May 5, 1988 - infection. Braude and Siemienenski (4), using a rat model of pyelonephritis, demonstrated that urease-positive represent- atives of the Proteeae ...
JOURNAL
OF BACTERIOLOGY, Aug. 1988, p. 3342-3349 0021-9193/88/083342-08$02.00/0 Copyright © 1988, American Society for Microbiology
Vol. 170, No. 8
Proteus mirabilis Urease: Genetic Organization, Regulation, and Expression of Structural Genes BRADLEY D. JONES AND HARRY L. T. MOBLEY*
Division of Infectious Diseases, Department of Medicine, University of Maryland School Baltimore, Maryland 21201 Received 21 January 1988/Accepted
5
of
Medicine, 10 S. Pine Street,
May 1988
Proteus mirabilis, a cause of serious urinary tract infection, produces urease, an important virulence factor for this species. The enzyme hydrolyzes urea to CO2 and NH3, which initiates struvite or apatite stone formation. Genes encoding urease were localized on a P. mirabilis chromosomal DNA gene bank clone in Escherichia coli by deletion analysis, subcloning, Bal31 nuclease digestion, transposon TnS mutagenesis, and in vitro transcription-translation. A region of DNA between 4.0 and 5.4 kilobases (kb) in length was necessary for urease activity and was located within an 18.5-kb EcoRI fragment. The operon was induced by urea and encoded a multimeric, cytoplasmic enzyme comprising subunit polypeptides of 8,000, 10,000, and 73,000 daltons that were encoded by a single polycistronic mRNA and transcribed in that order. Seventeen urease-negative transposon insertions were isolated that synthesized either none of the structural subunit polypeptides, the 8,000-dalton polypeptide alone, or both the 8,000- and 10,000-dalton subunit polypeptides. The molecular weight of the native enzyme was estimated to be 212,000 by Superose-6 chromatography. Homologous sequences encoding the urease of Providencia stuartii synthesized subunit polypeptides of similar sizes and showed a similar genetic arrangement. However, restriction maps of the operons from the two species were distinct, indicating significant divergence.
Proteus mirabilis, a common cause of urinary tract infection in both catheterized and noncatheterized patients (9, 24, 34, 43), can result in serious complications, including cystitis, prostatitis, urolithiasis, pyelonephritis, and bacteremia (34, 42). The mechanism of pathogenesis is unclear, but several virulence properties have been cited (31) and include motility (28), a uroepithelial cell adhesin (45), fimbriae (1. 9, 27, 37-39), hemolysin production (15, 29, 44), ability to invade kidney epithelium (4, 19, 30), and production of urease (4, 10, 13, 18, 19). Animal models of pyelonephritis have been used to implicate urease as a contributing factor to the severity of infection. Braude and Siemienenski (4), using a rat model of pyelonephritis, demonstrated that urease-positive representatives of the Proteeae tribe colonized kidney epithelium more avidly than did Escherichia (oli and Pseudomonas aeruginosa and produced more severe histological damage than enterococci. MacLaren (18) used an ethylmethane sulfonate-generated urease mutant of P. mirabilis in a mouse model of pyelonephritis and found that renal failure and death were caused by the parent strain but only rarely by the urease-negative mutant. In addition, bacterial urease is recognized as a virulence factor because of its role in kidney and bladder stone formation (10). Alkalinization of the urine by hydrolysis of urea to ammonia and carbon dioxide facilitates precipitation of polyvalent cations and anions, primarily struvite, MgNH4PO4 .6H20 and carbonate-apatite, Ca1(PO04CO3 OH)6(OH). The proportion of stones formed due to infection has been estimated at between 20 and 40% of all urinary stones (10). An additional complication in catheterized patients is the encrustation and blockage of urinary catheters by struvite, which has been uniquely correlated with the presence of P. mirabilis but not other urease-producing species (24). *
Although urease has long been recognized as a virulence factor in this organism, the physical properties of the enzyme and the genetic organization of the genes that encode the protein are just beginning to be understood. Urease gene sequences have recently been cloned by Walz et al. (41) and appear to encode seven polypeptides. However, no function has yet been assigned to any of the gene products. We previously found that the urease genes of P. mirabilis show significant homology with those of Prov,idencia stuartii, as demonstrated by Southern and dot blot hybridization of chromosomal DNA with a Providencia stuiartii urease gene probe (13), but the enzymes reveal considerable differences in molecular size, isoelectric point, affinity for substrate, and activity of induced cell lysates (13). The enzyme itself is a high-molecular-weight protein with an estimated size ranging from 280,000 to 560,000 for enzymes derived from crude preparations from different strains (13, 33) and 200,000 + 30,000 for the highly purified protein derived from one isolate (5; J. M. Brietenbach and R. P. Hausinger, personal communication). The enzyme from induced cell lysates, compared with that from other Proteus, Providencia, and Mor£ganella species, was found to have a lower affinity for the substrate but hydrolyzed urea at a significantly faster rate (13). We report here the molecular cloning, expression, regulation, and genetic organization of the genes encoding the structural subunits of P. ini)abilis urease in E. coli as well as the cellular localization of the enzyme in the wild-type and recombinant hosts. MATERIALS AND METHODS Bacterial strains and growth conditions. P. mirabilis H14320 was isolated from the urine of a nursing home patient with a urinary catheter in place (24). The organism was identified from a pure culture by using the Minitek Enterobacteriaceae II system (BBL Microbiology Systems, Cockeysville, Md.) as described previously (43).
