in soybean (71) . In Aspergillis nidulans, four genes are necessary for urease activity; mutation of two different loci caused production of inactive urease, and growth in high nickel could restore activity for one of these (56,57). In addition, transposon or deletion analysis of the Providencia stuartii (79), Proteus mirabilis (46), Klebsiella pneumoniae (31), Klebsiella aerogenes (Chapter 5; 78), and Proteus vulgarus (77) operons has resulted in mutants that express inactive urease subunits. Further evidence that a nickel insertion step is essential for urease activation is that Klebsiella aerogenes urease apoenzyme, which was purified from cells grown in the absence‘of nickel, could not be reactivated by nickel in vitro. Urease apoenzyme could be reactivated in whole cells even after treatment with protein synthesis inhibitors (Chapter 4; 51). Aims of this thesis. At the time this thesis was started, bacterial ureases had been shown to consist of heterologous subunits and possess nickel. No urease genes had been cloned and it was not known if a heterologous host would express the urease protein, if the urease would be in an active or inactive form, or whether the nickel center could be correctly incorporated. This thesis reports on the first purification and characterization of a heterologously expressed urease from Providencia stuartii (Chapter 2), the cloning, regulation, overexpression, and cellular localization of Klebsiella aerogenes urease (Chapter 3), the in vivo reconstitution of urease apoenzyme (Chapter 4), and the sequence of the six open reading frames of the Klebsiella aerogenes urease operon and 12 indication that one or more genes are required for nickel incorporation (Chapter 5). 10. 11. 12. l3. 14. 13 REFERENCES Andrews, R. 1., R. L. Blakeley, and B. Zerner. 1984. Urea and urease. Vol. 6, p. 245-283. In G.L. Eichhorn and L.G. Marzilli (eds.), Advances in inorganic chemistry. Elsevier Scientific Publishing Co. , New York. Aransan, M., O. 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Single-step purification of urease by affinity chromatography. Can. J. Microbiol. 20:623-630. CHAPTER 2 Purification and Characterization of Recombinant Pravidamcia stuartii Urease Expressed by Escherichia coli 22 23 ABSTRACT Recombinant urease from Pravidencia stuartii has been purified from Escherichia coli. Urease expression was induced by urea and repressed by nitrogen-rich components in the medium. The urease protein was purified 331-fold by DEAE-Sepharose, phenyl-Sepharose, Mano-Q, and phenyl-Superose chromatographies with a 7.3% yield. The enzyme possessed a K, for urea of 9.3 mM and hydrolyzed urea at a Van of 7,100 pmol/min per mg. P. stuartii urease is composed of three polypeptides (M,s, 73,000, 10,000, and 9,000) denoted as a, B, and r. The native enzyme is best described as (olfizrzh, based on a native M, of 230,000, obtained by gel filtration chromatography, and on the Coomassie blue staining intensities of the individual subunits. Atomic absorption analysis of the pure protein revealed 1.9 i 0.1 nickel ions per 0118er unit. 24 INTRODUCTION Urease, a nickel-containing enzyme that catalyzes the hydrolysis of urea to carbon dioxide and ammonia, is synthesized by a wide variety of bacteria, fungi, and plants (1,2,9,1l). Bacterial urease may be a contributing factor in the development of pyelonephritis (4,16,24), hyperammonemia (25) , and catheter encrustation (20), as well as kidney and bladder stone formation (7,23). In the last case, ammonia generated from urea hydrolysis alkalinizes urine, resulting in the precipitation of polyvalent anions and cations in the form of struvite and apatite salts (7) . Although stones may develop from other causes, it has been estimated that 20-40% of all urinary stones are due to urease-positive bacteriuria (23). In chronically catheterized patients, there is a high incidence of bacteriuria, with the organism Pravidencia stuartii a prevalent isolate (28,29). The urease genes of this microbe are located on a large conjugative plasmid in a number of isolates (6,18) and have been cloned and expressed in Escherichia coli (19). Preliminary minicell analysis of insertion and transposon mutants of the recombinant urease revealed that a region comprising 3.0 to 6.2 kilobase pairs (kb) of DNA was necessary for urease activity and encoded at least twa polypeptides (Mrs, 73,000 and 25,500) (19). The kinetic parameters of the recombinant enzyme were similar to those of the native strain (19). In addition to the urease genes of P. stuartii, those of Bacillus pasteurii (l3), Proteus mirabilis (12), Klebsiella aerogenes (22.; Chapter 3), Klebsiella pneumoniae (5), and Morganella morganii (10) have been 25 cloned in E. coli by selecting for a urease-positive phenotype. However, no detailed studies of urease expression, enzyme characterization or protein purification have been reported for any recombinant urease. It was recently shown that the purified ureases of several bacterial species are composed of one large and two small polypeptides (27), which contrasts with the homomeric structure of the jack bean enzyme (1,2) . The multimeric structure may be a general property of bacterial ureases. This report extends the work of Mobley et a1. (19) in which P. stuartii urease genes were cloned and some aspects of urease expression in E. coli were investigated. A purification scheme for recombinant urease is provided, factors affecting urease expression are examined, and the properties of the recombinant enzyme are described. MATERIALS AND METHODS Bacterial strains and growth conditions. E. coli HBlOl (recA pro leu rpsL hst) containing pMID201 (ure* tet) , which possesses urease genes cloned from a conjugative plasmid of P. stuartii B82467 (19), were used for these experiments. The medium used (17) was either LB broth or ammonia-free M9 minimal medium supplemented with 5 or 10$ (vol/vol) LB broth. When required, 1 ml of trace mineral solution (26) and 15 ml of filter-sterilized 1 M urea per liter were added. For growth of E. coli MBlOl(pMID201), all cultures contained 10 pg of tetracycline per ml. Regulation of urease expression. For urease expression studies, 125- ml Erlenmeyer flasks containing 20 ml of medium were inoculated with 5 pl of a stationary LB culture and were grown overnight in a 37’C water bath with rapid shaking. Aliquots (5 ml) of the cultures were chilled on ice, 26 centrifuged, washed twice with an equal volume of ammonia-free M9 salts (M9 without glucose and ammonium chloride), and suspended in a final volume of 2.5 ml. The cells were sonicated four times for 30 5 each time with a sonicator (Sonic Dismembranator; Ficher Scientific Co., Livonia, Mich.) by using a small probe (4-mm diameter tip) at 30% power. A l-ml portion of the extract was centrifuged for 5 min at 4°C in a microcentrifuge (Eppendorf; Brinkmann Instruments, Inc., Westbury, N.Y.), and the supernatant was assayed for urease activity. Assays. Urease activity was determined by monitoring the rate of ammonia released from urea by formation of indophenol, which was measured at 625 nm (30). The assay buffer consisted of 25 mM HEPES (N-2-hydroxy- ethylpiperazine-N'-2-ethanesulfonic acid; Sigma Chemical Co., St. Louis, Mo.), 50 mM urea, and 0.5 mM EDTA (pH 7.75). One unit of urease activity is defined as the amount of enzyme required to hydrolyze 1 umol of urea per min at 37'C under the assay conditions described above. Protein was measured by the method of Lowry et a1. (15) by using bovine serum albumin as the standard. Polyacrylamide gel electrophoresis. All electrophoresis was carried out by using the buffers of Laemmli (14), except that sodium dodecyl sulfate was omitted for native gels. Denaturing gels were run by using a 10 to 15% polyacrylamide gradient (bisacrylamide/acrylamide, 1:32) resolving gel with a 4.5% stacking gel. Samples were run after denaturing at 100'C for 5 min. The gels were stained with Coomassie brilliant blue (Sigma) and scanned by using a Gilford Respense spectrophotometer (Gilfard Instrument Laboratories, Inc., Oberlin, Ohio) at 562 nm. Nondenaturing gels were run by loading 1 U of urease activity on a 3% stacking gel and a 6‘ running gel, which was then stained for activity by using a phenol 27 red indicator, similar to the method of Blattler et al. (3). Large-scale growth and urease purification. E. coli HBlOl(pMID201) was grown in mania-free M9 minimal medium supplemented with 10% (v\v) LB broth, 15 mM urea, 10 ug tetracycline per m1, and 1 ml trace mineral solution per liter. Cultures were grown at 37'C in either a 25-liter Microferm fermentor (New Brunswick Scientific Co., Edison, N.J.)(15 l cultures) with rapid mixing and aeration, or in a 20 1 bottle (containing a 10-1iter culture) immersed in a 37°C bath without shaking but with vigorous sparging. Cells were harvested by using a Pellicon concentrator (Millipore Corp., Bedford, Mass.), washed once with PEB buffer (20 mM phosphate, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.0 ), resuspended in an equal volume of PEB buffer and frozen at -20’C. The cells were thawed, disrupted by two passes through a French pressure cell (American Instrument Co. , Silver Spring, Md.) at 16,000 lb/inz, and centrifuged at 100,000 x g for 60 min at 4'0. DEAE-Sepharose and phenyl-Sepharose chromatographies were performed on conventional columns at 4’C. All subsequent purification steps were carried out on a Fast Protein Liquid Chromatography system (Pharmacia, Uppsala, Sweden) at room temperature. All resins and columns were purchased from Pharmacia. PEB buffer with the stated additions was used in all phases of the purification. Urease characterization. The reaction rates for purified urease were measured as the concentration of urea was varied from 1 to 100 mM, and the data were analyzed by the method of Wilkinson (31). The molecular weight for native P. stuartii urease was estimated by using Superose 6 gel filtration chromatography in PEB buffer containing 0.1 M RCl. The column (1.0 X 30 cm) was standardized by using thyroglobulin, gamma globulin, ovalbumin, myoglobin, and vitamin B-12 (Mrs, 28 670,000, 158,000, 44,000, 17,000, and 1,350; Bio-Rad Laboratories, Richmond, Calif .). The nickel content of the purified urease was determined by using a PE 5000 atomic absorption spectrophotometer (Perkin-Elmer Corp. , Norwalk, Conn.) equipped with an RCA 500 graphite furnace and an AS-l autosampler. Samples were hydrolyzed in 1 M HNOa, evaporated, and dissolved in 50 mM HNOa. Nickel standards, prepared with and without bovine serum albumin (to mimic the enzyme matrix), were treated identically to the urease samples. Aliquots (20 pl) were dried at 120’C, charred at 1,200'C, atomized at 2,700'C, and quantitated for nickel by integrating the peak area while using the backround correction mode. RESULTS Optimization of cloned urease expression. The specific activities of crude cell extracts grown under various conditions are shown in Table l. Urease expression in E. coli was greatest when urea was present, consistent with regulation by urea induction. In addition, urease expression was repressed in very rich medium (LB broth), but the enzyme was synthesized when the amount of nitrogen-rich constituents in the medium decreased to 5 or 10% the amount in LB broth. The presence or absence of trace minerals had little effect. When a growth curve was performed by using the medium described for large scale growth, the specific activity of the culture was highest during the early exponential phase, after which it gradually subsided (data not shown). Purification of urease. The crude extract from 31 g (wet weight) of cells was applied to a DEAE-Sepharose column (2.5 x 15 cm) equilibrated 29 with PEB buffer. The urease was eluted with a 400 m1 linear gradient of 0 to 1.0 M KCl in PEB buffer, resulting in a single peak of activity at 350 mM MCl. Peak fractions were adjusted to 1.0 M KCl and loaded onto a pre- equilibrated phenyl-Sepharase column (1.5 x 14 cm) . After washing with 80 ml of 1.0 M KCl in PEB buffer, the urease was removed with a single step elution using 80 ml of PEB buffer. Washing the column with 20% dimethyl- sulfoxide in PEB buffer eluted only trace amounts of additional urease activity. Peak phenyl-Sepharose fractions were combined, diluted with an equal volume of PEB buffer and applied to a Mono-Q MR 5/5 Fast Protein Liquid Chromatography column. The activity eluted as a doublet at 350 mM KCl by using a linear KCl gradient in PEB buffer. The active fractions were pooled, adjusted to 2.0 M KCl, and loaded onto a phenyl-Superose MR 5/5 Fast Protein Liquid Chromatography column. The protein was eluted with a descending 2.0 to 0 M KCl gradient in PEB buffer yielding a major activity peak at 1.1 M KCl and a minor activity peak at 0.3 M [(01 (Fig. 1). The latter peak was composed of both urease and contaminating protein, presumably as an aggregate. Fractions of the major peak were pooled and concentrated by ultrafiltration (Centricon .10; Amicon Corp. , Lexington, Mass.). The purification procedure and results are summarized in Table 2. Analysis of urease by gel electrophoresis. Denatured samples of purified urease were electrophoresed by using a sodium dodecyl sulfate-10 to 15% polyacrylamide gradient gel (Fig. 2). Three polypeptides were observed in the pure recombinant protein; they were similar to those found in several other bacterial ureases (27). Subunit ratios derived from seaming densitometry of the Coomassie blue-stained bands after normalization for molecular weights were 1:1.7:1.9 for the bands with Mrs of 73,000, 10,000, and 9,000, respectively. These peptides, in decreasing 30 0.20" -\\\ \\\\ \\\\ i a \\ \\\ \ g \\ 8 “x N 0.10J \ In] \x L) r E 1 , m l I: . o . (I) 9 0.00- , o 25 FRACTION Fig. l. Phenyl-Superose FPLC chromatography of recombinant P. urease expressed in E. coli(pMID201). -1000 -2.0 -800 ’ T .J -600 2 ’ 'E b1.02 : . .400 5 '5; a: 5.. ’ D tn -200 3 O _ 2 3. h0.0 50 stuartii Active fractions from Mono-Q chromatography were pooled, adjusted to 2M KCl, and chromatographed as described in the text by using a KCl gradient (---) . Aliquots of the 1.0 ml fractions were assayed for urease activity (II) , and absorbence was monitored (—) . 