Morganella morganii Urease: Purification, Characterization, andIsolation of Gene Sequences
LI-TAI HU, ERIC B. NICHOLSON, BRADLEY D. JONES, MARTHA J. LYNCH,AND HARRY L. T. MOBLEY*
Division of Infectious Diseases, Department of Medicine, University of MarylandSchool of Medicine, 10 South Pine Street, Baltimore, Maryland 21201
Received 3 November 1989/Accepted 7 March 1990
Morganella morganui, a very common cause of catheter-associated bacteriuria, was previously classified withthe genus Proteus on the basis of urease production. M. morganui constitutively synthesizes a urease distinctfrom that of other uropathogens. The enzyme, purified 175-fold by passage through DEAE-Sepharose,phenyl-Sepharose, Mono-Q, and Superose 6 chromatography resins, was found to have a native molecular sizeof 590 kilodaltons and was composed of three distinct subunits with apparent molecular sizes of 63, 15, and 6kilodaltons, respectively. Amino-terminal analysis of the subunit polypeptides revealed a high degree ofconservation of amino acid sequence between jack bean and Proteus mirabils ureases. Km for urea equalled 0.8mM. Antiserum prepared against purified enzyme inhibited activity by 43% at a 1:2 dilution after 1 h ofincubation. AD urease activity was immunoprecipitated from cytosol by a 1:16 dilution. Antiserum did notprecipitate ureases of other species except for one Providencia rettgeri strain but did recognize the large subunitsof tireases of Providencia and Proteus species on Western blots (immunoblots). Thirteen urease-positive cosmidclones of Morganella chromosomal DNA shared a 3.5-kilobase (kb) BamHI fragment. Urease gene sequenceswere localized to a 7.1-kb EcoRI-SaII fragment. TnS mutagenesis revealed that between 3.3 and 6.6 kb ofDNAwere necessary for enzyme activity. A Morganella urease DNA probe did not hybridize with gene sequences ofother species tested. Morganella urease antiserum recognized identical subunit polypeptides on Western blotsof cytosol from the wild-type strain and Escherichia coli bearing the recombinant clone which corresponded tothose seen in denatured urease. Although the wild-type strain and recombinant clone produced equal amountsof urease protein, the clone produced less than 1% of the enzyme activity of the wild-type strain.
Morganella morganii, a gram-negative enteric bacterium,is an increasingly prevalent cause of nosocomial and cathe-ter-associated bacteriuria (25, 35). Most (97%) isolates areurease positive (8) and are multiply antibiotic resistant (6,10). Senior (30) found that 34% of 220 routine fecal speci-mens contained M. morganii, suggesting that the source ofinfecting organisms is the gastrointestinal tract. In the samestudy, this species was also implicated as a cause of gastro-enteritis.
In the human urinary tract, the ability of M. morganii tohydrolyze urea has been linked to formation of xanthinecalculi (28) as well as the more typical struvite and carbonite-apatite stones (32). Management of infection stones hastypically included a urease inhibitor and broad-spectrumantibiotic (1, 5, 18).Based on urease production, M. morganii was previously
classified as Proteus morganii. However, DNA-DNA hy-bridization studies (4) revealed that this species representeda relatively homogeneous genus that was quite distinct,exhibiting only 13 to 26% DNA relatedness to Proteus andProvidencia species.These differences are also reflected at the level of the
urease itself. Antibodies directed against crude preparationsof the Proteus mirabilis enzyme did not cross-react withcrude preparaiions of the Morganella enzyme (11). As well,we and other investigators have found that the native Mor-ganella enzyme has a much larger apparent molecularweight than that of other species as assayed on molecularsieve columns and nondenaturing polyacrylamide enzymeactivity gels (13, 29, 31). In addition, urease gene probes
* Corresponding author.
from Providencia stuartii (24) and Proteus mirabilis (B.Jones and H. Mobley, unpublished observation) did nothybridize with Southern blots of Morganella chromosomalDNA, even under conditions of low stringency. The Mor-ganella enzyme also appears to have a higher affinity forurea (i.e., lower Kn) than the Proteus and Providenciaenzymes (13, 29) and is produced constitutively (29).We report here that the M. morganii urease appears
distinct from the enzymes produced by Proteus mirabilis andProvidencia stuartii by virtue of apparent molecular weightsof native enzyme and subunit polypeptides, affinity forsubstrate, regulation of enzyme synthesis, and DNA homol-ogy of genes encoding the enzyme. However, some strongsimilarities are revealed by comparison of N-terminal aminoacid sequences of subunit polypeptides. As well, someantigenic determinants of the native and denatured enzymeare apparently conserved between Morganella, Proteus, andProvidencia species.