Corresponding author. 3342
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Escherichia coli HB101 (F- hsdR hsdM supE44 proA2 leuB6 rpsL20 recAJ3 lacYl galK2 thi-J ara-14) was the recipient for transformation with recombinant plasmids (20). E. coli P678-54 was used for minicell isolation. E. coli HB101(pMID204) encodes the urease of Providencia stiuartii and has been described previously (23). Bacteria were maintained on Luria agar (20) and transferred weekly. Cultures were grown in Luria broth (20) at 37°C with aeration (200 rpm). A modification of urea segregation agar (8) was used to select urease-positive recombinant clones. Sterile urea was added to a final concentration of 0.1% (wt/vol) after the medium was autoclaved. Gene bank preparation. A gene bank of P. mirabilis H14320 chromosomal DNA was constructed in E. coli by the method of Maniatis et al. (20). Briefly, isolated chromosomal DNA (21) was partially digested with Sau3A, ligated into the BamHI site of plasmid vector pHC79 (12), and packaged in vitro by using the Gigapack lambda packaging kit (Stratagen Cloning Systems, San Diego, Calif.) according to the manufacturer's instructions. This preparation was used to infect E. coli HB101 following growth in Luria broth supplemented with 0.2% (wt/vol) maltose. Transformants were selected on Luria agar containing ampicillin (200 jig/ml). Urease-positive clones were detected by replica-plating onto modified urea segregation agar. Plasmid isolation. Plasmid DNA was isolated by alkaline sodium dodecyl sulfate (SDS) extraction (3) from cultures (200 ml) of E. coli HB101 grown in Luria broth. DNA was purified by centrifugation to equilibrium in cesium chlorideethidium bromide density gradients (20). BaI31 nuclease digestion. Plasmid pMID901 (10 p.g) was digested to completion with EcoRI, precipitated, dried, and dissolved in 100 RI of Bal3l reaction buffer (20). Bal31 (3 U; Bethesda Research Laboratories, Gaithersburg, Md.) was added, and the solution was incubated for 15 min at 32°C. The reaction was stopped by addition of 15 ,u1 of 0.2 M EGTA [ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'tetraacetic acid]) and heating to 70°C for 10 min. The DNA was precipitated, and the ends were rendered flush by filling in with DNA polymerase I Klenow fragment (20). Transposon mutagenesis. E. coli HB101(pMID1003) was infected with bacteriophage lambda 467 (which carries transposon Tn5) by the method of deBruijn and Lupski (7). Plasmid DNA was isolated from pooled kanamycin-resistant transformants and used to transform E. coli HB101. Kanamycin-resistant transformants were screened for urease activity by replica-plating onto modified urea segregation agar. The site and orientation of the transposed element in pMID1003 were estimated by single digests of plasmid DNA with Sall and either BamHI or XhoI. Nondenaturing gel electrophoresis. Urease preparations (10 p.l), prepared as described previously (13, 23), were mixed with equal volumes of 50% sucrose-0.1% bromphenol blue and loaded onto a 7% polyacrylamide gel (8 by 8 by 0.15 cm; N,N'-methylene-bisacrylamide-acrylamide [1:32]; U.S. Biochemical Co., Cleveland, Ohio) with a 4% stacking gel and electrophoresed with the buffers of Senior et al. (36) for 1 h at 200 V. The gel was equilibrated in 0.2% (wt/vol) cresol red-0.1% (wt/vol) EDTA (pH 6.5) until the gel remained yellow (pH c 6.8) (36). The solution was well drained, and the gel was overlaid with a 1.5% (wt/vol) urea solution. Enzymatic activity was visualized by development of a red band at the point of migration of urease. The gel was photographed with Kodachrome 64 slide film (Eastman Kodak, Rochester, N.Y.). In vitro transcription-translation. Purified plasmid DNA (5
P. MIRABILIS UREASE OPERON
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j.g) was transcribed and translated by using [35S]methionine (specific activity, 1,106 Ci/mmol; New England Nuclear Corp., Boston, Mass.) in a volume of 25 RI with an in vitro transcription-translation system according to the instructions of the manufacturer (Digene, College Park, Md.). The reaction was stopped by dilution and addition of 1 ml of cold acetone. Precipitated polypeptides were concentrated by centrifugation and washed once with cold acetone in preparation for electrophoresis. Minicell isolation and labeling. Minicells were isolated from cultures (250 ml of M9 salts medium [20] plus 0.5% Casamino Acids [Difco]) of E. coli P678-54 containing pBR322, pMID1003, or pMID1010 by differential centrifugation and passage through three sequential sucrose step gradients (4.5 ml of 5 to 20% [wt/vol] sucrose) as described previously (22). Minicells were suspended in 0.1 