31 TABLE 1. Optimization of recombinant P. stuartii urease expression by E. coli(pMID201)‘ Presence of Medium” Aw,‘l Sp act' 15mM Trace urea minerals° LB - - 0.79 0.0 L8 + - 0.69 0.8 LB + + 0.66 3.4 M9 10%LB - - 0.53 0.0 M9 10%LB - + 0.51 0.0 M9 10%LB + + 0.51 68.0 M9 10%LB .+ 3+‘ 0.80 75.0 M9 5%LB - - 0.54 2.5 M9 5%LB - + 0.53 2.7 M9 5%LB + + 0.40 54.0 ' E. coli NBlOl is nonureolytic. b c d e f See reference 17. See reference 26. A.“ for the culture at time of harvest. Specific urease activity of extracts from disrupted cells expressed in pmol urea per min per mg. Three times the normal amount of trace minerals was added. All cultures contained tetracycline (10 pg/ml). 32 — 92,500 . — 66,200 - 45,000 — - 3|,000 - 2|,500 l6,950 = : :' 14,400 : 8,|60 6,2l0 l 1 W Urease Stds Fig. 2. SDS polyacrylamide gradient gel of purified recombinant P. stuartii urease expressed in E. coli. A sample of the purified enzyme was run as described in the text using 8ug of protein, and the gel was stained with Coomassie Brilliant Blue. The standards were: phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, lysozyme (Bio-Rad, Richmond, CA) , myoglobin, and myoglobin cyanogen bromide fragments I+II, I, and II (Sigma). 33 «.5 m.o omm.e Ann oun.m oucuomzmuaacoam «.mm o.» ooe.aa and om~.~ oioeox o.mv o.mn oom.nn mm Hem oncogenes Assess m.ee mam oom.m¢ as can ououeaaou mama oou oma.e coo.mo A , h.oa uoeuuxo ensue A705 raga .mav Aftfia Hoaav _ ooh: Hoaav and chances sua>auoa .oaou. aua>auoa >uo>ooam Houoa Houoa coauooauwunm owuwoonm noun toduoowuwuom Adam 4w aouu mucous Admummmm 4N acetaneooou no tawuoo«uqusm .N mamas 34 order of size are designated a, B, and 7. Samples of P. stuartii urease were run on nondenaturing gels and stained for activity. The final pure protein gave a single sharp band, whereas samples of the preparation taken at earlier stages in the purification showed several additional faint, slower-migrating bands (data not shown). Kinetic parameters. A K, of 9.3 i 1.2 mM and a Vm of 7,100 i 300 pmol of urea per min per mg were obtained for purified P. stuartii urease. This R. value compares quite well with that reported for crude cell extracts from P. stuartii or from E. coli expressing recombinant urease (19). Native molecular weight. The native molecular weight for the purified P. stuartii urease was estimated as 230,000 1 20,000 by Superose 6 gel filtration chromatography. This value is significantly less than the value of 337,000 reported for crude cell extracts (19). This discrepancy may be due to association with other proteins in the more crude preparations. Nickel analysis. Atomic absorption analysis showed that urease had 1.9 i 0.1 mol of nickel per mol of olfizrz structural unit when compared with a standard curve prepared as described in Materials and Methods. DISCUSSION Growth of E. coli containing the P. stuartii urease genes shows that urease expression is induced by urea, as reported for wild-type P. stuartii (18). This result differs from that of Mobley et a1. (19), who reported that the expression of cloned urease gene sequences appeared to 35 be constitutive for recombinant cells grown in L broth. Nitrogen-rich conditions (e.g., LB medium) repressed urease expression in the experiments described above. Urease regulation by induction and repression indicates that the cloned segment must still possess regions necessary for regulating urease expression. In addition, E. coli regulatory components are probably necessary. The fact that omission of the trace mineral solution still resulted in high specific activities shows that there is enough trace nickel in the medium constituents to support active urease production. An ammonia-free M9 medium supplemented with 10% LB broth, trace minerals, and 15 mM urea was selected for large scale culturing; it was low enough in available nitrogen sources to promote high levels of urease, yet allowed the cells to grow rapidly for fast culturing of cells. The early purification steps for cloned P. stuartii urease followed a protocol similar to that used for K. aerogenes (27), and Selenomonas ruminantium (8), indicating that the ureases may have similar physical properties. The presence af'a single band, when stained either for protein or for activity, after native polyacrylamide gel electrophoresis demonstrated that a single isozyme of homogeneous urease was obtained. Sodium dodecyl sulfate-polyacrylamide gels showed that the recombinant P. stuartii urease'has 3 polypeptides, which.is consistent‘with the structure of several other bacterial ureases. The subunit ratio for P. stuartii urease is very close to the 1:2:2 ratio reported for the K. aerogenes enzyme (27). The native molecular mass of 230,000 kDa is consistent with s ((118212); structure, as has been suggested for the K. aerogenes enzyme. Each clam unit was demonstrated to contain two nickel ions, a metal which has been found in a variety of plant, fungal, and bacterial ureases (9). Recent transposon mutagenesis experiments using cloned P. stuartii 36 DNA gave data consistent'with the above findings in which expression of at least three polypeptides are required for urease activity (21). These polypeptides may arise either from three separate genes, or a larger precursor protein which is subsequently processed. 10. 11. 12. l3. 14. 37 REFERENCES Andrews, R.R., R.L. Blakeley, and B. Zerner. 1984. Urea and urease. Vol. 6, p. 245-283. In G.L. Eichhorn and L.G. Marzilli (eds.), Advances in inorganic chemistry. Elsevier Scientific Publishing Co., New York. Blakeley, R.L., and B. Zerner. 1984. Jack bean urease: the first nickel enzyme. J. Mol. Catal. 23: 263-292. Blattler, D.P., C.C. Contaxis, and B.J. Reithel. 1967. Dissociation of urease by glycol and glycerol. Nature(London). 216:274-275. Braude, AuI., and. J. Siemienski. 1960. Role of 'bacterial urease in experimental pyelonephritis. J. Bacteriol. 80:171-179. Gerlach, G.-F., S. Class. and W. A, Nichols. 1988. Characterization of the genes encoding urease activity of Klebsiella pneumoniae. FEMS Microbiol. Lett. 50:131-135. Grant, R.B., J.L. Penner, J.N. Hennessy, and B.J. Jakowski. 1981. Transferable urease activity in Pravidencia stuartii. J. Clin. Microbiol. 13:561-565. Griffith, D.P., D.M. Musher, and C. Itin. 1976. Urease: the primary cause of infection-induced urinary stones. Invest. Ural. 13:346-350. Mausinger, R. P. 1986. Purification of a nickel-containing urease from the rumen anaerobe Selenamonas ruminantium. J. Biol. Chem. 261:7866-7870. Hausinger, R. P. 1987. Nickel utilization by microorganisms. Microbiol. Rev. 51:22-42. Mu, L.-T., E. B. Nicholson, B. D. Jones, M. J. Lynch, and R. L. T. Mobley. 1990. Morganella morganii urease: Purification, characterization, and isolation of gene sequences. J. Bacteriol. 172:3073-3050. Jones, B. D., and M. L. T. Mobley. 1987. Genetic and biochemical diversity of ureases of Proteus, Pravidencia, and Morganella species isolated from urinary tract infection. Infect. Immun. 55:2198-2203. Jones, B. D., and M. L. T. Mobley. 1988. Proteus.mirabilis urease: genetic organization, regulation, and expression of structural genes. J. Bacteriol. 170:3342-3349. Rim. S.-D., and J. Spizizen. 1985. Mblecular cloning and expression of Bacillus pasteurii urease gene in Escherichia coli. Mar. J. Appl. Microbiol. Bioeng. 13:297-302. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature(London). 227:680-685. 15. l6. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 38 Lowry, G.R., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. MacLauren, D.M. 1969. The significance of urease in Proteus pyelonephritis: a histological and biochemical study. J. Pathol. 97:43-49. Maniatis, T., E.F. Frisch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Mobley, M.L.T., G.R. Chippendale, M.B. Fraiman, J.N. Tenney, and J.W. Warren. 1985. Variable phenotypes of Pravidencia stuartii due to plasmid encoded traits. J. Clin. Microbiol. 22:851-853. Mobley, R. L. T., B. D. Jones, and A. E. Jerse. 1986. Cloning of urease gene sequences from Pravidencia stuartii. Infect. Immun. 54:161-169. Mobley, H. L. T., and J. W. Warren. 1987. Urease-positive bacteriuria and obstruction of long-term urinary catheters. J. Clin. Microbiol. 25:2216- 2217. Mulrooney, s. B., M. J. Lynch, 1!. L. T. Mobley, and R. P. Hausinger. 1988. Purification, Characterization, and Genetic Organization of Recombinant Pravidencia stuartii Urease Expressed by Escherichia coli. J. Bacteriol. 170:2202-2207 . Mulrooney, S. B., M. S. Pankratz, and R. P. Msusinger. 1989. Regulation of gene expression and cellular localization of cloned Klebsiella aerogenes (K. pneumoniae) urease. J. Gen. Microbiol. 135:1769-1776. Rosenstein, I. J. M., and J. M. T. Hamilton-Miller. 1984. Inhibitors of urease as chemotherapeutic agents. Crit. Rev. Microbiol. 11:1-12. Rubin, R. B., N. E. Tolkoff-Rubin, and R. S. Cotran. 1986. Urinary tract infection, pyelonephritis, and reflux neuropathy, p. 1085-1141. In The Kidney, vol. 11. 3rd ed., Brenner, B.M., and F.C. Rector, Jr. (eds.), W.B. Saunders Company, New York. Samtoy, B. and M. M. DeBeukelaer. 1980. Ammonia encephalopathy secondary to urinary tract infection with Proteus mirabilis. Pediatrics. 15:294-297. Smith, C. J., R. B. Bespell, and M. P. Bryant. 1980. Ammonia assimilation and glutamate formation in the anaerobe Selenomonas ruminantium. J. Bacteriol. 141:593-602. Todd, M. J., and R. P. Rausinger. 1987. Purification and characterization of the nickel-containing multicomponent urease from Klebsiella aerogenes. J. Biol. Chem. 262:5963-5967. Warren, J. W. 1986. Pravidencia stuartii: a common cause of antibiotic resistant bacteriuria in patients with long-term indwelling catheters. Rev. Infect. Dis. 8:61-67. 29. 30. 31. 39 Warren, J. W., J. B. Tenney, J. M. Boopes, R. L. Muncie, and W. C. Anthony. 1982. A prospective microbiologic study of bacteriuria in patients with chronic indwelling urethral catheters. J. Infect. Dis. 146:719-723. Weatherburn, M. W. 1967. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 39:971-974. Wilkinson. G. N. 1961. Statistical estimations in enzyme kinetics.Biochem. J. 80:324-332. CHAPTER 3 Regulation of Gene Expression and Cellular Localization of Cloned Klebsiella aerogenes (K. pneumoniae) Urease 40 41 ABSTRACT The genes for Klebsiella aerogenes (K. pneumoniae) urease were cloned and the protein was overexpressed (up to 18% of total protein consisted of this enzyme) in several hosts. The restriction map of the DNA encoding urease and the regulation of enzyme expression directed by the recombinant plasmid are distinct from other cloned ureases. Nickel concentration did not affect urease gene expression, as demonstrated by the high levels of apoenzyme measured in cells grown in nickel-free media. However, nickel was required for urease activity. The overproducing recombinant strain was used for immunogold electron microscopic localization studies to demonstrate that urease is a cytoplasmic enzyme. 