MATERIALS AND METHODSBacterial strains and growth conditions. Clinical isolates of
M. morganii, Proteus mirabilis, Proteus vulgaris, Providen-cia stuartii, Providencia rettgeri, and Klebsiella pneumoniawere isolated at concentrations of i105 CFU/ml from theurine of long-term-catheterized patients as previously de-scribed (25, 35). Helicobacter pylori strains were isolated aspreviously described (22).
Escherichia coli HB101 (F- hsdR hsdM supE44 proA2leuB6 rpsL20 recA13 lac Y1 galK2 thi-1 ara-14) was therecipient for transformation with recombinant plasmids (19).
Purification. M. morganui was grown in 10 liters of Luriabroth for 18 h with aeration (200 rpm) at 37°C. Cells were
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harvested by centrifugation (10,000 x g, 10 min, 4°C),washed two times in 20 mM sodium phosphate (pH 6.8), andstored as a cell pellet at -20°C. Cells (30 g [wet weight])were suspended in 30 ml of 20 mM potassium phosphate (pH6.8)-i mM EDTA-1 mM P-mercaptoethanol (PEB) andruptured by passage through a precooled French pressurecell (Aminco-Bowman) at 20,000 lb/in2. Unbroken cells wereremoved by centrifugation (10,000 x g, 10 min, 4°C), and thesupernatant was centrifuged (100,000 x g, 60 min, 4°C) toremove membrane. Soluble protein was used for columnapplication. DEAE-Sepharose chromatography was per-formed on a conventional column (1.5 by 7.5 cm). Phenyl-Sepharose (1.5 by 12 cm), Mono-Q (HR 5/5), and Superose 6(1.0 by 29.5 cm) purification steps were done on a fastprotein liquid chromatography system (Pharmacia FineChemicals, Piscataway, N.J.) at room temperature. Frac-tions were screened for urease activity by the phenol redspectrophotometric assay (24). Specific activity of pooledfractions was determined by the indophenol reaction (37).Protein concentration was determined by the method ofLowry et al. (17) with bovine serum albumin as a standard.Samples were electrophoresed on sodium dodecyl sulfate
(SDS)-polyacrylamide gels by the method of Hames (12).Molecular weight standards are listed in the legend to Fig. 2.
N-terminal analysis. The N-terminal amino acid sequencewas determined by a modification (R. Hall and J. Coffins,unpublished data) of the procedure of Moos et al. (26).Purified urease (10 jig) denatured in SDS gel sample buffer(16) for 5 min at 100°C was electrophoresed on a linear 10 to20o polyacrylamide gradient gel. Polypeptides were trans-ferred to polyvinylidene difluoride membrane (Immobilon-P;Millipore Corp., Bedford, Mass.) and stained with 0.1%Coomassie brilliant blue (Bio-Rad Laboratories, Richmond,Calif.) in 50% methanol. Membranes were destained with10o acetic acid-50%o methanol. Stained protein bands wereexcised, and N-terminal amino acid sequences were deter-mined with a pulsed liquid-phase sequenator (model 477A;Applied Biosystems).
Urease assays. Urease hydrolysis was quantitated by twomethods. In a continuous assay, enzyme was added to acuvette containing 2.8 ml of 3 mM sodium phosphate (pH6.8)-120 mM urea-phenol red (7 p,g/ml). Reactions wereperformed at 23°C, and optical density was monitored at 560nm. Rate of change of optical density was correlated withammonia production as previously described (24).
In an endpoint assay, ammonia production was measuredby indophenol formation by using the phenol-hypochloritereaction (phenyl nitroprusside and alkali hypochlorite) (37).Optical density was measured at 625 nm with ammoniumchloride as a standard.