ml of M9 salts and labeled for 1 h at 37°C with 50 ,uCi of [35S] methionine (specific activity, 1,151 Ci/mmol, New England Nuclear). The 19 other common amino acids were included in the labeling mix at 50 jig/ml each. For induction, 0.1% (wtl vol) urea was added just prior to isotope addition. SDS-polyacrylamide gel electrophoresis. The labeled polypeptide products of in vitro transcription-translation or minicells were treated for 5 min at 100°C in SDS gel sample buffer (16). Solubilized proteins (4 x 105 cpm of trichloroacetic acid-precipitable activity per lane) were electrophoresed on a 14% polyacrylamide gel (16 by 16 by 0.15 cm) with a 4% stacking gel and the buffers and method of Laemmli (16). After electrophoresis, gels were fixed in 50% ethanol-10% acetic acid, soaked in 1% glycerol-10% acetic acid, dried onto filter paper under vacuum, and autoradiographed with XAR-2 X-ray film (Eastman Kodak) for 18 h. Prestained molecular weight markers (Sigma Chemical Co., St. Louis, Mo.; Diversified Biotech, Newton Centre, Mass.) were used to estimate molecular size. Spectrophotometric urease assay. Rates of urea hydrolysis were measured by the spectrophotometric assay of Hamilton-Miller and Gargan (11) and as described previously (13). Column chromatography. The urease preparations (13), 0.2 ml containing 2 to 4 mg of protein, derived from either P. mirabilis HI4320 grown in Luria broth supplemented with 0.1% (wt/vol) urea or E. coli HB101(pMID1003) grown in the same medium with ampicillin (200 ,ug/ml), were loaded onto a Superose-6 (Pharmacia/LKB, Piscataway, N.J.) column (1 by 30 cm) previously equilibrated with 20 mM sodium phosphate (pH 6.8)-0.02% (wt/vol) sodium azide or the same buffer with 100 mM KCI. Fractions (0.35 ml) were collected at a flow rate of 0.5 ml/min. Urease activity was determined for each fraction with the spectrophotometric assay. The column was calibrated with blue dextran (2,000 kilodaltons [kDa]; Pharmacia) for void volume determination. Thyroglobulin (660 kDa), ferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa) were used as standards (Pharmacia) of known molecular size. Molecular weight estimations were done in triplicate. Cell fractionation. Cells from stationary-phase cultures (1 liter) of P. mirabilis H14320 and E. coli HB101(pMID801) grown at 37°C with aeration (200 rpm) in Luria broth supplemented with 0.1% urea and, for E. coli, ampicillin (200 jig/ml), were harvested by centrifugation (10,000 x g, 10 min, 4°C). Portions of the supernatant were adjusted to pH 6.8 and placed on ice. Cells were washed two times with 10 mM Tris hydrochloride (pH 8.0) and subjected to the cold osmotic shock procedure of Rosen and Heppel (32). The shock fluid was adjusted to pH 6.8 and placed on ice. Cell pellets (5 g [wet weight]) were suspended in 20 ml of cold 20
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FIG. 1. Localization of P. mirabilis urease genes from a gene bank clone by subcloning, deletion, nuclease digestion, and transposon mutagenesis. A urease-encoding clone (pMID701) was digested with EcoRI and religated, resulting in pMID801. The 18.5-kb EcoRI fragment was subcloned to create pMID901. This plasmid was cleaved with EcoRI and digested with Bal31 nuclease. Blunt ends were prepared with Klenow fragment, and the fragments were ligated into the EcoRV site of pBR322. A urease-positive plasmid containing the smallest DNA insert (pMID1003) was subjected to transposon mutagenesis by the method of deBruijn and Lupski (7). Unique sites of insertion are shown by solid triangles, the urease activity of each insertion mutant is shown by a + or -, and plasmid designations (pMID numbers) are shown below the triangles. Restriction endonuclease sites are indicated above the line. ori, Origin of replication; El, EcoRI; EV, EcoRV.
mM sodium phosphate, pH 6.8, and ruptured in a precooled French pressure cell at 20,000 lb/in2. The cell lysates were centrifuged (10,000 x g, 10 min, 4°C) to remove cellular debris and whole cells, and the supernatant fluid was centrifuged (100,000 x g, 60 min, 4°C). The supernatant (cytosolic fraction) was placed on ice, and the pellet (membrane fraction) was washed twice with 20 mM sodium phosphate buffer, pH 6.8, and suspended in 3 ml of buffer. All fractions were assayed for urease activity. Enzymes known to partition with osmotic shock fluid, cytosol, or membrane were assayed spectrophotometrically for each cell fraction (see footnotes to Table 1). All assays were done in triplicate and standardized for protein content by the method of Lowry et al. (17). Specific activity is expressed as micromoles of substrate hydrolyzed per minute per milligram of protein.