42 INTRODUCTION Nickel-containing ureases (EC 3.5.1.5), which hydrolyze urea to ammonia and carbon dioxide, play an important role in nitrogen metabolism of plants and microorganisms (11). One of the best-studied bacterial ureases is that from Klebsiella aerogenes [currently Klebsiella pneumoniae (30)]; its regulation has been characterized (8,20) and the three-subunit enzyme has been purified and shown to contain four nickel ions per native molecule (35) . Our efforts are geared toward elucidating the structure and function of the K. aerogenes urease nickel center by using chemical, biophysical, and spectroscopic approaches which require large amounts of enzyme. Typical yields of urease are only 0.1 mg per liter of culture. Therefore, we sought to clone the K. aerogenes urease genes and to define conditions needed to optimize enzyme overexpressian. In addition, the effect of nickel concentration on urease activity and expression was characterized and the cellular localization of the recombinant urease was defined . MATERIALS AND METHODS Gene cloning. K. aerogenes CG253 was obtained from Boris Magasanik and Alexander Ninfa (Massachusetts Institute of Technology). Chromosomal DNA of K. aerogenes was isolated (26) and partially digested with Sau3A to yield fragments approximately 40 kb in length. After phenol extraction and ethanol precipitation, the digestion mixture was ligated to Banal-cleaved, phosphatase-treated, cosmid vector pWH4 (12) and the resulting DNA was packaged into lambda phage by using an in vitro packaging system (Boehringer Mannheim) according to the manufacturer's instructions. The 43 phages were used to transfect Escherichia coli strain VCSZS7 (Strategene) and kanamycin-resistant (50 pg ml") colonies were screened on urease indicator plates, which consisted of ammonia-free M9 minimal agar (21) adjusted to pH 6.8 and supplemented with 10% (v/v) LB medium, 20 mM urea, 20 pg phenol red m1”1 and 1 ml trace mineral solution 1’1 (32). One of the 102 colonies tested positive, as shown by the development of a red halo after 24 h. The cosmid which encoded urease (designated pKAUl) was isolated (14) and transformed into E. coli D111 (10); this plasmid conferred bath kanamycin-resistance and urease-positive phenotypes. Purified cosmid pKAUl was digested with BamHl, and the fragments were ligated into BamHl-cleaved vector pBR328 (33). One ampicillin-resistant, tetracycline-sensitive transformant was positive on urease indicator plates, and was found to contain a 10 kb insert in a plasmid derivative of pBR328 which was termed pKAU2687. Restriction fragments of the K. aerogenes insert in pKAU2687 were isolated (7) , subcloned into vector pUC8 (36) , transformed into E. coli JMlOl (25) and screened on urease indicator plates. Assays. Culture samples of cells grown in various media (0.5 ml) were centrifuged for 2 min in a microcentrifuge at 4'C, washed twice with 10 mM potassium phosphate, 1 mM EDTA, 1 mM _2-mercaptoethanol buffer (pH 7.5) and resuspended in the same buffer plus 0.5 mM phenylmethanesulphonyl fluoride. Cells were disrupted with a Fisher Sonic Dismembranator (micro probe) using three 20 s bursts at 30% power. Crude cell extracts were then centrifuged 15 min and the supernatant solutions were assayed for urease activity by converting released ammonia to indophenol, which was quantified spectrophotometrically (35) . Protein was assayed by the Lowry method, with bovine serum albumin as the standard. 44 Nickel dependence studies. The effect of nickel concentration on the expression of recombinant urease protein and enzyme activity was determined by growing cultures to late exponential phase in ammonia-free MOPS minimal medium (29) containing defined nickel levels. Glutamine (10 mM) was used as the sole nitrogen source in order to derepress urease (see below); however, under these conditions urease activity was not required for microbial growth. Immunological methods. Antibodies that recognized. urease were generated in a New Zealand rabbit after injection with homogeneous enzyme, and the IgG fraction.‘was purified from serum (22). For immunoblot analyses, samples were denatured, electrophoresed on an sodium dodecyl sulfate-10 to 15% polyacrylamide gradient gel (17), and blotted onto nitrocellulose. The blot was probed ‘with anti-K. aerogenes urease antibodies and developed by using anti-rabbit IgG-alkaline phosphatase conjugates (4). Immunagold electron microscopy. Wild-type K. aerogenes and K. aerogenes(pKAUl9) (see below) were grown to stationary phase in ammonia- free MOPS medium supplemented with 10 mM arginine plus 100 pM NiClz. After centrifugation, the cells were washed once in 10 mM potassium phosphate, 1 mM EDTA (pH 7), and fixed in 0.1 M potassium phosphate (pH 7.2) containing 1% (v/v) glutaraldehyde for 30-60 min at room temperature. The fixed cells were resuspended in 1% (w/v) Nobel agar, dehydrated in ethanol, and embedded in Lowacryl K4M (1). Polymerization was carried out for 2 d at room or cold room temperatures under UV irradiation. Thin sections were cut by using an LKB'Ultratome III microtome, and placed on Butvar B-98-coated nickel grids. Sections were floated first on a drop of TBST (Tris-buffered saline, pH 7.4, with 0.05% Tween 20) for 5 min and 45 transferred to 1% (w/v) bovine serum albumin in TBST for 15 min in order to block non-specific sites. The samples were transferred to the anti- urease IgG (35 pg ml'1) in TBST for l h, washed three times for 5-15 min each in TBST, and floated on gold particles that were attached to anti- rabbit IgG (15 min, Jansen) for l h (3) . After washing in TBST and H20, the samples were stained with uranyl acetate and lead citrate. Sections were observed with a Philips CM-10 electron microscope. RESULTS AND DISCUSSION Gene cloning. The K. aerogenes urease genes were localized to a 10 kb DNA fragment which possesses the restriction map shown in Fig. 1. This region included two Bali fragnents of 7.0 and 3.0 kb, indicating that incomplete digestion had occurred in the subcloning of pKAU2687. When cloned individually, neither fragment conferred urease activity, indicating that sequences spanning the internal BamHl site are required. A PvuII fragnent (6.2 kb) overlapped this Bani-ll site, yet no urease activity was detected in transformations containing this subcloned fragment (pKAUl3) . Partial Sau3A digestion of pKAUlS was used to generate a clone containing 5.7 kb (pKAUl7) which conferred urease activity. Urease genes have been cloned from Bacillus pasteurii (16), a urease-positive E. coli (5), Proteus mirabilis (13,37), and Pravidencia stuartii (27,28). The restriction maps of these clones all differ significantly from that of the K. aerogenes urease gene fragnent. Klebsiella pneumoniae urease genes also have been cloned by Gerlach et al. (9); however, the mechanism of gene regulation and properties of the recombinant enzyme were not studied. The restriction pattern, and the 46 ups pBR328 r ”V mm Elg- pKAUlS pKAU2687 =3 ._ El 3 UREASE l//’ / ACT—II—lTY ----- pKAU11 '- \SQJ ELLA“ ~—S_st| ll QQI BamHl Se" ism ELLA“ ----- pKAU12 _ ----- pKAU13 pKAU17 pKAU19 + 1 kb ____ Fig. 1. Restriction map and summary of cloned K. aerogenes urease gene frsgsents. A restriction map is presented of a 10 kb DNA fragnent containing the K. aerogenes urease genes. HindIII, EcoRI, and Xbal did not cleave this fragment. Subclones of the K. aerogenes DNA fragment were generated and tested for the presence (+) or absence (-) of urease activity based on results from indicator plates. 47 associated polypeptide sizes for the K. pneumoniae urease genes studied by Gerlach et a1. (9) differ from that of the K. aerogenes DNA cloned in the present study. Urease regulation. To increase their expression, the cloned urease genes were transformed into three hosts and the enzyme levels monitored under varied growth conditions. The 5.7 kb fragment was transferred from pUCB to pBR328, which had more desirable antibiotic markers: pKAUl7 was digested with EcoRI and HindIII, end-filled with polymerase I Klenow fragnent, and ligated into SmaI-digested pBR328 to yield pKAU19. This plasmid was transformed into E. coli DHl, K. aerogenes CG253 (10), and into Salmonella typhimurium LT-2 (19). Since pKAUl9 carried sequences homologous to the K. aerogenes host (which could lead to plasmid loss due to homologous recombination), colonies obtained directly from the transformation were used to inoculate starter cultures for overnight incubation. Specific activities were determined for cells of these strains and wild-type K. aerogenes grown to early exponential and stationary phase in different media that contained individual nitrogen sources; typical values are shown in Table 1. For comparison, pure K. aerogenes urease has a specific activity of 2200 pmol urea min'1 mg'1 (35). Among the hosts tested, urease activity was regulated by nitrogen repression (20) as originally reported in the wild-type microbe (8). Low enzyme levels were observed during growth in nitrogen-rich medium (LB, or MOPS+CA+N, Table 1), whereas nitrogen limitation (MOPS+N, Gln, or Arg) led to derepression of urease activity. Enzyme levels exceeded 7% of the soluble protein in stationary-phase cultures of strains containing the cloned urease genes, and accounted for 18% in the case of S. typhimurium grown in MOPS+Arg calculated from the data in Table 1. However, the 48 masseuse use .xmnv whd now own who: .Hoowcoamaouoaso one eomwz zoooa outwnucoo .Ho no roman no not» an ooooamou mo3 sagasdon Heuocwa ooouu can one .chADouucoosoo oouoowosw can no noouson savanna: msofiuo> ma ooooamou one momma umoa cw oouuwso on: aver: "mcoauaoaueeoe measoaaou on» sea; own: was Anna assume fineness mac: 0 .ocwcwmuo zaoa .mu< anofioe ocfiaouoo.nm.o .40 «mafiaousam mad mHN mac: amend muocowuoum zaoa .cHo “anvmz SEoH .z “Amv assume «couuomioausn .mn uncofiuow>ounn< u uno>uoz no ofiuu as aloow< n .Hnmaaicwa can: sodas: :« oounoumxo one nude: a who: hOH mac: H.m N.HH v.0 OmQOZ v.H m.~ d.o amend susoum Hoautotomxm Hmnpasda are “Hoe .m Hmabdxnm asfiusfifinnhu .m Hmdbdxmg notomauoe .M as nocomouoo .x muaudzo wanton: mmmmmmmmm Mdflmwmdem utotfinaooom mo tawnuoumxm .H manna 49 highest activity per ml of culture was obtained for K. aerogenes(pKAUl9). Regulation of other recombinant ureases has only been reported for urea- inducible, nitrogen-repressible Pravidencia stuartii (28) and urea- inducible Proteus mirabilis (13,37). Characterization of the urease made by strains containing pKAU19. Crude cell extracts of E. coliDHl(pKAUl9) and S. typhimurium(pKAUl9) were examined by using immunoblot analysis (Fig. 2). Three urease subunits of the expected size were shown to be expressed in each case. Furthermore, enzyme purified.from.K..aerogenes(pKAUl9) was demonstrated to be identical to wild-type enzyme in subunit composition, nickel content, specific activity, and inhibitor sensitivity (data not shown). Routine purification of urease is now carried our from stationary-phase cultures of K. serogenes(pKAUl9), resulting in 100- to ZOO-fold increased in yield over wild-type levels. These results demonstrate that urease-processing or nickel-insertion activities, if'required, are encoded either on the 5.7 kb pKAU19 fragment or in the heterologous hosts. Effects of nickel concentration on urease gene expression and urease activity. Derepressed, stationary-phase K. aerogenes(pKAUl9) cultures grown in media of different nickel concentrations yielded identical intensities of anti-urease antibody crass-reactive material (Fig. 3). Hence, nickel does not affect expression of urease in strains with this plasmid. The urease activities, however, were greatly affected by nickel concentration, and 100 to 200 pM nickel was required for maximum activity (Fig. 3). The high requirement: may reflect the ability of' medium components to bind nickel. The production of inactive urease apoenzyme by this enteric bacterium is similar to the case of soybean, where apoenzyme was shown to be synthesized in the absence of nickel (38). Furthermore, 50 U—" 7 --92,500 '—"I " T- ——66,200 ——45,000 I. --- —-—31,000 A -—21,500 <. 1 f ——14,400 0% A ' --4 ! 