Cell fractionation. Cells of M. morganii TA43 were har-vested by centrifugation from 8-h-old Luria broth cultures (1liter) grown at 37°C with aeration (200 rpm). Supernatantculture medium (50 ml) was saved for later assay, and cellpellets (4 g) were washed once with 10 mM Tris hydrochlo-ride (pH 8.0) and subjected to the cold osmotic shockprocedure as previously described (24). Osmotic shock fluidrepresenting the periplasmic proteins was placed on ice forlater assay. Cells were suspended in 10 ml of 20 mM sodiumphosphate (pH 6.8) and ruptured by one passage through aprecooled French pressure cell at 20,000 lb/in2. Wholeunbroken cells were removed by centrifugation (8,000 x g,4°C, 10 min). Supernatant was centrifuged at 100,000 x g at4°C for 60 min. Supernatant from this centrifugation repre-sented the cytosol fraction and was placed on ice for laterassay. The membrane pellet was washed two times with 20
mM sodium phosphate (pH 6.8) and then resuspended in 5ml of 20 mM sodium phosphate (pH 6.8) and used directlyfor assay.
Spectrophotometric assays were used to measure enzymeactivities. Urease was measured by the phenol red spectro-photometric assay. Catalase was measured by using H202 asa substrate at 240 nm. Alkaline phosphatase was measuredby using p-nitrophenylphosphate as a substrate at 420 nm.NADH dehydrogenase activity was measured at 340 nm withNADH as the substrate. Protein concentration was deter-mined by the method of Lowry et al. (17) with bovine serumalbumin as a standard.Gene bank preparation. Chromosomal DNA was isolated
from M. morganii TA43 by the method of Marmur (21) andused for the preparation of a gene bank in E. coli by standardmethods (20). DNA was partially digested with Sau3A,ligated into the BamHI site of pHC79, and packaged in vitrointo lambda bacteriophage particles which were used totransfect E. coli HB101. Cells were plated onto Luria agarcontaining ampicillin at 200 jig/ml. Ampicillin-resistant col-onies were screened for urease activity by replicating ontomodified urea segregation agar (9) which was prepared bycombining component A (4 g of yeast extract, 4 g of peptone,0.34 g of NaH2PO4, 1.03 g of Na2HPO4, 1 g of gelatin, 5 g ofNaCl, 0.90 g of KH2PO4, 1.10 g of K2HPO4, and 15 g of agarin 900 ml of distilled H20, autoclave sterilized) with compo-nent B (9 g of D-glucose, 6 g of urea, and 0.035 g of phenolred in 100 ml of distilled H20, filter sterilized). After over-night incubation, urease-positive (red) colonies were pickedand used for subsequent experiments.DNA hybridization. Dot blots of whole-cell DNA from
enteric species were prepared by spotting overnight culturelysates onto nitrocellulose filters as described by Maniatis etal. (20). A 3.5-kilobase (kb) BamHI DNA fragment fromwithin the urease operon was isolated by elution of thefragment from a preparative 0.7% agarose gel and waslabeled with [a-32P]ATP by random primer extension andused for hybridization under stringent (50% formamide, 650Cwash) and nonstringent (20% formamide, 510C wash) hybrid-ization conditions (20). Blots were washed, dried, and auto-radiographed.Transposon mutagenesis. TnS mutagenesis was performed
by the method of deBruijn and Lupski (7). Briefly, E. coliHB101(pMOM203) was infected with lambda::TnS and se-lected on Luria agar containing kanamycin (50 ,ug/ml).Kanamycin-resistant cells were pooled and incubated over-night in Luria broth. Plasmid DNA was isolated by themethod of Birnboim and Doly (3), purified by centrifugationto equilibrium on CsCl gradients, and used to transformcompetent cells of E. coli HB101. Kanamycin-resistantcolonies were screened for urease activity, and sites oftransposon insertion were determined by double digestion ofplasmid DNA with EcoRI and BamHI.
In vitro transcription-translation. Purified plasmid DNA (5p.g) isolated from cells containing Tn5 insertions of plasmidpMOM203 was used for in vitro transcription-translation inthe presence of [35S]methionine (>600 Ci/mmol; Dupont,NEN Research Products, Boston, Mass.). Reagents wereobtained from Digene (College Park, Md.), and labeling wasdone according to the instructions of the manufacturer.Labeled polypeptides were solubilized in gel sample buffer(16) and electrophoresed on SDS-15% polyacrylamide gels.