RESULTS Molecular cloning of urease genes. A gene bank of chromosomal DNA from P. mirabilis H14320 was screened for urease activity. Ampicillin-resistant colonies were replicaplated onto modified urea segregation agar, and one ureasepositive clone was identified from among approximately 1.3 x 104 colonies. Plasmid DNA isolated from this clone, designated pMID701, was found to contain an insert of 37 kilobases (kb) of P. mirabilis DNA in pHC79 (Fig. 1). Digestion of pMID701 with EcoRI and religation resulted in the deletion of an 11-kb fragment to yield pMID801, containing a 26-kb insert. This plasmid was digested with EcoRI, and the 18.5-kb fragment was electroeluted from a preparative agarose gel, phenol extracted, and cloned into the EcoRI site of pBR322 to create pMID901. To localize urease gene sequences further, pMID901 was cut to completion with
EcoRI and then subjected to Bal3l nuclease digestion. Blunt ends were prepared by filling in with DNA polymerase I Klenow fragment, and the resulting population of fragments were ligated into the EcoRV site of pBR322. Plasmid DNA was isolated from 12 urease-positive transformants, and the sizes of the inserts were determined. A 12-kb plasmid (pMID1003) was found to have the smallest insert (7.6 kb) that retained urease activity. Transposon mutagenesis of urease genes. E. coli HB101 (pMID1003) was subjected to transposon mutagenesis with TnS. The unique sites of 33 TnS insertions were mapped, and the strains carrying them were screened for urease activity (Fig. 1). Eighteen insertions that resulted in loss of activity covered a 4.0-kb region. Insertions that did not inactivate urease (pMID1433 and pMID1426) and flanked sites of inactivation were separated by 5.4 kb. Urease subunits. Plasmid DNA was isolated from all mutants carrying urease-inactivating transposon insertions and from selected urease-positive mutants. Plasmid-encoded polypeptides were labeled with [35S]methionine in an in vitro transcription-translation system, denatured, and electrophoresed on an SDS-polyacrylamide gel. An autoradiograph of the gel (Fig. 2) on which representative samples were run revealed that phenotypic inactivation of urease was accompanied by loss or truncation of one, two, or three polypeptides with apparent molecular sizes of 8, 10, and 73 kDa. Four insertion mutants produced none of the three polypeptides (pMID1407, -1416, -1417, and -1408). One insertion mutant produced only the 8-kDa polypeptide (pMID1418). Twelve mutants synthesized the 8- and 10-kDa products (pMID1409, -1419, -1420, -1410, -1411, -1421, -1422, -1424, -1412, -1413, -1425, and -1414). In 4 of these 12 insertion
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FIG. 3. Comparison of wild-type and recombinant ureases by gel filtration and gel electrophoresis. Soluble protein from urea-induced cell lysates was electrophoresed on a nondenaturing 7% polyacrylamide gel with a 4% stacking gel and stained for urease activity (inset) and fractionated on a Superose-6 column in the presence (solid symbols) and absence (open symbols) of 100 mM KCI. Fractions were tested for urease activity by the spectrophotometric assay. Symbols and lanes: O, * and A, P. mirabilis H14320; 0, 0 and B, E. coli(pMID1003). Arrows indicate points of elution of standard proteins. Molecular sizes are in kilodaltons.