1' l .. Lm/~—-—/o - ' M Fig. 2. Immunoblot analysis of recombinant urease expressed in E. coli DHl and S. typhimurium. Samples of crude extracts of E. coli DH1(pKAU19) (lane 1) and S. typhimurium(pKAUl9) (lane 2) containing 0.1 units (50 ng) of urease were analyzed. A standard of purified K. aerogenes urease (lane 3) and M, standards (lane 4) were also run and stained for total protein with Amido black. The migration position of M, markers (phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, lysozyme; Bio-Rad) are indicated to the right of the figure. 51 I (a) _ 160 's E, 120 g: Z 25 80 O 5; ti .0 m B O 2 3‘ 1 l L l l l 1 l A l r l g1 ; I so 100 150 200 250 300 350 400 [N1]. MM (b) 66.200— — — -— — — -__. .— ’ l 2 3 4 5 5 f' a Q Fig. 3. Effect of nickel concentra'ti’on‘ofi r'fimbinant urease expression and activity. (a) Urease specific activity was determined for K. aerogenes(pKAUl9) after growth in media containing the indicated nickel concentrations. (b) The level of urease protein expression was quantified for each sample by innnunoblot staining. Lanes 1 and 2 are Amido black- stained M, standards and purified urease, respectively. Lanes 3-9 represent the samples (2 pg total protein) obtained from cultures containing 0, 12.5, 25, 50, 100, 200, and 400 pM nickel respectively. 52 the algae Phaeodactylum tricornutum and Tetraselmis subcordiformis (31) , the cyanobacterium Anabaena cylindrica (18) , and the purple sulphur bacterium Thiocapsa roseopersicina (2) have also been suggested to synthesize urease apoenzyme in the absence of nickel. The microbial studies were based on reconstitution of urease activity for cells grown under nickel-free conditions when nickel was added, even in the presence of protein-synthesis inhibitors. In contrast to the lack of nickel- dependent regulation of ureases, nickel-containing hydrogenases from Bradyrhizobium japonicum and Alcaligenes latus have been shown to exhibit nickel - dependent express ion (6 , 34) . Immmogold localization. The urease in cells of K. aerogenes containing pKAU19 is a cytoplasmic enzyme as shown by immunogold electron microscopy localization (Fig. 4). This result is consistent with the observed enzyme behavior during purification. Urease from wild-type K. aerogenes behaved identically to the recombinant enzyme during isolation, which is consistent with the reported cytoplasmic location from cell fractionation studies (8). Wild-type cells which contain 1-5 M urease [calculated as in (15)] , were insufficiently labeled by the immunogold technique to allow localization, as expected from the findings of Kellenberger et a1. (15), who state that a cytoplasmic protein cannot be detected by this method at concentrations less than 10-100 pM. Our results contrast with two previous electron microscopic localization studies involving urease from a Staphylococcus species (23) and from Proteus mirabilis (24). The earlier workers utilized tetraphenylboron, a compound which reacts with ammonia to form a precipitate; ammonia was exchanged for electron-dense silver ions to allow visualization by electron microscopy. after thin ' sectioning. The 53 Fig. 4. Immunogold localization of recombinant K. aerogenes urease. (a) Thin sections of K. aerogenes(pKAUl9) expressing recombinant urease (274 pmol urea hydrolyzed min'1 mg”) were reacted with anti-urease antibodies and labeled with anti-rabbit IgG-gold particles. Urease was localized to the cytoplasmic portion of the cell. (b) Similar experiments were carried out with wild-type K. aerogenes (2.0 pmol urea hydrolyzed.minq-mg”). Bar, 0.2 pm. 54 precipitated metal was observed on the membrane of the Gram-positive Staphylococcus sp. (23) or in the periplasmic space and on the outer membrane of the Gram-negative P. mirabilis (24). The discrepancy between these earlier studies and our findings may be due to the inability of tetraphenylboron to cross the cytoplasmic membrane, thus it could only react with external ammonia. .Acknowledgements. I wish. to thank Stuart Pankratz for sample preparation and electron microscopy used in the immunogold labeling experiments. 10. ll. 12. 13. 55 REFERENCES Armbruster. B. L., E. Carlemalm, R. Chiovetti, R. M. Garavity, J. A. Robot, E. Kellenberger, and W. Villiger. 1982. Specimin preparation for electron microscopy using low temperature embedding resins. Journal de Microscopic. 126:77-85. Bast, E. 1988. Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae). Arch. Microbiol. 150:6- 10. Bendayan, M. 1984. Protein A-gold electron microscopic immunochemistry: methods, applications, and limitations. J. of Electron Microscopy . 1:243 - 270 . Blake, M. 8., K. H. Johnston, G. L. Rullell-Jones, and E. C. Gotschlich. 1984. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on western blots. Anal. Biochem. 136:175-179. Collins, C. M., and S. Falkow. 1988. Genetic analysis of an Escherichia coli urease locus: evidence of DNA rearrangement. J. Bacteriol. 170:1041-1045. Doyle, C. M., and D. J. Arp. 1988. Nickel affects expression of the nickel-containing hydrogenase of Alcaligenes latus. J. Bacteriol. 170:3891-3896. Dretzen, C. M., P. Bellard, P. Sassone-Corse, and P. Chambon. 1981. A reliable method for recovery of DNA fragnents from agarose and acrylamide gels. Anal. Biochem. 112:295-298. Friedrich, B. and B. Magasanik. 1977. Urease of Klebsiella aerogenes: control of its synthesis by glutamine synthetase. J . Bacterial. 131:446-452. Gerlach, G.-F., S. Clegg, and W. A. Nichols. 1988. Characterization of the genes encoding urease activity of Klebsiella pneumoniae. FEMS Microbiol. Lett. 50:131-135. Harmahan. D. 1983. Studies on transformation of Eschericia coli with plasmids. J. Mol. Biol. 166:557-580. Heusinger, R. P. 1987. Nickel utilization by microorganisms. Microbiol. Rev. 51:22-42. Herrera, A., J. Elhai, B. Hahn, and C. P. Walk. 1984. Infrequent cleavage of cloned Anabaena variabilis DNA by restriction endonucleases from A. variabilis. J. Bacteriol. 160:781-785. Jones, B. D., and B. L. T. Mobley. 1988. Proteus mirabilis urease: 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 56 genetic organization, regulation, and expression of structural genes. J. Bacteriol. 170:3342-3349. Redo, C. I., and S.-T. Liu. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145:1365-1373. Kellenberger, E. , M. Durrenberger, W. Villiger, E. Carlemslm, and M. Wurtz. 1987. The efficiency of immunolabel on Lowicryl sections compared to theoretical predictions. J. of Histochem. Cytochem. 35:959-969. Kim, S.-D. , and J. Spizizen. 1985. Molecular cloning and expression of Bacillus pasteurii urease in Escherichia coli. Korean J. Appl. Microbiol . Bioeng . 13: 297 - 302 . Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriOphage T4. Nature(London) . 227:680-685. Mackerras, A. M., and G. D. Smith. 1986. Urease activity of the cyanobacterium Anabaena cylindrica. J. Gen. Microbiol. 132:2749- 2752. MacLechlan, P. R., and K. E. Sanderson. 1985. Transformation of Salmonella typhimurium with plasmid DNA: differences between rough and smooth strains. J. Bacteriol. 161:442-445. Magasanik, B. 1982. Genetic control of nitrogen assimilation in bacteria. Ann. Rev. Gen. 16:135-168. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N .Y. McKinney, M. M., and A. Parkinson. 1987. A simple non- chromatographic procedure to purify immunoglobulins from serum and ascities fluid. J. Immunol. Meth. 96:271-278. McLean, R. J. C., K.-J. Chang, W. D. Gould, J. W. Costerton. 1985. Cytochemical localization of urease in a rumen Staphylococcus sp. by electron microscopy. Appl. Environ. Microbiol. 49:253-255. McLean, R. J. C., K.-J. Chang, W. D. Gould, J. C. Nickel, and J. W. Costerton. 1986. Histochemical and biochemical urease localization in the periplasm and outer membrane of two Proteus mirabilis strains. Can. J. Microbiol. 32:772-778. Messing, J., R. Crea, and P. H. Seaburg. 1981. A system for shotgun DNA sequencing. Nuc. Acids Res. 9:309-321. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N .Y. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 57 Mobley, H. L. T., B. D. Jones, and A. E. Jerse. 1986. Cloning of urease gene sequences from Pravidencia stuartii. Infect. Immun. 54:161-169. ‘Mulrooney, S. B., M.J. Lynch, H. L. T. Mobley, and R. P. Hausinger. 1988. Purification, characterization, and genetic organization of recombinant Pravidencia stuartii urease expressed by Escherichia coli. J. Bacteriol. 170:2202-2207. Neidhardt, F. C., P. L. Bloch, and D. F. Smith. 1974. Culture media for enterobacteria. J. Bacteriol. 119:736-747. Orskov, I. 1974. Genus VI. Klebsiella Trevisan 1885. In Bergey's manual of determinative Bacteriology, 8th ed. R. E. Buchanan and.N. E. Gibbons, eds., pp. 321-324. Rees, T. A. V., and I. A. Bekheet. 1982. The role of nickel in urea assimilation by algae. Plants 156:385-387. Smith C. J., R. B. Hespell, and M. P. Bryant. 1980. Ammonia assimilation and glutamate formation in the anaerobe Selenomonas ruminantium. J. Bacteriol. 141:593-602. Soberon,.x., L. Covarrubias, and F. Boliver. 1980. Construction and characterization of new cloning vehicles, IV. Deletion derivatives of pBR322 and pBR325. Gene 9:287-305. Stults, L. W., W. A. Spray, and R. J. Meier. 1986. Regulation of hydrogenase biosynthesis by nickel in Bradyrhizobium japonicum. Arch. Microbiol. 146:280-283. Todd, M. J., and R. P. Hausinger. 1987. Purification and characterization of the nickel-containing multicomponent urease from Klebsiella aerogenes. J. Biol. Chem. 262:5963-5967. Vieira, J., and J. Messing. 1982. The pUC plasmids, and M13mp7- derived system for insertion. mutagenesis and sequencing *with universal primers. Gene. 19:259-268. Wklz, S. B., S. K. Wray, S. I. Hull, and.R. A. Hull. 1988. Multiple proteins encoded within the urease gene complex of Proteus .mirabilis. J. Bacteriol. 170:1027-1033. Winkler, R. G., J. C. Polacco, D. L. Eskew, and R. M. Walsh. 1983. Nickel is not required for apourease synthesis in soybean seeds. Plant Physiol. 72:262-263. CHAPTER 4 In Vivo Reconstitution of Klebsiella aerogenes urease apo-enzyme 58 59 Abstract Recombinant Klebsiella aerogenes cells overexpressing urease protein were grown in the absence of nickel and reactivation of urease activity upon restoration of nickel to the growth medium was examined in control cells and cells treated with protein synthesis and energy uncoupling inhibitors. Control cultures gradually gained activity after nickel addition primarily due to new urease synthesis, and this increase continued for several hours after culture growth had ceased. In the presence of a protein synthesis inhibitor, nickel addition also led to an increase in urease activity, although at reduced rates when compared to untreated cultures. This activity arose from activation of pre-formed apo- protein. Energy dependence of nickel incorporation into spa-urease was demonstrated by the failure to generate urease activity in dicyclohexyl- carbodiimide and dinitrophenol-treated cells. Sonicsted cells also lost the ability to activate urease spa-enzyme. These results indicate that nickel is incorporated into pre-formed spa-enzyme in an energy-dependent process which is destroyed by cell disruption. 60 INTRODUCTION Urease, a nickel-containing enzyme found in many plants and microorganisms, hydrolyzes urea to yield carbonic acid and ammonia (1,14) . Whereas the role of urease in plants is poorly understood (23), the microbial enzyme plays important roles in human and animal pathogenic states, in ruminant metabolism, and in environmental transformations of certain nitrogenous compounds ( 14) . The best characterized plant urease is that isolated from jack bean; it was the first enzyme ever crystallized (l8) and the first enzyme shown to possess nickel (4). This hexameric protein contains two Ni ions per subunit (M. - 90,770), the sequence of which was recently reported (19). The most studied bacterial urease is that from Klebsiella aerogenes (currently K. pneumoniae) which possesses three subunits [Mrs - 72,000 (a), 11,000 (E), and 9,000 (r)] in an ozfiu, stoichiometry (20). The native enzyme contains two catalytic sites, each of which is associated with 2 Ni ions (21). The genes for K. aerogenes urease were recently cloned and overexpressed such that urease accounted for over 10% of the cellular protein (Chapter 3; 16). Although the enzyme requires Ni for activity, no Ni-dependent regulation of gene expression was observed. This chapter describes the in viva reconstitution of spa-urease in K. aerogenes cells. Experiments were designed to ascertain whether Ni ions are incorporated into the enzyme during synthesis, or if the Ni is incorporated into pro-formed spa-enzyme. Possible energy requirements of Ni-incorporation are also explored. 