Preparation of antisera. Two female New Zealand White-SPF rabbits (Hazelton Dutchland, Inc.) were injected sub-cutaneously with column-purified M. morganii urease inFreund complete adjuvant at a concentration of 100 ,ug/ml
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MORGANELLA MORGANII UREASE 3075
TABLE 1. Purification of urease from M. morganii
Sp act ofPurification (,umol of Purifi- Total Total Enzyme
step urea/min per cation activity protein recoverystep mg of pro- (fold) (,umol/min) (mg) (%)tein)
(four injections of 0.25 ml each). Blood was tested at 2weeks, and a second set of injections was administered at 3weeks. Blood was taken again at 4 to 5 weeks. Rabbits wereterminally bled at 44 days.Western blot (immunoblot). Crude soluble protein frac-
tions of E. coli HB101(pMOM203) and M. morganii TA43were electrophoresed on SDS-20% polyacrylamide gel bythe method of Laemmli (16) and blotted onto nitrocellulose.Western blots were developed as described by Towbin et al.(34) with the modifications of Batteiger et al. (2). Rabbitantiserum was diluted in 0.5% Tween 20 in 20 mM sodiumphosphate (pH 7.4)-120 mM NaCl (phosphate-buffered sa-line) and incubated with nitrocellulose filters for 2 h at 37°C.Filters were washed three times for 10 min each in diluentand agitated in 1:1,000 goat anti-rabbit immunoglobulin Gconjugated to alkaline phosphatase (Sigma Chemical Co., St.Louis, Mo.) overnight at 4°C. Filters were thoroughlywashed again and developed with Nitro Blue Tetrazoliumand 5-bromo-4-chloro-3-indolyl-phosphate.
Neutralization and immunoprecipitation. Twofold dilutionsof serum were made in PEB buffer. Cytosol (100 ,ul contain-ing 1 mg of protein; obtained as described above) was mixedwith an equal volume of serum dilution incubated for 1 h at4°C. A portion (75 ,ul) was assayed for urease activitydirectly. The remaining portion was centrifuged in a micro-centrifuge (15,600 x g, 3 min, 4°C), and the supernatant (75,ul) was assayed for urease by the phenol red spectrophoto-metric assay in a reaction volume of 2.70 ml of 3 mM sodiumphosphate (pH 6.8)-120 mM urea containing phenol red at 7,ug/ml.
RESULTS
Purification. Urease was purified from a French press celllysate from 30 g (wet weight) of cells by using the four-stepscheme outlined in Table 1. Crude lysate was applied to aDEAE-Sepharose column, and urease was eluted at 395 mMKCI within a 0 to 500 mM KCl gradient. Peak fractions werepooled and adjusted to 1.0 M KCI and loaded onto aphenyl-Sepharose column. Urease was not retained on thecolumn and eluted at 70 ml, prior to the elution of themajority of cell protein and before the running of the 1.0 to0.0 M KCl gradient, which released no additional urease.Peak fractions were pooled, dialyzed against PEB buffer,and loaded onto a Mono-Q anion-exchange column. Ureaseeluted in a single fraction at 330 mM KCl. Protein from theactive fraction was concentrated from 2.5 to 0.5 ml by acentrifugal concentrator (Centrisart I; Sartorius, Hayward,Calif.) and loaded onto a Superose 6 molecular sieve column(Fig. 1); activity eluted at 12.1 ml, which corresponded to anapparent molecular size of 590 kilodaltons (kDa). Proteinfrom the active fractions of each purification step wasdenatured and electrophoresed on SDS-polyacrylamide gels
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FIG. 1. Superose 6 chromatography of M. morganii urease.Pooled fractions with peak urease activity from Mono-Q anion-exchange chromatography were dialyzed against PEB buffer andchromatographed on a Superose 6 column as described in the text.Fractions (0.5 ml) were assayed for urease activity by the phenol redspectrophotometric assay. Active fractions with elution volumesbetween 11.5 and 13.0 ml were pooled.