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FIG. 2. Autoradiograph of electrophoresed 35S-labeled polypeptides from in vitro transcription-translation of transposon mutants of pMID1003. Plasmid DNA was purified from isolates of E. coli HB101(pMID1003) containing transposon Tn5 insertions. Proteins were translated in the presence of [35S]methionine in an in vitro transcription-translation preparation. Solubilized proteins (4 x 105 acid-precipitable cpm per lane) were electrophoresed on a 14% polyacrylamide gel. The gel was fixed, dried, and autoradiographed. Lanes: A, pBR322; B, pMID204 (encodes urease of Providencia stuartii); C, pMID1003, urease+; D, pMID1417, urease-; E, pMID1418, urease-; F, pMID1419, urease-; G, pMID1425, urease-; H, pMID1426, urease+; I, pMID1003, urease+. Prestained molecular size standards were fructose-6-phosphate kinase (84 kDa), pyruvate kinase (68 kDa), fumarase (48.5 kDa), lactate dehydrogenase (36.5 kDa), triosephosphate isomerase (26.6 kDa), trypsin inhibitor (20.4 kDa), myoglobin (16.9 kDa), myoglobin fragment IV (14.4 kDa), myoglobin fragment III (8.2 kDa), and myoglobin fragment 11 (6.2 kDa). Estimated molecular sizes of urease subunit polypeptides are shown at the left (in kilodaltons). Note truncated version of the 73-kDa polypeptide in lane G. a truncated version of the 73-kDa polypeptide was observed (pMID1412, -1413, -1425, and -1414). All of the urease-deficient insertion mutants also failed to produce a faint 21-kDa polypeptide that was produced by pMID1003. This polypeptide has not been observed in purified urease protein (40). Plasmids pMID1003, encoding active P. mirabilis urease, and pMID204, encoding active Providencia stuartii urease, produced the three subunit polypeptides in similar sizes. Enzyme characterization. Cell lysates derived from wildtype and recombinant strains were fractionated on a Superose-6 column, and fractions were tested for urease activity. Peak activities eluted at 14.7 ml, corresponding to an apparent molecular weight of 275,000, when eluted at low ionic strength (Fig. 3). However, when 100 mM KCI was included in the elution buffer to prevent aggregate formation, the enzymes eluted at 15.4 ml, which corresponded to an apparent molecular weight of 212,000. When wild-type and recombinant urease preparations (cell lysates) were electro-
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phoresed and stained for urease activity, identical electrophoretic mobilities were observed (Fig. 3, inset). The Kms for urea did not differ significantly for the wild type and recombinant urease and were consistent with previously reported values (11). Inducibility of urease. Inducibility of the uirease operon was found to depend on the presence of specific gene sequences. A series of 12 Bal31 deletion mutants, derived from pMID901 as shown in Fig. 1, were tested for induction of urease synthesis by urea. Four representative patterns of induction are shown in Fig. 4. Plasmid pMID1003 carried the smallest insert that allowed synthesis of urease and production of a basal amount of enzyme activity. However, this plasmid was not inducible by urea. pMID1001 synthesized urease constitutively at levels sixfold higher than that of pMID1003. The presence of additional DNA sequences in pMID1010 and pMID1004 conferred inducibility on the recombinant plasmids. Plasmid pMID1010 produced basal amounts of enzyme when uninduced but was induced 16-fold when grown in the presence of 0.1% urea. Plasmid pMID1004, the deletion mutant with the largest insert, produced a basal level of enzyme, but its production level was reproducibly increased 1,000-fold when it was grown in the presence of urea. A comparison of the ability of urea to induce urease synthesis in wild-type P. mirabilis and E. coli HB101 (pMID1004) was made. The enzyme activity of cells was measured at various stages of growth in either the presence or absence of urea (Fig. 5). Urea hydrolysis was significantly faster in P. mirabilis H14320 grown with urea than in uninduced cultures at all times tested. Maximal urease production occurred at mid-exponential phase, when the optical density at 660 nm was 0.35 to 0.40 (Fig. 5A). At this point urease production was induced approximately 10-fold over the level in uninduced cultures. Regulatory regions apparently were cloned along with the structural genes for urease, since urease induction could also be demonstrated in E. coli HB101(pMID1004) (Fig. 5B). In this recombinant strain, urease was induced to a level comparable to that in the wild-type strain. Induction was manifested in vivo by increased synthesis
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FIG. 4. Inducible and constitutive urease synthesis by Bal31 deletion mutants. The 18.5-kb EcoRI fragment of pMID901 was digested with Bal31. Ends were rendered flush with DNA polymerase I Klenow fragment, and DNA fragments were ligated into the EcoRV site of pBR322. Plasmid DNA from urease-positive transformants was mapped, and lysates from cells grown in the presence or absence of 0.1% urea were tested for urease activity by the spectrophotometric assay. P. mirabilis gene sequences are represented as thin lines; DNA length is shown in kilobases. The arrow represents the direction of transcription of the structural subunits and spans the length of DNA necessary for urease activity as determined for pMID1003. Orientation of the restriction map is the same as in Fig. 1. Urease activities of uninduced (hatched bars) and induced (solid bars) cells are shown.