61 ‘MATERIALS AND METHODS Bacterial strains and growth conditions. K. aerogenes 06253 was transformed with plasmid pKAUl9 (16), which possesses the urease genes from this microorganism. The recombinant bacterium was grown at 37°C in MOPS-glutamine medium (16) containing 30 pg/ml chloramphenicol. Ni-free cultures were grown without added Ni, and puratronic grade metal salts (Alpha Products, Danvers, MA) were then added. Assays. Urease activity was determined by monitoring the rate of ammonia release from urea by formation of indophenol, which was measured at 625 nm (22). The assay ‘buffer consisted of' 25 mMI HEPES (N-2- hydroxyethylpiperszine-N'-2-ethanesu1fonic acid; Sigma Chemical Co., St. Louis, MO), 50 mM urea, and 0.5 mM EDTA (pH 7.75). One unit of urease activity is defined as the amount of enzyme required to hydrolyze l pmole of urea per min at 37°C under the assay conditions described above. Protein was measured by the method of Lowry et a1. (10) with bovine serum albumin as the standard. In vivo activation of apo-urease. Early exponential phase Ni-free cultures of K. aerogenes CG253(pKAUl9) were treated with an inhibitor of protein synthesis (50 pg/ml spectinomycin), an uncoupler of electron transport phosphorylation (2 mM dinitrophenol), or an.ATPase inhibitor [5 mM (DCCD)]. In the latter case, 10 mM ammonium chloride was used as the nitrogen source and 20 mM malate was used as the sole carbon source to preclude the possibility of substrate level phosphorylation (6). After 30 minutes, NiSO. was added (50 pM final concentration) to these treated cultures and to an untreated Ni-free control. Portions of the cultures were taken at several time points, washed, disrupted by sanitation, and 62 assayed for protein and urease activity as previously described (15). To investigate whether protein synthesis was completely abolished in the presence of spectinomycin, 1 mM leucine (supplemented with 1 pCi/ml of l- [3,4,5-3H(N)]-leucine at 153 Ci/mmole; New England Nuclear, Boston, MA) was added to control and inhibited cultures, and aliquots were removed at selected timepoints. The aliquots were mixed with an equal volume of 30% trichloroacetic acid, placed on ice for 30-60 min, heated to 80°C for 30 min, and filtered on thtman GF/C filters. After washing and drying, the filters were counted in a toluene based scintilstion solution. RESULTS Addition of Ni to Ni-free K. aerogenes CG253(pKAU19) cultures led to an increase in urease specific activity which may represent both newly synthesized enzyme and activation of previously formed spa-protein (Fig. 1). Following Ni addition there was a delay of 30 min, then urease activity continuously increased until two hours after protein synthesis ceased. This result is consistent with a slow incorporation of Ni into previously synthesized spa-enzyme. Furthermore, activation of pre-formed spa-urease was observed in cultures treated with spectinomycin, an inhibitor of protein synthesis. [am-leucine uptake studies were carried out (Fig. 10) to confirm that protein synthesis was fully inhibited in these cultures, e. g., that small amounts of protein turnover did not occur. Similar to the control culture, in viva activation of urease apo- protein was a very slow process and exhibited a 30 min lag. After 4 hr, the urease specific activity for spectinomycin-trested cultures was approximately 1/3 that of the control culture, perhaps due to non-specific effects of long-term exposure to the inhibitor. Similar results were 63 Fig. 1. In vivo reconstitution of urease spa-protein. Exponentially growing nickel-free cultures of K. aerogenes(pKAUl9) in MOPS-10 mM glutamine were treated with no inhibitor (0), 50 pM spectinomycin (I), or 2 mM dinitrophenol (A) followed after 30 min by addition of Ni (50 pM) as indicated by the arrow. Aliquots were assayed for urease specific activity (panel A), total protein (panel B), and level of protein synthesis (panel C). In the latter case, [am-leucine was added to the cultures where indicated and the incorporation of labeled leucine into trichloroacetic acid-precipitable protein was assessed. In addition, cells were grown in MOPS-10 mM NH.C1 with 20 mM malate as the sole carbon source (Panel D). The cultures were treated with no inhibitor (0), or with 5 mM DCCD (A). The low specific activities in the malate control culture are due to the non-optimal meditun conditions which were required in order to insure a minimum of substrate level phosphorylation. 64 240 180 120 “my 03 '39" 00"" room adv-5 j""""Y*1'v v 1 v If] vfi ‘fi: 8 8 ° 0 N O V o " e- — wdo co; l—QW t..le vaun sa'lowfi 150- ' V V t T-w w 1“ I v < m i? § 3 °§§§§§ how hum vaan $310M Nlaioud Inn on 100 9 MINUTES MINUTES 65 observed for wild-type K. aerogenes (not shown). In contrast, cells treated with dinitrophenol or DCCD gave no urease activity upon addition of nickel (Fig. 1). Furthermore, if the cells were disrupted prior to adding Ni no urease activity was generated. DISCUSSION Ni has recently been shown to be an essential trace metal for several microorganisms and is specifically incorporated into four types of Ni-dependent enzymes (7) . Although bacterial ureases contain tightly bound Ni at their active sites (14, 21), the mechanism by which Ni is incorporated into urease, the identity of the Ni ligands, the metallocenter structure, and the role for Ni are unclear. Purified K. aerogenes urease spa-enzyme could not be activated by simple addition of Ni ions, hence, a cellular factor may be required for incorporation of the metal center. Apo-urease synthesized in Ni-free K. aerogenes cultures could be activated by Ni addition to whole cells even in the presence of protein synthesis inhibitors; similar results have been previously reported for ureases from soybean (24), algae (17), a cyanobacterium (12), and purple sulfur bacteria (2). In contrast, apo- enzyme was not activated in dinitrophenol-or DCCD—treated cells. This result could be explained by an energy dependence for Ni transport or for Ni incorporation into protein. We prefer the latter explanation because spa-urease could not be activated in disrupted cells where Ni transport is not a consideration. The energy dependence of reconstitution is not simply a requirement for ATP as shown by experiments where MgATP was added to purified spa-protein or to Ni-free cell extracts: Ni addition did not yield urease activity (9). Other studies have also shown the inability to 66 generate urease activity by addition of Ni to disrupted, Ni—free cells of soybean (24), jack been (5), and a cyanobacterium (12). Preliminary studies in both plants and microorganisms are consistent with the requirement for urease-related accessory factors which activate spa-enzyme. In soybean for example, Meyer-Bothling et a1. (13) reported the isolation.of pleiotropic mutants that were totally defective in.urease activity yet expressed both urease isozymes. They concluded that the mutations, which mapped distant from the urease genes, led to a defect in a urease maturation factor. Similarly, a urease-deficient mutant of Aspergillus nidulans is thought to be defective in the production or incorporation of a nickel cofactor essential for urease activity (11) . The addition of 0.1 mM Ni restored the ability of this mutant fungus to grow on.urea and enhanced the urease activity to 5-8% of the wild-type levels, but Ni addition to cell extracts did not result in urease activity. Furthermore, the number of urease loci in Neurospora crassa (3) and Schizosaccharomyces pombe (8) exceeds that required for the urease structural genes. The function of the additional genes is unknown. but one could speculate that they may function in Ni incorporation or other maturation event. In this regard, DNA sequence analysis has established the presence of six genes in the K. aerogenes urease operon (Chapter 4), which is three more than is required to encode the urease subunits. 10. ll. 12. 13. 67 REFERENCES Andrews, R. R., R. L. Blakeley, and B. Zerner. 1988. Urease -- a Ni(II) metalloenzyme. In J. R. Lancaster, Jr. (ed.) The bioinorganic chemistry of nickel, 141-165. VCH Pub., Inc., New York, NY. Bast, E. 1988. Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatisceae). Arch. Microbiol. 150:6- 10. Benson, E. W., and H. B. Howe, Jr. 1978. Reversion and interallelic complementation at four urease loci in Neurospora crassa. Molec. Gen. Genet. 165:277-282. Dixon, N. E., C. Gazzola, R. L. Blakeley, and B. Zerner. 1975. Jack bean urease (EC 3.5.1.5). A metalloenzyme. A simple biological role for nickel? J. Am. Chem. Soc. 97:4131-4233. Dixon, N. E., C. Gazzola, C. J. Asher, D. S. W. Lee, R. L. Blakeley, and B. Zerner. 1980. Jack bean urease (EC 3.5.1.5). II. The relationship between nickel, enzymatic activity, and the “abnormal" ultraviolet spectrum. The nickel content of jack beans. Can. J. Biochem. 58:474-480. Fillingame, R. H. 1975. Identification of the adenosine 5’- triphosphate transducing system of Escherichia coli. J. Bacteriol. 124:870-883. Hausinger, R. P. l987.~ Nickel utilization by microorganisms. Microbiol. Rev. 51:22-42. Kingharn, J. E., and R. Fluri. 1984. Genetic studies of purine breakdown in the fission yeast Schizosaccharomyces pombe. Curr. Genet. 8:99-105. Lee, M., S. B. Mulrooney, and R. P. Hausinger. 1990. Purification, characterization, and in-vivo reconstitution of Klebsiella aerogenes urease apoenzyme. J. Bacteriol. 172: in press. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randell. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Mackay, E. M., and J. A. Pateman. 1980. Nickel requirement of a urease-deficient mutant in Aspergillus nidulans. J. Gen. Microbiol. 116: 249-251 . Mackerras, A. H., and G. D. Smith. 1986. Urease activity of the cyanobacterium Anabaena cylindrica. J. Gen. Microbiol. 132:2749- 2752. Meyer-Bothling, L. E., J. C. Polacco, and S. R. Cianzio. 1987. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 68 Pleiotrapic soybean mutants defective in both urease isozymes. Molec. Gen. Genet. 209:432-438. Mobley, H. L. T., and R. P. Hausinger. 1989. Microbial urease: significance, regulation, and molecular characterization. Microbiol. Rev. 53:85-108. Mulrooney, S. B., M. J. Lynch, H. L. T. Mobley, and R. P. Hausinger. 1988. Purification, characterization, and genetic organization of recombinant Pravidencia stuartii urease expressed by Escherichia coli. J. Bacteriol. 170:2202-2207. Mulrooney, S. B., H. S. Pankratz, and R. P. Hausinger. 1989. Regulation of gene expression and cellular localization of cloned Klebsiella aerogenes (K. pneumoniae) urease. J. Gen. Microbiol. 135 : 1769-1776 . Rees, T. A. V., and I. A. Bekheet. 1982. The role of nickel in urea assimilation by algae. Plants 156:385-387. Sumner, J. B. 1926. The isolation and crystallization of the enzyme urease. J. Biol. Chem. 69:435-441. Takashima, K. T. Suga, and G. Mamiya. 1988. The structure of jack bean urease. The complete amino acid sequence, limited proteolysis and reactive cysteine residues. Eur. J. Biochem. 175:151-165. Todd, M. J., and R. P. Hausinger. 1987. Purification and characterization of the nickel-containing multicomponent urease from Klebsiella aerogenes. J. Biol. Chem. 262:5963-5967. Todd, M. J., and R. P. Hausinger. 1989. Competitive inhibitors of Klebsiella aerogenes urease: mechanisms of interaction with the nickel active site. J. Biol. Chem. 264:15835-15842. Weatherburn, M. W. 1967. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 39:971-974. Winkler, R. G., D. G. Blevins, J. C. Polacco, and D. D. Randall. 1988. Ureide catsbolism in nitrogen-fixing legumes. Trends Biochem. Sci. 13:97-100. Winkler, R. G., J. C. Polacco, D. L. Eskew, and R. M. Walsh. 1983. Nickel is not required for apourease synthesis in soybean seeds. Plant Physiol. 72:262-263. 