(Fig. 2). A critical step in the purification appeared to bephenyl-Sepharose (Fig. 2, lane D), eliminating greater than80% of the contaminating protein. Some inactivation ofenzyme occurred during the purification procedure.The pure enzyme (Fig. 2, lanes F and I) was electro-
phoresed on both gradient (8 to 15%; left panel) and high-percentage acrylamide (15%; right panel) gels and was foundto be composed of three polypeptide subunits with apparent
A B C D E F G H I J
4_
FIG. 2. SDS-polyacrylamide gel electrophoresis of purified M.morganii urease. Protein samples from fractions with peak ureaseactivity from each purification step were denatured in SDS gelsample buffer and electrophoresed on either an 8 to 15% (lanes A toH) or a 15% (lanes I and J) SDS-polyacrylamide gel. Lanes A, H,and J, High-range molecular-size markers: rabbit muscle phospho-rylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; hen egg whiteovalbumin, 42.7 kDa; bovine carbonic anhydrase; 31.0 kDa; soy-bean trypsin inhibitor, 21.5 kDa; and hen egg white lysozyme, 14.4kDa; lane B, whole-cell lysate; lane C, DEAE-Sepharose eluate;lane D, phenyl-Sepharose eluate; lane E, Mono-Q eluate; lanes Fand I, Superose 6 eluate. Lane G, Low-molecular-size markers:myoglobin, 17.0 kDa; myoglobin fragments I and II, 14.4 kDa;myoglobin fragment I, 8.2 kDa; myoglobin fragment II, 6.2 kDa.
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63 kDa Subunit
M morgani
P. mirabils (61kDa)
Jack bean
1 5 10 15Pro Gin lie Smr Arg Gln Glu Tyr Gly Gly Lou Phe Gly Pro Thr2 * * * * * * * * *Lys Thr lie Smr Arg Gin Ala Tyr Ala Asp Met Phe Gly Pro Thr272 * * * *Thr Phe lie His Arg Lys Glu Tyr AJa Asn Lys Tyr Gly Pro Thr
Matches with M morganji
Conservative %Exact Replacements Similarity
9/15 3/15 80
7/15 1/15 53
1 5 10 15Ser Asn Thr Lys Gin Pro Thr Pro Lou Gly Gly Val le Phe Ala
6 kDa Subunit
M morgaMni
P. ,rwabNs (11 kDa)
Jack bean
1 5 10Met Gin Leu Thr Pro Pro Glu Val Glu Lys
Met Glu Leu Thr Pro Arg Glu Lys Asp Lys
Met Lys Lou Ser Pro Arg Glu VaW Glu Lys
6/10 1/10 70
7/10 1/10 80
FIG. 3. N-terminal amino acid analysis ofM. morganii urease and comparison with ureases from Proteus mirabilis andjack bean. Purifiedurease was electrophoresed under denaturing conditions and transferred to polyvinylidene difluoride membrane, and the three subunits weresubjected to N-terminal analysis as described in the text. M. morganii sequences were compared with the Proteus mirabilis sequencepredicted from the complete nucleotide sequence (15) and with the amino acid sequence ofjack bean urease (18). Numbers above the aminoacid sequence are the residue numbers within the respective subunit polypeptide. Exact matches (*) are defined as residues identical to theM. morganii residues per total number of M. morganii residues determined. Conservative replacements are defined as a substitution foranother amino acid within the same group (i.e., acidic, basic, neutral-polar, or neutral-nonpolar [36]) per total number ofM. morganii residuesdetermined. Percent similarity is based on (exact matches + conservative replacements)/total number of residues compared.
molecular weights of 63,000, 15,000, and 6,000 when com-pared with migration of standards (Fig. 2, lanes A, G, H, andJ).
Cellular localization. Cells of M. morganii were fraction-ated into periplasmic, membrane, and cytoplasmic fractions.Unlike the ureases of Providencia stuartii and Proteusmirabilis which are clearly cytoplasmic, Morganella ureasepartitioned in both the membrane (2.5 ,umol of urea hydro-lyzed per min per mg of protein; 59% of total activity) andcytosol (1.76 ,umol of urea per min per mg of protein; 41% oftotal activity). Fractionation of control enzymes indicatedthat membrane and cytosol were well separated; 93% ofNADH dehydrogenase activity partitioned with the mem-brane, and 100% of catalase activity was found in thecytosol.