of urease structural subunits. Plasmid-encoded polypeptides were radiolabeled in minicells isolated from E. coli P678-54 harboring either the uninducible pMID1003 or the inducible pMID1010. Denatured proteins were electrophoresed on an
H14320 and E. coli HB1O1(pMID801) were fractionated. Urease assays were done for each fraction along with assays for control enzymes known to partition with osmotic shock fluid, membrane, or cytosol (Table 1). For P. mirabilis, 87% of urease activity and 73% of catalase activity, a known cytosolic protein (14), fractionated with the cytosolic fraction. None of the urease activity and 13% of the urease activity were associated with membrane and periplasmic fractions, respectively. As controls, 98% of NADH dehydrogenase was membrane associated and 96% of alkaline phosphatase fractionated with the osmotic shock fluid, rep-
SDS-polyacrylamide gel and autoradiographed (Fig. 6). The of urea for pMID1003 did not influence the relative amounts of the largest urease structural polypeptide (73 kDa) synthesized; whereas for pMID1010, exposure to urea synthesis of this subunit polypeptide approximately 10-fold. Cellular localization of urease. To determine the cellular compartment in which urease resides, cells of P. mirabilis
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24 5 3 4 0 24 3 5 4 l 2 TIME (HR) TIME (HR) FIG. 5. Induction of wild-type and recombinant ureases. Suspensions of P. mirabilis H14320 (A) or E. coli HB101(pMID1004) (B) from overnight cultures were inoculated into'Luria broth with and without 0.1% urea and grown at 37°C with aeration. Growth was monitored at 660 nm, and cells were periodically harvested, washed, and lysed by passage through a French pressure cell. Urease activity was determined in the soluble protein by the spectrophotometric assay. Symbols: Ol, *, induced cultures; 0, 0, uninduced cultures; 0, O, optical density; 0, E, urease activity. 0
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FIG. 6. Autoradiograph of electrophoresed [35S]methioninelabeled plasmid-encoded polypeptides from minicell lysates. Minicells were isolated from E. coli P678-54 containing urease-encoding or vector plasmids. Proteins were labeled with [35S]methionine in the presence (+) or absence (-) of urea, denatured in gel sample buffer, and electrophoresed on a 14% SDS-polyacrylamide gel. The gel was dried and autoradiographed. (A) pBR322; (B) pMID1003; (C) pMID1010. Arrow, position of the 73-kDa subunit polypeptide. Molecular size standards are given (in kilodaltons).
resentative of periplasmic proteins. Recombinant urease in E. coli partitioned with the soluble protein fractions (cytosol and periplasm); however, the majority (75%) of the activity was found in the osmotic shock fluid. Control enzymes partitioned as expected.
DISCUSSION Between 4.0 and 5.4 kb of P. mirabilis chromosomal DNA is necessary to encode an active urease. Additional DNA sequences are necessary for regulation of enzyme synthesis. The operon, inducible by urea, encodes a multimeric enzyme comprising subunit polypeptides with apparent molecular sizes of 8, 10, and 73 kDa. The subunits are encoded on a single polycistronic mRNA and are transcribed in that order. The gene products appear to be similar in size to urease subunits of several other bacterial species that have been identified directly by purification of the proteins. The ureases of Selenomonas ruminantium, Klebsiella aerogenes, and Sporosarcina ureae, as well as those of P. mirabilis, were described by Todd and Hausinger (40) as each having one large subunit of 65 to 72 kDa and two smaller subunits, each between 8 and 11 kDa in size. This is in agreement with the subunit structure of the urease from the closely related organism Providencia stuartii, which also has been determined by enzyme purification and by transposon mutagenesis of cloned gene sequences (25).
3347
With the terms alpha, beta, and gamma (40) used to designate the 73-, 10-, and 8-kDa polypeptides, respectively, and by assuming that the subunits are present in the same ratio found for K. aerogenes (40) and Providencia stuartii (25), the P. mirabilis native urease structure is consistent with an (at12Y2)2 configuration. This formula would yield an apparent molecular size of 218 kDa, which is consistent with the observed estimations of 212 and 200 kDa native molecular size obtained by using Superose 6 columns equilibrated with 100 mM KCl (Fig. 3; J. M. Brietenbach and R. P. Hausinger, personal communication). However, additional data on subunit ratios are required to support this hypothesized native structure. Previous estimations made under conditions of low ionic strength (13) yielded higher values for native molecular size, possibly owing to aggregation with additional subunits or other proteins. The P. mirabilis genes show homology with the urease operon of the related species Providencia stuartii (13), which also encodes three subunit polypeptides of very similar sizes. These subunits were seen both on a Coomassie blue-stained SDS-polyacrylamide gel of purified urease and by in vitro transcription and translation of plasmid DNA bearing the urease genes (24). Transposon mutagenesis revealed a similar organization of the subunit genes for both Providencia stuartii and P. mirabilis, that is, the genes for the 8-, 10-, and 73-kDa polypeptides transcribe a single mRNA encoding subunits of increasing size (Fig. 7). Although these gene sequences are clearly homologous, as determined previously by DNA hybridization experiments (13), the restriction maps of the two operons and biochemical characteristics of the enzymes of the two species indicate significant divergence. Our data are also consistent with the recent report of Walz et al. (41), who delineated the polypeptides encoded by the P. mirabilis urease operon. Although no function was assigned to any of the polypeptides, three gene products of 5.2, 7.5, and 68 kDa were described and would be encoded by adjacent genes. By aligning the restriction map shown in Fig. 1 with that of Walz and colleagues (Fig. 4 in reference 41; map pictured in opposite orientation), these three peptides probably also represent the three structural subunits of the P. mirabilis urease. In the only other report to have appeared that describes the genetic organization of genes encoding a bacterial urease, Collins and Falkow (6) demonstrated that a transposon mutation of the E. coli urease locus that resulted in loss of synthesis of a 67-kDa polypeptide also led to a urease-negative phenotype. This polypeptide probably represents the largest of three subunits of the E. coli urease.