69 CHAPTER 5 Sequence of the Klebsiella aerogenes Urease Genes and Evidence for Accessory Proteins Facilitating Nickel Incorporation 70 ABSTRACT A 4.8-kilobase-pair region of cloned DNA encoding the genes of the Klebsiella aerogenes urease operon has been sequenced. A total of six closely-spaced open reading frames were found: ureA (encoding,s peptide of 11.1 kilodaltons, kDa), ureB (11.7-kDa), ureC (60.3-kDa), ureE (17.6-kDa), ureF (25.2-kDa), and ureG (21.9-kDa) . Immediately following the ureG gene is a putative rho-dependent transcription terminator. The three subunits of the nickel-containing enzyme are encoded by ureA, ureB, and ureC based on protein structural studies and sequence homology to jack bean urease. Potential roles for ureE, ureF, and ureG were explored by deleting these accessory genes from the operon. The deletion mutant produced inactive urease which was partially purified and found to have the same subunit stoichiometry and native size as the active enzyme, but contained no significant levels of nickel. The three accessory genes were able to activate spa-urease in viva when they were cloned into a compatible expression vector and co-transformed into cells carrying the plasmid containing ureA, ureB, and ureC. Thus, one or more of the ureE, ureF, or ureG gene products are involved in nickel incorporation into urease. 71 INTRODUCTION Urease (EC 3.5.1.5) was the first enzyme to be crystallized (37) as well as the first shown to contain nickel (6). Early studies involving urease made use of enzyme purified from jack bean; this homohexameric plant enzyme has continued to be extensively studied, as reviewed by Andrews et a1. (1). Recently, interest has focused on ureases from bacterial sources, which differ from the plant enzyme in subunit composition and native molecular weight (30). The urease of Klebsiella aerogenes (currently K. pneumoniae) is similar to those of several other bacteria; it contains 4 tightly-bound nickel ions distributed among two active sites per native molecule and consists of three subunits in an 0128.1. stoichiometry (39, 40). The genes for K. aerogenes urease were recently cloned, and the enzyme was overexpressed in E. coli (Chapter 3; 33). Inactive urease spa-enzyme was synthesized when recombinant cells were grown in the absence of nickel ion (33) , ruling out nickel-dependent transcriptional regulation. Subsequent addition of nickel to whole cells led to spa-enzyme activation, even after treatment with protein synthesis inhibitors (Chapter 4; 23). In contrast, purified spa-urease could not be activated in vitro by addition of nickel indicating that some cellular component may be required for nickel incorporation into the enzyme (23). This report presents the DNA sequence of the six open reading frames in the K. aerogenes urease operon. Three of these genes are shown to encode the urease subunits. The precise role of the remaining three genes is unknown; however, evidence is presented that these accessory genes function in activating urease ape-enzyme by incorporating nickel. 72 MATERIALS AND METHODS Bacterial strains and growth conditions. Escherichia coli strains JM109 (43) or XL-l-B (2), grown at 37°C in 2xYT medium (28), served as hosts for M13 clones used for sequencing. E. coli DHl (14), used as the recipient for all studies involving urease expression, was grown at 37°C in MOPS-glutamine medium supplemented with 100 pM NiSO, and appropriate antibiotics as previously described (33). DNA sequencing. All restriction enzyme digestions, end-fillings, and other common DNA manipulations, unless otherwise stated, were performed according to standard procedures (27, 34). Sequencing was carried out on a portion of the urease operon from the upstream SstI site to the downstream HindIII site of plasmid pKAUl7 (33), illustrated in Fig. 1. This fragment contains all of the urease operon genes but lacks urease activity due to partial deletion of the upstream promotor region. Portions of the SstI-HindIII fragment were cloned into phage vector M13mpl9 (43) and a series of unidirectional deletions were constructed for each strand by using exonuclease 111 according to the method of Henikoff (15). Phage DNA from selected deletion clones was purified (28) and sequenced with Sequenase enzyme (U.S. Biochemicals, Cleveland, OH) by using [358] dATP according to the manufacturer's instructions. Duplicate reaction sets were made for each clone: one with dGTP and the other with dITP. Sequencing gels consisted of 6% acrylamide (19:1 acrylamide:bisacrylsmide) and were either wedged shaped (0.4-1.2 mm) or straight (0.4 mm) , in which case they were run by making the bottom buffer reservoir 1 M sodium acetate before electrophoresis (35). Sequence analysis was carried.out for both strands. Alignments of overlapping sequence data were made by using the GENEPRO program (Riverside Scientific, Seattle, WA). Database searches and open 73 reading frame assignments were performed with the Wisconsin Genetic Computer Group software package version 6.1 (5) . The Profilesearch program was used to look for homologous proteins in the National Biomedical Research Foundation (NBRF) protein sequence database. The sequences determined here have been deposited in Genbank under the accession number (M36068) . Construction of plasmids pKAU601 and pKAU506. Plasmid pKAUl7 was partially digested with AatI, the DNA fragments were electrophoresed in 1% sgarose, and the band corresponding to a 1.5-kilobase-pair (kb) deletion was isolated by interception on DEAE paper (thtman DE81). The DNA was eluted, ethanol precipitated, recircularized by T4 DNA ligase, and transformed into E. coli DHl. The resulting plasmid, pKAU520, was digested by several restriction enzymes to verify the proper deletion. In order to take advantage of more appropriate antibiotic markers, the pKAU520 fragment was cleaved from the pUC8 vector by digestion with EcoRI and HindIII. After treatment with Klenow fragnent of E. coli DNA polymerase to produce blunt ends, the fragment was ligated into PstI cut and Klenow- treated pBR328 (36) to yield pKAU601 (Fig. 1). A second subclone was made by digesting pKAU2687 (33), a precursor to pKAUl7, with Basin. The resulting 2.9-kb fragment, which contains the ureE, ureF, and ureG region, was isolated as described above and ligated into BamHI-cleaved pMMB66HE (10, obtained from Michael Bsgdasarian, Michigan Biotechnology Institute). The desired recombinant plasmid was verified by restriction analysis and designated pKAU506 (Fig. l). Assays. Urease activity was measured by quantitating the rate of ammonia released from urea by formation of indophenol which was monitored at 625 nm (42). The assay buffer consisted of 25 mM HEPES (N-2- 74 I 2 3 4 4.8 l I l l l J E $5 9" {E3 g i 3 as- H * C "’ ~03” pKAU1 m5” ”7'? . . 7‘ ’77 7 : x” m : a ii: a E «is W ’////////z 'r‘ '7‘ n '1‘ ‘- 7' 7m E if: M "‘ T‘M' ‘weeseopenongenes Mr C 1}] F I E J m V0010? m m vector pUm vector Fig. 1. Structure of the urease operon and two subclones. The restrictionmap for pKAU17, containing the cloned urease operon, is shown along with two subclones derived from this plasmid. As described in the text and indicated at the bottom of the figure, the complete operon is comprised of six genes. The first three genes encode the urease structural subunits, whereas the function of the last three accessory genes may be related to nickel incorporation into urease. The structural genes and urease promoter region were subcloned into the ampicillin resistance gene of vector pBR328, yielding pKAU601. Similarly, the accessory genes were cloned under control of the tac promoter of expression vector pMMB66HE to give pKAU506 . 75 hydroxyethylpiperazine-N'-2-ethanesu1fonic acid; Signs Chemical Co. , St. Louis, MO), 50 mM urea, and 0.5 mM EDTA (pH 7.75). One unit of urease activity is defined as the amount of enzyme required to hydrolyze 1 pmole of urea per min at 37°C under the assay conditions described above. When urease activity was determined in cultures, cells were disrupted as previously described (33) . Protein content was determined by the method of Lowry et a1. (24) by using bovine serum albumin as the standard. SDS-Polyacrylamide gel electrophoresis. All gels for protein analysis were prepared by using the buffers of Lsenunli (21) and consisted of either a 15% polyacrylamide (acrylamide:bisacrylamide, 32:1) or s 10- 15% polyacrylamide gradient resolving gel with a 4.5% stacking gel. Gels were stained with Coomassie brilliant blue (Sigma) and scanned by using a Gilford Response spectrophotometer (Gilford Instrument Laboratories , Inc. , Oberlin, Ohio) at 540 nm. Purification and characterization of pKAU601-derivad urease. A 2- 1iter stationary phase culture of E. coli DHl containing plasmid pKAU601 was grown as described above. The cells were collected by centrifugation, resuspended in 80 mL PEB buffer (20 mM phosphate, 1 mM EDTA, 1 mM H- mercaptoethanol, pH 7.0) containing 0.5 mM phenylmethylsulfonyl fluoride, and sonicated 3 min at 50% duty and 30% power using a Branson Sonifier (Danbury, CT) equipped with a 0.5 in diameter tip. Cell debris was removed by sedimentation at 100,000 x g for 60 min at 4°C. Further purification of the pKAU601-derived urease from the cell extracts made use of room temperature chromatography on DEAE-Sepharose, Superase 6, and Mono-Q resins obtained from Pharmacia (Uppsala, Sweden) by using methods which were previously described for urease isolated from cells grown in the absence of nickel ion (23). Because the urease from this strain was 76 enzymatically inactive, its presence was assayed by SDS-polyacrylamide gel electrophoresis and by comparison to authentic urease enzyme. The nickel content of the urease preparation was assessed by using atomic absorption spectrometry as previously described (32) . RESULTS AND DISCUSSION Sequence analysis of the urease operon. The sequence of a 4.8-kb region encoding the urease operon is shown in Fig. 2. Analysis of the sequence revealed six open reading frames that are designated ureA, ureB, ureC, ureE, ureF, and ureG. These genes are all transcribed in the same direction and are predicted to encode peptides of 101, 106, 567, 158, 224, and 205 amino acids with M,- 11,086, 11,695, 60,304, 17,558, 25,221, and 21,943, respectively. The very close spacing of the genes results in 3 cases where the ribosome binding site for one gene overlaps the coding region of the previous gene. Furthermore, the end of ureB overlaps the beginning of ureG by 8 nucleotides. K. aerogenes urease is expressed under nitrogen limited conditions by the global Ntr system (9,26). Detailed characterization of the urease upstream regulatory region will be described separately (Markawicz, Mulrooney, and Hausinger, in preparation) , and the sequence is provided as an appendix to this thesis. P. mirabilis contains an additional urease gene (ureD) located immediately upstream of ureA (20); the ureD gene is thought to function in urea induction, a mode of regulation which does not occur in K. aerogenes. A potential rho-dependent transcriptional termination site has been identified immediately after the ureG gene. In addition, the first 75 amino acids of another open reading frame was detected starting at base 77 Fig. 2. Nucleotide sequence of the urease genes. The deduced amino acid sequences for the six open reading frames are shown for ureA (bp 264-566) , ureB (bp 576-896), ureC (bp 889-2592), ureE (2602-3078), ureF (bp 3080- 3754), and urea (bp 3763-4380). Putative Shine-Dalgarno sites are underlined and a possible rho-dependent transcription termination sequence is indicated by arrows. If 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 78 CTCTCGCCGAACGTCCCTGGGTCGGCACTTTGCTGTGCTATCCGGCTACCGATGCCCTGC TCGACGGGGTGCGCCACGCGCTGGCGCCGCTCGGTCTCTACGCCGGCGCCAGCCTGACCG ACCGCCTGCTGACGGTGCGTTTCCTCAGTGACGATAATCTGATTTGCCACCGGGTGATGC GCGACGTATGGCACTTTCTGCGCCCTCATCTCACCGGTAAATCTCCCGTACTTCCCCGAA TCTGGCTGACTTAAQAQAACCTTATGGAACTGACCCCCCGAGAAAAAGACAAGCTCTTGC M E L T P R E K D K L L L TGTTTACCGCCGCGCTGGTGGCGGAGCGTCGCCTGGCCCGCGGCCTGAAGCTCAACTATC F T A A L V A E R R L A R G L K L N Y P CGGACTCCGTGGCCCTGATCAGCGCCTTTATTATGGAAGGCGCTCGGGACGGCAAAAGCG E S V A L I S A F I M E G A R D G K S V TGGCCTCCCTGATGGAGGAAGGCCGTCACGTCCTGACCCGCGAGCAGGTGATGGAGCGCG A S L M E E G R H V L T R E Q V M E G V TCCCGCAAATCATCCCGGATATCCAGGTCGAAGCCACCTTCCCGGACGGCTCGAAGCTGG P E M I P D I Q V E A T F P D G S K L V TCACCCTTCACAACCCGATTATCTQAQQTAGCGCCATGATCCCCGGTGAATATCACCTTA T V M N P I I * M I P G E Y H V K AGCCCGGTCAGATAGCCCTGAATACCGGCCGGGCAACCTGTCGCGTGGTCGTTGAGAACC P C Q I A L N T G R A T C R V V V E N H ACGCCGATCGGCCGATTCAGGTCCGTTCGCACTACCATTTCGCCGAGGTTAACCCGGCGC G D R P I Q V G S H Y H F A E V N P A L TGAAGTTCGACCGTCACCAGGCCGCCGGCTATCGCCTGAATATCCCGGCGGGCACGGCGG K F D R Q Q A A G Y R L N I P A G T A V TACGCTTTGAACCCGGCCAGAAACGCGAGGTCGAGCTGCTGGCCTTCGCCGGTCACCGCC R F E P C Q K R E V E L V A F A C H R A CCGTCTTCGGCTTCCGCGGCCACGTCATGGGCCCTCTQQAQQTAAACGATGAGTAATATT V F G F R G E V M G P L E V N D E * M S N I