N-terminal analysis. The results of N-terminal analysis areshown in Fig. 3 with the relevant amino acid sequences ofProteus mirabilis (15) and jack bean (19) ureases. Compari-son of M. morganii urease subunits with those ofjack beanor Proteus mirabilis revealed a high percentage of sequencesimilarity, defined as the percentage of exactly matching andconserved amino acid residues between the compared seg-ments. The first 15 amino acids of the 63-kDa subunitshowed 80% similarity to amino acids 2 to 16 of the 61-kDaProteus subunit and 53% similarity to an internal 15-amino-acid segment (amino acids 272 to 286) of jack bean urease.Likewise, the first 10 amino acids of the 6-kDa subunitshowed a high degree of conservation with the first 10 aminoacids of the jack bean urease (80%) and the 11-kDa subunitof Proteus urease (70%). The N-terminal 15 amino acids ofthe 15-kDa subunit shared no amino acid sequence similaritywith either the Proteus or jack bean urease.
Neutralization and precipitation. Antiserum preparedagainst M. morganii urease was able to neutralize approxi-mately 60% of urease activity when incubated with cellcytosol from M. morganii TA43 for 1 h. Preimmunizationserum had no effect. Immune serum did not significantlyinhibit the urease activities in cytosol of other Proteus,Providencia, or Klebsiella species.
M. morganii urease activity was completely precipitatedby centrifugation at a titer of 1:32; that is, no activityremained in the supernatant (Fig. 4). Activity was reduced57% at a titer of 1:64. Jack bean, Proteus, and Klebsiellaureases were apparently not precipitable by the antiserum.However, Providencia rettgeri showed a 40% reduction inactivity at a titer of 1:8 and was the only urease other thanMorganella urease to show a significant reduction in activityupon precipitation with antiserum.Antiserum specifically recognized the three urease sub-
units on Western blots of whole-cell soluble protein from M.morganii electrophoresed under denaturing conditions (Fig.5). The large subunit of the ureases from representativeProteus and Providencia species was also recognized if cellswere grown in urea-containing medium, that is, if urease wasinduced. When culture medium did not contain urea, onlythe Morganella urease, which is constitutively produced,was recognized by the antisera. When uninduced, the Pro-teus and Providencia species did not produce sufficientenzyme to give a strong signal on the Western blot. Evenwhen induced, however, the two minor subunits of theselatter species went unrecognized by specific antisera.
Molecular cloning of urease genes. Of approximately 1,000gene bank clones of M. morganii chromosomal DNA, 13expressed urease activity in E. coli HB101 when assayed onurea segregation agar. All plasmids (approximately 45 kb insize) shared a common 3.5-kb BamHI fragment later local-ized within urease gene sequences. One gene bank clone,pMOM101, was selected, and genes were subcloned byHindIII deletion to produce the 20-kb pMOM202. To local-ize the urease genes, we mutagenized the plasmid with TnS.All urease-negative insertions mapped to a 7.1-kb EcoRI-SalI fragment which contained the 3.5-kb BamHI fragment.Urease genes were further localized by digesting pMOM202with EcoRI and Sall, religating, transforming, and screeningfor the smallest plasmid capable of urease activity by elec-trophoretic sizing. Plasmid pMOM203 (Fig. 6) contained the7.1-kb fragment and the remaining 5.8-kb EcoRI-SalI frag-ment of vector pHC79. Although the clone produced urease,
15 kDa Subunit
M morganii
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1:8 1; 16 1:32
1:8 1:16 1:32 1:64
1:64
1;128DILUTION OF SERUM
FIG. 4. Immunoprecipitation of bacterial and plant ureases by anti-M. morganii urease antiserum. Soluble protein derived from wholebacterial cell lysates was incubated for 60 min with an equal volume of twofold dilutions of preimmune (A) or immune (B) serum directedagainst purified M. morganella urease. Immunoprecipitates were removed by centrifugation, and urease activity was determined for thesupernatant fraction. Control activity representing 100%o was determined in the absence of antiserum.
the activity was extremely weak. To further localize thegenes, TnS mutagenesis was repeated on pMOM203. Inser-tions resulting in inactivation were separated by 3.3 kb(TnS-7 and TnS-31); the maximum size of fragment neces-sary for activity spanned 6.6 kb (from TnS-34 to the EcoRIsites at the vector-insert junction).