TABLE 1. Enzyme activity of cell fractions Sp act (p.mol of substrate/min per mg of protein) Enzyme assayeda
P. mirabilis HI4320
Cytosol
Urease Catalase
Membrane
E. coli HB101(pMID801)
Periplasm
2.0 55.3
0.0 0.6
0.3 8.1
9.0 110.0
680.0 88.0
4.0 4,089.0
P-Galactosidase NADH dehydrogenase Alkaline phosphatase
Cytosol
0.14 26.8 84.0 2.0
Membrane
0.0 0.0 1,398.0 1.0
Periplasm
0.41 1.8 9.0 44.0
a Activity was measured as follows. Urease: Urea hydrolysis measured in the presence of phenol red monitored at 560 nm. Catalase: H202 absorbance monitored at 240 nm (14). P-Galactosidase: o-Nitrophenylgalactoside hydrolysis monitored at 420 nm (35). NADH dehydrogenase: NADH absorbance monitored at 340 nm (2). Alkaline phosphatase: p-Nitrophenylphosphate hydrolysis monitored at 420 nm (26).
3348
J.BACTERIOL.
JONES AND MOBLEY P. mirabilis 0
'
2
0-Iu
c
0.
X
a
I l
X-
II
I II
El M 1
ACKNOWLEDGMENTS This work was supported in part by Public Health Service grants A123328 and AG04393 from the National Institutes of Health. We thank Merrill Snyder and Robert Hausinger for editorial
-
t
review.
1
73
LITERATURE CITED
I
1. P.
stuartil a
-=
%.
>
>
=W
--
-
)(W0
ti
-.
la a
l am
k
a
l
6
Rs
q
Q).
i-'
l
2.
Ir
s
I1iL
73
i
FIG. 7. Proposed genetic organization of the urease operons of P. mirabilis and Providencia stuartii. Restriction maps of the DNA sequences found to encode urease in P. mirabilis and Providencia stuartii are shown with approximate locations of structural subunit genes indicated as rectangles. The arrow indicates the direction of transcription and covers the sequences essential for urease activity. Polypeptide products are assigned apparent molecular sizes (in kilodaltons).
3.
4. 5. 6. 7.
As demonstrated for Providencia stuartii (23), the P. mirabilis urease is a cytoplasmic protein, as determined by cell fractionation (Table 1). This assignment is consistent with its multimeric native structure, as only a very few multiple-subunit proteins are secreted from the bacterial cell. In addition, we saw no evidence of protein processing during minicell analysis. Our observation is not in agreement with the work of McLean et al. (22), who found the majority of enzyme in the periplasm and associated with the outer
membrane of a P. mirabilis strain. However, the activities of control enzymes known to partition with cell fractions were not reported. In the recombinant host E. coli HB101
8.
9.
10.
(pMID801), however, a significant percentage of the urease activity partitioned with the periplasmic fraction. Except that urease is a foreign protein in E. coli, we have no explanation for this observation. Regions of the urease operon not associated with synthesis of the three structural subunit polypeptides nevertheless modulate enzyme activity. First, regulatory regions appear to lie upstream from the structural subunit genes. pMID1001 (Fig. 4) synthesized urease constitutively, apparently because it lacked the sequences that encode a putative repressor. pMID1010 is regulated and, when exposed to urea as inducer, synthesizes urease at the constitutive level. This plasmid contains an additional 1.45 kb of DNA upstream compared with pMID1001 and 0.6 kb less DNA downstream than the constitutive mutant. Although we show clear evidence of induction, these results must be interpreted with caution because the DNA lengths varied at both the 5' and 3' ends. In addition, we can provide no adequate explanation of why pMID1004 bestowed a "superinducible" phenotype on the host bacterium, since this activity far exceeded the urease activity encoded by the parent plasmid (pMID901, Fig. 1) from which it was derived. Second, DNA sequences
downstream from the structural genes are necessary for synthesis of an active urease. Plasmid pMID1434 carries a transposon insertion in this region that eliminates enzyme
11. 12.
13.
14.