TCACGCCAGGCCTATGCCGATATGTTCGGCCCCACCGTCGGCGACAAGGTGCGCCTGGCA S R Q A Y A D M F G P T V G D K V R L A GATACCGAGCTCTGGATCGAGCTCGACGACGATTTCACCACCTACGGCGAAGAGGTCAAA D T E L W I E V E D D L T T Y C E E V K TTCGGCCGCGGCAAAGTGATCCGCGACGGCATGGGCCAGGGACAGATGCTGGCCGCCGAC F G G G K V I R D G M C Q C Q M. L A A D 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 79 TGTCTCGACCTGCTGCTCACCAACGCGTTGATCCTCGATCACTGGGGGATCGTTAAGGCC C V D L V L T N A L I V D H W G I V K A GATATCGGCGTCAACGACGGCCGGATCTTCGCCATCGGCAAAGCCGGCAACCCCGACATC D I G V K D G R I F A I G K A G N P D I CAGCCCAACGTCACCATCCCCATCGGCCCTGCGACGGAACTGATCGCCGCCGAAGGAAAA Q P N V T I P I G A A T E V I A A E G K ATTGTCACCGCCGGCGGGATCGATACCCATATTCACTGGATCTGTCCGCAGCAGGCGGAA I V T A G G I D T H I H W I C P Q Q A E CAGGCCCTGGTCTCTGGCCTGACCACCATCGTCGGCGGCGGCACCGGCCCGGCCGCGGGC E A L V S G V T T M V G G G T G P A A G ACCCATGCCACCACCTGCACCCCGGGCCCGTGGTATATCTCACGCATGCTGCAGGCGGCC T M A T T C T P G P W Y I S R M L Q A A GACAGCCTGCCGGTCAATATCGGCCTGCTGGGCAAGGGAAACGTTTCTCAGCCGGATGCC D S L P V N I G L L G K G N V S Q P D A CTGCGCGAGCAGGTGGCCGCAGGCGTTATTGGCCTGAAGATCCATGAGGACTGGGGCGCC L R E Q V A A G V I G L K I H E D W G A ACCCCCGCGGCCATCGACTGTGCGTTAACCGTCGCCCATGAAATGGACATCCAGGTCGCC T P A A I D C A L T V A D E M D I Q V A CTGCACAGCCACACCCTGAATGAATCCGGTTTTGTGGAACACACCCTCGCCGCCATCGGC L M S D T L N E S G F V E D T L A A I G GGGCGCACCATCCACACCTTCCATACCGAAGGGGCCGGCGGCGGCCATGCGCCGGACATC C R T I H T F H T E G A G G G H A P D I ATCACCGCCTGCGCCCACCCGAACATTTTCCCGTCGTCCACCAACCCAACGCTGCCCTAC I T A C A M P N I L P S S T N P T L P Y ACCCTCAACACCATCGATGAACATCTCGATATGCTGATGCTCTGCCACCATCTGGACCCG T L N T I D E M L D M L M V C H H L D P GACATCGCCGAGGACCTGCCCTTTGCCGACTCGCGCATTCGCCGGCAAACCATCGCTCCG D I A E D V A F A E S R I R R E T I A A GAAGACCTCCTGCACCATCTCGGCGCCTTCTCGCTCACCTCCTCCGATTCCCAGGCCATG E D V L M D L G A F S L T S S D S Q A M GGCCGCCTCGGCGAAGTCATTCTCCCCACCTGGCAGGTGGCGCATCGCATGAAGGTGCAG G R V G E V I L R T W Q V A M R M K. V Q CCCGGAGCGCTGCCGGAGGACACCGGGGATAACGACAACTTCCGCGTGAAGCGCTACATC R G A. L .A E E T G D N D N F R V K R Y I CCCAAATACACCATCAACCCGGCGCTGACCCACGGCATCGCACACGAAGTCGGATCCATT .A K ‘Y T I N P A L T H G I A. H E V G S I 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 80 GAGGTCGCTAAGCTGGCTGACCTCGTGCTCTGGTCACCAGCCTTCTTCGCCGTGAAACCG E V G K L A D L V V W S P A F F C V K P GCCACCGTGATCAAACGCGCCATGATCGCCATCGCGCCGATGCGCGATATCAATCCCTCT A T V I K G G M I A I A P M G D I N A S ATTCCGACCCCCCAGCCGCTCCACTACCGCCCGATGTTTGGCCCGCTGGGCAGCGCCCGC I P T P Q P V B Y R P M F G A L G S A R CATCACTGCCGCCTCACCTTCCTGTCGCAGGCGGCGGCAGCCAATGGCGTTGCCGAGCGG HHCRLTFLSQAAAANGVAER CTCAACCTGCGCAGCGCCATCCCCGTGGTGAAAGGCTGCCCTACGGTGCAGAAAGCCGAC L N L R S A I A V V K G C R T V Q K A D ATGCTGCACAACAGTCTGCAGCCTAACATCACCCTCCACGCCCAGACCTATCAGCTGCGG M V H N S L Q P N I T V D A Q T Y E V R GTGGATGGCGAACTTATCACCAGCCAGCCGGCAGACCTTCTCCCGATGGCGCAACGATAT V D C E L I T S E P A D V L P M A Q R Y TTTCTGTTTTAAQQAQAGCGGATGCTTTATTTAACTCAACCTCTGGAGATCCCCGCCGCC F L F * M L Y L T Q R L E I P A A GCGACCGCCAGCGTTACGCTGCCGATTCATGTTCGCGTCAAAAGCCGGGTTAAGGTCACC A T A S V T L P I D V R V K S R V K V T CTCAACGATGGCCGGGATCCCGCCCTCCTGCTCCCCCGCGGCCTGCTACTACCCGCCGGC L N D C R D A C L L L P R G L L L R C C GATCTCCTCAGCAACGAACAACGCACCGACTTTGTCCACCTGATTCCCCCTCATCAACAG D V L S N E E G T E F V Q V I A A D E E CTCTCGGTAGTCCGCTGCCACGATCCGTTTATGCTGCCGAAGCCCTGCTACCACCTCCGC V S V V R C D D P F M L {A K A C Y M L C AACCGTCACGTGCCGCTGCACATCATGCCGGGCGACCTGCGCTACCATCACGATCACGTC N R H V P L Q I M P G E L R Y H H D H V CTGGACGATATCCTGCGCCACTTCGGCCTGACGGTGACCTTTGCCCACCTGCCGTTCGAG L D D M L R Q F C L T V T F C Q L P F E CCGGAACCCCGCCCTTACGCCACCGACACCCACGGTCATCATCATGCTCATCATCACCAC P E A C A Y A S E S H G H H H A H H D H CACGCTCACAGCCACTAGCATCTCCACACCCGAACAACGCCTGCGGCTGATGCAGCTGGC H A H S M * M S T A E Q R L R L M Q L A CAGCACCAACCTCCCCCTACGCGGTTACACCTGGTCCCACGGCCTGGACTGGGCTGTGGA S S N L P V G G Y S W S Q G L E W A V E AGCCCGCTCCCTGCTGGACGTCGCCGCCTTCGACCGCTGGCAGCGACGCCACATGACCGA A G U’ V L D V A A F E R V Q R R Q M T E 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 81 AGGCTTTTTTACCGTTGACCTGCCGCTCTTCGCCCGCCTGTACCCCGCCTCCGAACAAGG G F F T V D L P L F A R L Y R A C E Q G CGATATCGCTGCCGCCCACCGCTGGACCCCCTATCTGCTGCCCTGCCGGGAAACTCGTGA D I A A A Q R W T A Y L L A C R E T R E ACTGCGGGAGGAACAGCGCAACCGCCGCGCGGCCTTTGCCCGTCTGCTGACCGACTGCCA L R E E E R N R C A A F A R L L S D W Q GCCGGACTGTCCGCCGCCGTGGCGCTCCCTGTGCCAGCAAAGCCAGCTCGCCGGGATGGC PDCPPPWRSLCQQSQLAGMA CTGCCTCGGCCTCCGCTGGCGTATCGCCCTGCCCGACATGGCCCTCAGCCTGGGCTATAG W L G V R W R I A L P E M A L S L C Y S CTGGATTCAGAGCGCCGTGATGGCCGGCGTCAACCTGCTCCCCTTCGGCCAGCAGGCCGC W I E S A V M A G V K L V P F C Q Q A A CCAGCAGCTGATTTTACGTCTTTGTGACCACTACGCGCCCGAGATGCCCCGCGCGCTGGC Q Q L I L R L C D H Y A A E M P R A L A CGCGCCGCACGCCGATATCGGATCGGCCACCCCGCTCGCCGCCATCGCCTCTGCCCGGCA A P D C D I C S A T P L A A I A S A R H TGAAACCCAATACTCTCGATTATTCCGTTCCTAQQAQAAGCCATGAACTCTTATAAACAC E T Q Y S R L F R S * M N S Y K H CCGCTGCGCGTCGGCGTCGGCGGCCCGGTCCGCTCCGCTAAAACCGCTCTGCTGGAACCG P L R V G V G G P -V G S C K T A L L E A CTGTCTAAAGCGATCCGCGATACCTGGCAGCTGGCGGTGGTCACTAACGACATCTATACC L C K A M R D T U Q L A V V T N D I Y T AAAGAAGATCACCCCATCCTCACCGAAGCGCGCCCCCTCCCGCCTGAACGCATCGTCGGT K E D Q R I L T E A G A L A P E R I V G CTGGAAACCGGCGGCTGCCCGCATACGGCGATCCGCGAAGATGCCTCAATGAACCTCCCC V E T C G C P H T A I R E D A S M N L A GCCGTGCAACCGCTCACTCAAAAGTTCGGTAACCTCGACCTTATCTTCGTGGAAAGCGCC A V E A L S E K F G N L D L I F V E S G GGCGATAACCTGAGCGCCACCTTCAGCCCGGACCTGGCGGATCTGACCATCTACGTCATC C D N L S A T F S P E L A D L T I Y V I GATGTGCCCGAACCCCAGAAGATCCCCCGCAAACGCGGACCGCGGATCACCAAATCCGAT D V A E G E K I P R K G C P G I T K S D TTCCTGGTGATCAATAAAACCGACCTTGCCCCCTATCTCGGCGCGTCGCTGGAGGTGATG F L V I N K T D L A P Y V C A S L E V M CCGACCGATACCCAGCGTATGCGCCCCCATCGCCCATGGACCTTCACCAATCTCAAGCAC A S D T Q R M R G D R P W T F T N L K Q 4321 4381 4441 4501 4561 4621 4681 4741 82 CCCGACCGCCTGACCACCATTATCGCCTTCCTCCAAGACAAAGCCATGCTTGGCAAATAG C D C L S T I I A F L E D K C M L G K * GCWWAGCWCTCNCTWCTCTNA TATCATCCTCCCTCCACCTCCGCCCCACCCCTGCCCTGCAATATGGCATAAGGTTTGCTA ATTCAACTCATGCCTAACCATTAACGAATCACTATGTCATCACTGGATCTTAACCCTGAA TTACCCGCGACAACGCGGACTTCCGGTACCCGCGAAACCTTAGAAGATTACACCTTACGT TACCCCCCGCTGAGCTTCCCCCGCTGGGGTCCGCGCCTCCTCCCGGTCACCGCGCTCGCC CGCATCCCCTATCTGGCCCACTTTTCCATCGGCCCCAGCATCGGTATGGCCTGGGGCACC ACCAACGCCATCTATTCGATC 4761 . 7' -~ .‘ZILLHL‘Ifl 83 4534. It is preceded by two potential Ntrc binding sites (4388-4402, 4407- 4421) and an NtrA binding site (4483-4499) consistent with nitrogen- dependent regulation. The partial open reading frame was not homologous to any sequence in the DNA or protein database and is not part of the urease operon. Homology comparisons. The predicted sequences for the first three genes of the K. aerogenes urease operon display significant homology to the reported amino acid sequence of jack bean urease (38): UreA is 59% identical to residues l-101, UreB is 52% identical to residues 132-237, and UreC is 60% identical to residues 271-840 of the plant protein. The amino-terminal protein sequence of the large subunit of K. aerogenes urease (30) confirmed the assignment of the ureG gene to this polypeptide. Furthermore, the gene sequences encoding the K. aerogenes urease subunits are 72%, 71%, and 58% identical to recently reported sequences encoding urease subunits from Proteus mirabilis (20), Proteus vulgaris (31), and Helicobacter pylori (3). Indeed, 42% of the K. aerogenes amino acid residues are present in all three bacterial ureases and the jack bean enzyme. No significant homology was detected between the urease structural genes and any non-urease sequences in the NBRF data bank. Gene fusion or gene disruption may have occurred during evolution to explain the single subunit plant protein, the two subunit H. pylori sequence, and the three subunit ureases found in other bacteria. The predicted sequences for UreE, UreF, and UreG displayed no homology to the amino acid sequence of jack bean urease. Furthermore, little homology was observed when these sequences were compared to other sequences in the NBRF data bank. By contrast, sequences determined for the ureE and ureF genes of the P. mirabilis urease operon (20) were 53% and 84 38% identical to the K. aerogenes genes. Moreover, the reported P. mirabilis sequence included the start of an open reading frame (located 60 nucleotides beyond the termination of ureF) which was homologous to ureG from K. aerogenes. In addition, the limited DNA sequence data reported for the urease operons from P. vulgaris (31) and H. pylori (3) included regions corresponding to the start of ureE. Complementation analysis of the urease operon genes. When a region extending from the middle of ureE to the end of ureG was deleted from the urease operon (plasmid pKAU601, Fig. 1), no activity could be detected in transformed cells (Table l). SDS-polyacrylamide gel analysis showed that the ureA, ureB, and ureC gene products were being expressed, although at lower levels than are seen with the intact operon (results not shown). A fragment containing the missing ureE, ureF, and ureG genes was subcloned behind the tac promoter of the compatible expression vector pMMB66HE to yield pKAUSO6 (Fig. 1). Expression of the genes was verified by SDS- polyacrylamide gel analysis; polypeptides corresponding to the predicted sizes for UreE (17-kDa), UreF (ZS-kDa), and UreG (22-kDa) were clearly seen upon induction with 1 mM IPTG (Fig. 3). Furthermore, centrifugation of the extracts resulted in a partial loss of the ureE gene product and complete disappearance of the ureG gene product. Extraction of the washed pellet with SDS showed the presence of the UreG protein (Fig. 3). When plasmids pKAUSO6 and pKAU601 were cotransformed into the E. coli host, the cotransformant was ureolytic and IPTG enhanced urease activity 2.4 fold (Table 1). The high levels of urease activity in the uninduced controls may indicate that small amounts of the accessory gene products are able to activate large amounts of inactive urease. Thus, ureE, ureF, and urea gene products could act in trans with ureA, ureB and 85 F i TABLE 1. Urease specific activities of recombinant E. coli cultures. Specific activity' 4:“ (mole urea min'1 mg") 921531; .2139 $1219 E. coli DH1(pKAUSO6) < 0.2 < 0.2 E. coli DHl(pKAUGOl) < 0.06 < 0.06 E. coli DHl(pKAUSO6 + pKAU60l) 12.9 31.2 ' For comparison, E. coli DH1(pKAUl7) typically has a specific activity of 120 U mg"1 ’ 86 1234.5 0* swat” . 925 K’". I- 662 K *‘fi E , C 45 Kn-g.‘ Q‘s?! ’* I»- --— 31 K - Q- [mt .. “'U’PG 21.5 K .- 33% Fig. 3. Polyacrylamide gel analysis of UreE, UreF, and UreG. Sonicsted cell extracts of E. coli containing pKAU506 were compared for uninduced (lane 2) and IPTG-induced (lane 3) cells by SDS-polyacrylamide (15%) gel electrophoresis. Extracts from the induced culture were centrifuged at 12,000 x g for 45 min and analyzed (lane 4). The pellet was washed, resuspended in SDS sample buffer and run (lane 5). Standards (Bio Rad low MW, Richmond, CA) are shown for comparison (lane 1). All samples were boiled 5 min before running. 