In vitro transcription-translation of pMOM202 andpMOM203 revealed the synthesis of polypeptides corre-sponding to 63,000, 15,000, and 6,000 molecular weight (datanot shown). In addition, cell cytosol was isolated, denaturedin gel sample buffer, and electrophoresed on an SDS-poly-acrylamide gel, and proteins were transferred to nitrocellu-lose. Antisera raised against purified M. morganii ureaserecognized subunits with an apparent molecular weight of63,000, 15,000, and 6,000 in cytosol from both M. morganiiTA43 wild-type strain and recombinant clone E. coliHB101(pMOM203) (Fig. 7). Despite the weak urease activityof the recombinant clone, approximately equal amounts ofsubunit polypeptides were produced by recombinant andwild-type strains.To determine whether DNA homology existed between
Morganella urease genes and urease genes of other species,we used the 3.5-kb BamHI fragment as a DNA probe. Dotblots of whole-cell DNA from urease-positive K. pneumo-niae (10 strains) and H. pylori (1 strain) were not recognizedby the probe under high or low stringency. We previouslydetermined that Morganella chromosomal DNA did nothybridize with urease gene probes from Providencia stuartii
(24) or Proteus mirabilis (B. Jones and H. Mobley, unpub-lished observation).
DISCUSSION
The urease of M. morganii, a very large protein with anestimated native molecular weight of 590,000, is composedof three subunit polypeptides with apparent molecularweights of 63,000, 15,000, and 6,000. The stoichiometry ofthe subunit is unknown. This subunit structure reflects ageneral pattern seen in other genera of the family Entero-bacteriaceae, including Proteus mirabilis (14), Providenciastuartii (27), and K. pneumoniae (33), which also have threeunique subunits: one large subunit 61 to 72 kDa in size andtwo smaller subunits 8 to 12 kDa in size. This contrasts withsingle-subunit-type ureases reported for several other bacte-ria (reviewed in reference 23) as well as that produced by thejack bean. We found that the M. morganii urease sharessome features with other ureases, but by other criteria, thisprotein is quite distinct from ureases that have been de-scribed previously.Two of the three enzyme subunits shared amino acid
similarity with subunits of the Proteus mirabilis urease aswell as with portions of the jack bean sequence (Fig. 3). Thefirst 15 amino acids of the large subunit showed 80% simi-larity to a 15-amino-acid sequence that begins at the secondamino acid of the largest subunit of the Proteus enzyme. Thefirst 10 amino acids of the 6-kDa subunit showed 70%
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A B C D E F G H I
J K LMN O P Q R
FIG. 6. Molecular cloning of M. morganii urease gene se-
quences. pMOM203 was constructed by digesting pMOM202 withEcoRI and SalI and religating (see text). pMOM203 contains the7.1-kb fragment of M. morganii chromosomal DNA able to encodean active urease and the 5.8-kb EcoRI-SalI fragment of vectorpHC79. Origin of replication (orn) and the 1-lactamase gene (bla) are
indicated by arrows. Symbols: *, locations of Tn5 insertionalmutations; -, inactivated urease; +, urease activity retained.
FIG. 5. Western blot of soluble protein from Proteus, Providen-cia, and Morganella species with anti-Morganella urease antiserum.Soluble protein (50 ,ug) derived from bacterial cell lysates was
electrophoresed on SDS-20% polyacrylamide gels and transferred tonitrocellulose by electroblotting. Blots were incubated with a 1:100dilution of rabbit antiserum derived from animals challenged withpurified enzyme, washed, incubated with goat anti-rabbit immuno-globulin G conjugated to alkaline phosphatase, washed, and devel-oped by the addition of Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Top panel, Soluble protein from cellsinduced with 0.5% urea; bottom panel, cells uninduced. Lanes: Aand R, M. morganii; B and J, Proteus mirabilis BU517; C and K,Proteus mirabilis BR2528; D and L, Proteus mirabilis HI4320; E andM, Proteus mirabilis BU7354; F and N, Providencia stuartiiBE2467; G and 0, Proteus vulgaris G01232; H and P, Providenciarettgeri TA1738; I and Q, Providencia rettgeri S15453. Prestainedmolecular size markers (Bio-Rad) are bovine serum albumin (75kDa), ovalbumin (50 kDa), carbonic anhydrase (39 kDa), soybeantrypsin inhibitor (27 kDa), and lysozyme (17 kDa).