Adegbola, R. A., D. C. Old, and B. W. Senior. 1983. The adhesins and fimbriae of Proteus mirabilis strains associated with high and low affinity for the urinary tract. J. Med. Microbiol. 16:427-431. Adler, L. W., T. Ichikawa, S. M. Hasan, T. Tsuchiya, and B. P. Rosen. 1977. Orientation of the protonmotive force in membrane vesicles of Escherichia coli. J. Supramol. Struct. 7:15-27. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. Braude, A. I., and J. Siemienenski. 1960. Role of bacterial urease in experimental pyelonephritis. J. Bacteriol. 80:171-179. Brietenbach, J. M., and R. P. Hausinger. 1988. Proteus mirabilis urease: partial purification and inhibition by boric and boronic acids. Biochem. J. 250:917-920. Collins, C. M., and S. Falkow. 1988. Genetic analysis of an
Escherichia coli urease locus: evidence of DNA rearrangement. J. Bacteriol. 170:1041-1045. deBruin, F. J., and J. R. Lupski. 1984. The use of transposon TnS mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids-a review. Gene 27:131-149. Farmer, J. J.,III, F. W. Hickman, D. J. Brenner, M. Schreiber, and D. G. Rickenbach. 1977. Unusual Enterobacteriaceae: "Proteus rettgeri" that "change" into Providencia stuartii. J. Clin. Microbiol. 6:373-378. Fowler, E. J., and T. A. Stamey. 1978. Studies of introital colonization in women with recurrent urinary infections. X. Adhesive properties of Escherichia coli and Proteus mirabilis: lack of correlation with urinary pathogenicity.J. Urol. 120:315318. Griffith, D. P., D. M. Musher, and C. Itin. 1976. Urease: the primary cause of infection-induced urinary stones.Invest. Urol. 13:346-350. Hamilton-Miller, J. M. T., and R. A. Gargan. 1977. Rapid screening for urease inhibitors. Invest. Urol. 16:327-328. Hohn, B., and J. Collins. 1980. A small cosmid for efficient cloning of large DNA fragments. Gene 11:291-298. Jones, B. D., and H. L. T. Mobley. 1987. Genetic and biochemical diversity of ureases of Proteus, Providencia, and Morganella species isolated from urinary tract infection. Infect. Immun. 55:2198-2203.
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properties of the
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activity even though the site of insertion is well outside the
19. MacLaren, D. M. 1969. The significance of urease in Proteus pyelonephritis: a histological and biochemical study. J. Pathol.
sequences for all three structural subunits. We can postulate
20.
that gene products encoded by this region may play a role in enzyme assembly, nickel transport, or nickel insertion and have yet to be elucidated.
21.
Bacteriol. 97:43-49. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular
cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
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VOL. 170, 1988
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34. Rubin, R. H., N. E. Tolkoff-Rubin, and R. S. Cotran. 1986. Urinary tract infection, pyelonephritis, and reflux nephropathy, p. 1085-1141. In B. M. Brenner and F. C. Rector (ed.), The kidney. W. B. Saunders Co., Philadelphia. 35. Schlammadinger, J., and G. Szabo. 1971. The effect of theophylline upon induced beta-galactosidase in Escherichia coli. Acta Microbiol. Acad. Sci. Hung. 18:55-59. 36. Senior, B. W., N. C. Bradford, and D. S. Simpson. 1980. The ureases of Proteus strains in relation to virulence for the urinary tract. J. Med. Microbiol. 13:507-512. 37. Silverblatt, F. J. 1974. Host-parasite interaction in the renal pelvis. A possible role for pili in the pathogenesis of pyelonephritis. J. Exp. Med. 140:1696-1711. 38. Silverblatt, F. J., and I. Ofek. 1978. Influence of pili on the virulence of Proteus mirabilis in experimental hematogenous pyelonephritis. J. Infect. Dis. 138:664-667. 39. Svanborg-Eden, C., P. Larsson, and H. Lomberg. 1980. Attachment of Proteus mirabilis to human urinary sediment epithelial cells in vitro is different from that of Escherichia coli. Infect. Immun. 27:804-807. 40. 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. 41. Walz, S. E., 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. 42. Warren, J. W., D. Damron, J. H. Tenney, J. M. Hoopes, B. Deforge, and H. L. Muncie, Jr. 1987. Fever, bacteremia, and death as complications of bacteriuria in women with long-term urethral catheters. J. Infect. Dis. 155:1151-1158. 43. Warren, J. W., J. H. Tenney, J. M. Hoopes, H. 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. 44. Welch, R. A. 1987. Identification of two different hemolysin determinants in uropathogenic Proteus isolates. Infect. Immun. 55:2183-2190. 45. Wray, S. K., S. I. Hull, R. G. Cook, J. Barrish, and R. A. Hull. 1986. Identification and characterization of a urinary cell adhesin from a uropathogenic isolate of Proteus mirabilis. Infect. Immun. 54:43-49.