87 ureC to give active urease and at least one of these accessory genes is required. In contrast, no activity was observed when sonicated cells containing pKAU601 were mixed with equal amounts of sonicated IPTG-induced cells containing pKAUSOG. Inclusion of 1 mM ATP in the 20 mM phosphate, 1 mM fi-mercaptoetanol, pH 7.0 reaction buffer had no effect. Lack of urease activation under these conditions may indicate a requirement for intact membranes, a need for an energy source other than ATP, or a temporal requirement for the accessory proteins during urease folding. A requirement for accessory genes has also been demonstrated for ureases from soybean (29), the fungus Aspergillus nidulans (25), Pravidencia stuartii (32), P. mirabilis (19, 41), a urease positive E. coli (4) , K. pneumoniae (13), P. vulgaris (31) , and Staphylococcus sapropbyticus (11). Genetic studies of soybean demonstrated the presence of two loci which are distinct from the embryo-specific or ubiquitous urease isozyme structural genes (29). Mutations in these loci result in the production of inactive urease protein, consistent with a possible role in maturation. A. nidulans has been shown to have four loci involved in urea utilization: ureA encoding a urea permease, ureB encoding urease, ureG of unknown function, and are!) suggested to participate in the synthesis or incorporation of a nickel cofactor (25). In the bacterial cases, transposon insertion mutants or deletion mutants downstream of the urease structural genes produced all three urease subunits but possessed little or no urease activity (4, ll, 13, 19, 31, 32, 41). Several of these mutants were defective in non-urease subunit peptides which may correspond to one or more of the accessory genes described here. Furthermore, the P. mirabilis urease operon included sequences that are analogous to ureE, ureE, and the start of ureG (20). A deletion of ureG and the very end of ll:— 88 are? led to substantial losses of activity in the P. mirabilis clone. Purification and characterisation of pKAUSOl-derived urease. Although plasmid pKAU601 contained the normal promoter region and the three urease subunit genes, cells containing this plasmid had no urease activity. In order to assess the role of the missing genes in this construct, inactive urease protein was purified by procedures which were nearly identical to that used for the native enzyme. Because no activity was present, the presence of urease protein in column fractions was assessed by SDS-polyacrylamide gel electrophoresis. The intensity of bands on the gels demonstrated that cell extracts contained greatly decreased levels of urease protein compared to that found in cells containing pKAUl7. Moreover, the amount of urease protein purified from this clone (approximately 1.5 mg) was less than 5% of that typically observed in cells containing pKAUl7. The final preparation of pKAU601-derived urease was only 51% homogeneous as estimated by densitometric analysis of Coomassie blue stained SDS-polyacrylamide gels, nevertheless, several properties of the protein were characterized. Although differing somewhat from the predicted sizes, the three urease subunits were identical in size to that found in native enzyme (apparent M, - 9, 11, and 72 kDa). Moreover, they clearly remained associated throughout the purification, which included ion exchange and gel filtration chromatography. Gel scanning demonstrated that the ratio of 60.3-kDa : 11.7-kDa : 11.1-kDa subunits was 1.1 : 1.8 : 1.7, nearly identical to that observed in the native enzyme (39). Furthermore, the chromatographic properties of the pKAUéOl-derived urease matched that of the native enzyme indicating a similar size [(60-kDa)2(12-kDa).(11- kDa).] and charge. The only observed distinction from the native urease was 89 in the nickel content. Whereas the native enzyme was shown to possess 4 moles of nickel per mole of enzyme (39), the pKAUGOl-derived protein had less than 0.25 moles of nickel per mole of enzyme. Thus, one or more of the accessory gene products is involved in facilitating assimilation of nickel into spa-urease. One possible role for the accessory genes involves nickel transport into the cell. However, we feel that this can not be the only role for the accessory genes because nickel can enter E. coli cells via the magnesium transport system (17) . Since our cells were grown in a medium containing 0.1 mM nickel, it seems probable that some nickel would enter the cell, yet we could not detect any measurable activity. A second possible role for the accessory genes involves nickel incorporation into ape-urease. We had previously reported that. K. aerogenes cells containing the recombinant urease plasmid pKAU19 grown in' nickel-free medium synthesized urease spa-enzyme, and that the ape-enzyme was activated upon addition of nickel even after treating the cells with protein synthesis inhibitors (Chapter 4; 23). The purified apo-urease could not be reactivated by addition of nickel, indicating that nickel incorporation does not occur by passive binding. It was proposed that some additional component was necessary to facilitate insertion of nickel into the apoenzyme. The accessory genes may participate in this process. In this regard, the carboxyl terminal sequence predicted for UreE is particularly interesting: ten of the last 15 amino acids are histidine residues. Histidine-rich regions are involved in metal binding sites for other proteins [e.g. , the zinc-binding protein from albacore tuna plasma (7) and the copper-containing hemocyanin (12)]; thus, such a sequence may bind nickel ions which are subsequently transferred to apo-urease. The 90 only other known function for a'histidine-rich region involves a 16 amino acid peptide containing 7 adjacent histidinyl residues; this sequence participates in regulation of the Salmonella histidine operon (18). In summary, the formation of active urease requires activation by accessory proteins which function in nickel incorporation. Urease is not the only metalloenzyme which requires accessory proteins for metal incorporation” For example, multiple gene products are required for proper incorporation of molybdenum into several enzymes, as reviewed by Hinton and Dean (16). Moreover, evidence has been presented for a copper- insertase required for N20 reductase biosynthesis (22). There is also evidence that in vivo nickel incorporation into Rhodospirillum rubrum carbon monoxide dehydrogenase may require nickel processing (8). Further characterization of the accessory proteins involved in activating urease may aid in elucidating some of the general incorporation mechanisms for metalloproteins. Future efforts will be directed at characterizing how the ureE, ureF, and ureG gene products are involved in this process. Acknowledgements. I would like to recognize the assistance of Robert Hausinger for purification of the pKAU60l-derived urease. 10. 11. 12. 91 REFERENCES Andrews, R. R., R. L. Blakeley, and B. Zerner. 1988. Urease -- A Ni(II) metalloenzyme, p. 141-165. In J. R. Lancaster, Jr. (ed.), The bioinorganic chemistry of nickel. VCH Publishers, New York. Bullock, W. 0., J. M. Fernandez, and J. M. Short. 1987. XLl-Blue: A high efficiency plasmid transforming recA Escherichia coli strain with beta- galactosidase selection. Biotechniques 5:376. Clayton, C. L., M. 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Improved M13 phage cloning vectors and host strains: nucleotide sequences of the Ml3mp8 and pUCl9 vectors . Gene: 103- 119 . CHAPTER 6 Conclusions and Future Prospects 95 96 There were three main goals established at the onset of the work described herein: to clone the Klebsiella aerogenes urease genes; to ascertain the best combination of plasmid vector, hast strain, and growth conditions to yield maximum production of urease; and to determine the sequence of the urease genes. As a prelude to the Klebsiella aerogenes investigations, experiments were carried out with recombinant Pravidencia stuartii urease. This work represents the first purification and analysis of a urease expressed in a heterologous host, demonstrating that E. coli could synthesize urease that was identical in every respect to the enzyme purified from the original strain. Furthermore, the regulation of enzyme expression observed in E. coli was identical to that of the parent Pravidencia stuartii. These results supported our expectation that the Klebsiella aerogenes urease genes could be successfully cloned and overexpressed. The Klebsiella aerogenes urease genes were successfully cloned by screening a cosmid library on modified urease indicator plates. Several subcloning steps reduced the size of the insert DNA to 5.7 kb. This fragment was cloned into vector pBR328 (pKAUl9) and urease expression was tested in several host strains. The optimal growth conditions were found to be a MOPS buffered minimal medium containing low nitrogen and high nickel concentrations. Greatest expression of urease was obtained by transforming pKAUl9 back into Klebsiella aerogenes; yields were 100 to 200-fold higher than in the wild-type organism carrying a single copy of the urease genes. Expression of these high levels greatly simplified purification providing a large stqaply of enzyme for biophysical studies. Furthermore, it allowed the use of inunogold localization to confirm that urease was a cytoplasmic enzyme. 97 Sequencing of the urease genes revealed an operon much larger and more complex than anticipated. Six open reading frames were present, three of which were the urease subunit genes: these genes show a high degree of homology to the ureases of jack bean and other bacteria. The three additional genes have unspecified functions, but their deletion from the urease operon results in the synthesis of inactive urease apoenzyme. In addition, these three genes can act in trans to produce active urease and thus may play a role in nickel incorporation. Additional information on the mechanism of nickel incorporation was obtained by growing cells in a nickel-free medium in the presence of various metabolic inhibitors: addition of nickel restored urease activity in cells inhibited in protein synthesis but not in cells inhibited in energy production or in sonicated cells. These results led to the proposal that urease is initially synthesized as an apoenzyme and is subsequently activated in an energy-dependent nickel incorporation step. Future directions. The results presented here lay a foundation for many new experimental paths. Several possibilities are summarized below: a) Characterization of UreE, UreF, and UreG proteins. These three accessory proteins are highly expressed from plasmid pKAUSO6, facilitating their purification and analysis. Metal chelation chromatography might be a good choice as a UreE purification step because of its unusual histidine-rich carboxy terminus. Antisera can be produced against all three peptides and immunogold electron microscopy used for localization using the same techniques as for Klebsiella aerogenes urease. thctional characterization of these peptides would include metal analysis. b) Complementation among urease genes of different species. Since it was e) 98 demonstrated that the Klebsiella aerogenes ureE, ureF, and ureG can complement, in trans, ureA, ureB, and ureG, it seems reasonable to try this with other cloned ureases. For example, there are mutants of Proteus mirabilis, Pravidencia stuartii, Klebsiella pneumoniae, and Proteus vulgarus which express inactive urease subunits. Furthermore, the bacterial accessory genes could be used to attempt complementation of the soybean urease structural gene. The ability of plasmid pKAU506 to restore activity in these mutants would indicate a common mechanism of nickel incorporation in these other ureases and would indicate conserved regions essential for maintaining the structure and function of the accessory proteins. The high production of urease described in chapter 2 should allow purification of large enough quantities for. biophysical and crystallization studies with the long term goals of determining the metallocenter function and the three dimensional structure of the enzyme . d) Site directed mutagenesis. Once the active site is located by labeling and peptide isolation, specific amino acids could be changed to test the effects on kinetic parameters. One obvious target would be the one cysteine that is conserved among all of the sequenced ureases: a cysteine is implicated in the active site of Klebsiella aerogenes and jack bean enzymes. Mutagenesis could also be performed on several of the highly conserved histidines, which could serve as nickel ligands. Other targets for mutagenesis would be the accessory proteins. For example, urease activity could be measured in UreE mutants containing amino acid substitutions or truncation of the histidine-rich carbaxy terminus . APPENDIX 99 Sequence of the urease upstream region from a Sau3A site to the SstI site (base 751 here is base 1 of Fig. 4, Chapter 5) 1 GATCACGATATTGACGGCAAACATTATCCCGGTGAATAATCCGGACCGGTTATGCCGCTT 60 TCCCCTCCGCGCCCCTCGCCACGCTCCGGCTTACGGATGACATAAGCCTTTCGTATGACC 120 GGGATAAACTCCCCCCGATCAATACTCATTGCTGCTGTTTTATCTTGATTTTGCACCGGC 180 GCAACATTCCACGGCACCGTGTTACCACCACTCAAAAAACGCTGCCACGCCACGCTGGAT 240 CTCCGCTTTCACCACGCCCCCCGCAAGACCGTTCTCGCCAGCGCCCAACACGTCGGCCCC 300 CTGACCGTCCACCGCCCCTTTTACCCCGAACAACAGACCTGTCACCTCTATCTGCTTCAC 360 CCCCCCGGCCGCATCCTCCCCCGTCATCACCTGACAATTACCGCGCACCTTCCCCCCGGC 420 TCCCATACCCTGATAACCATGCCTCGCGCCAGCAAGTTTTACCGCAGCACCGGCGCGCAA 480 CCGCTACTTCGCCACCAGTTGACCCTTGCCCCGCAGCCGACCCTCGAGTCGCTCCCGCAG 540 GATGCCATCTTCTTTCCCGCGGCCAATGCCCGGCTCTTCACCACCTTTCATCTTTGCGCC 600 TCCACCAGGCTGCTGGCCTCGGATCTCCTCTGCCTTGGCCGCCCGCTCATTCCCGAAACC 660 TTCAGCCACCCCACCCTCACCAACCGGCTGGACCTATGGGTCGACAATCAGCCGCTGCTG 720 CTCGACCGCCTGCACCTGCACCAGGGACAGC 751 N STATE UNIV w wmmm 008914 R HICHIG "WW 3'12 . NH 13