similarity to the first 10 amino acids of the 11-kDa Proteussubunit. Like the Proteus enzyme, the Morganella urease
showed marked similarity to the jack bean urease amino acidsequence. The 15-kDa subunit, however, showed no simi-larity to either Proteus or jack bean urease, a surprisingfinding in light of the high degree of conservation that existsfor the other subunits. No more than 4 of 15 amino acidmatches were found when the N-terminal sequence of the15-kDa subunit of the Morganella urease was searched alongthe entire jack bean and deduced Proteus amino acid se-
quences.Antisera which recognized the three Morganella subunits
on Western blots indicated a limited similarity with the large
subunits of the ureases from Providencia and Proteus spe-cies. The antisera were unable to precipitate ureases fromother species, with the single exception of partial precipita-tion of a Providencia rettgeri urease. That Providenciarettgeri is also reported to synthesize a high-molecular-weight enzyme (13) may account for the precipitation by theMorganella antiserum. Neutralization studies showed thatantiserum directed against Morganella urease would notinactivate heterologous enzymes from all strains tested,indicating that despite other similarities seen on Westernblot, the ureases differ enough in composition not to beaffected by heterologous antisera.We described the cloning of a fragment encoding the
urease gene from the chromosomal DNA ofM. morganii andthe expression of the enzyme activity in E. coli. Between 3.3and 6.6 kb of DNA was necessary for enzyme activity as
shown by subcloning and transposon mutagenesis. Theoperon constitutively produces an enzyme with a nativemolecular weight of 590,000, making the native Morganellaurease the largest bacterial urease purified to date andequalled in size only by that of jack bean (18). Most nativemicrobial ureases range in size from 200 to 250 kDa (13, 23).Genes encoding the subunit polypeptides appear to be well
expressed in E. coli as indicated by the amount of proteinrecognized on Western blot by specific antisera. However,because of the very low activity of the recombinant enzyme,we must conclude that either the subunits have not beenassembled into the native enzyme or the native urease hasbeen inactivated in the E. coli host. To a lesser degree thisinactivation was noted during the purification procedure, inwhich some activity was unaccountable between latter pu-rification steps.The Morganella urease is distinct from all other well-
studied bacterial ureases. Unlike Proteus or Providenciaurease, it is expressed constitutively. Antiserum directed
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MORGANELLA MORGANII UREASE 3079
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FIG. 7. Western blot of electrophoresed cell lysates of M. mor-
ganii TA43 and E. coli HB101(pMOM203) encoding cloned urease
gene sequences. Soluble protein (50 Rxg) derived from cell lysatesof M. morganii and E. coli harboring recombinant clones ofM. morganii urease was electrophoresed on an SDS-20o polyacryl-amide gel, transferred to nitrocellulose, and developed by usinganti-M. morganii urease antiserum as described in Materials andMethods. Lanes: A, M. morganii TA43; B, E. coli HB101(pMOM203). Polypeptides with mobilities consistent with those ofurease subunits (63, 15, and 6 kDa) were recognized with equalintensity in the wild-type strain and recombinant clone. Molecularsize markers are as described in the legend to Fig. 2.
against the Morganella urease neither recognized the smallersubunits of the Proteus or Providencia ureases nor was ableto precipitate or neutralize these enzymes. Furthermore,Proteus or Providencia urease gene probes did not hybridizewith Morganella chromosomal DNA (13). Evidence that theenzymes are different is also supported by the observationthat the first 15 amino acid residues of the 15-kDa subunitshowed no similarity with either Proteus or jack beanurease, although the other subunits revealed significantamounts of conservation. Finally, the large size of thepurified native enzyme sets Morganella urease apart fromother bacterial ureases purified to date.The urease synthesized by M. morganii displays some
similarities in subunit structure, antigenic determinants ofthe large subunit, and amino acid sequence with ureasesfrom other genera of the Enterobacteriaceae group. How-ever, it is indeed a distinct enzyme with subunit sizesvarying from those of other species, a higher native molec-ular weight, antigenic differences, and a divergent aminoacid sequence of the intermediate-size (15-kDa) subunit. Inaddition, Morganella urease gene sequences do not hybrid-ize with those of nearest relative Proteus species. Thesedifferences further support the division of Morganella andProteus genera.
ACKNOWLEDGMENTSWe thank Gwynn Chippendale, Cecille Andraos Selim, and Mau-
reen Fox for skilled technical assistance and Linda Home for expertmanuscript preparation.
This research was supported in part by Public Health Servicegrant A123328 from National Institutes of Health.
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