JOURNAL OF BACTERIOLOGY, Aug. 1976, p. 1032-1035 Copyright ©D 1976 American Society for Microbiology

Vol. 127, No. 2 Printed in U.S.A.

Cellular Localization of Uridine 5'-Diphosphate-N-Acetyl-DGlucosamine Oxidoreductase Activity in Citrobacter freundii ROBERT W. WHEAT,* ROBERT 0. BARROW,' AND GEOFFREY A. LAND2 Departments of Microbiology, Immunology, and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 Received for publication 27 February 1976

The uridine 5'-diphosphate-N-acetyl-D-glucosamine oxidoreductase activity of Citrobacter freundii ATCC 10053 was found to be located in the soluble cytoplasmic fraction of lysed spheroplasts. The occurrence in Citrobacter freundii ATCC 10053 of one or more uridine diphosphate-Nacetyl-D-glucosamine (UDPGlcNAc) oxidoreductase (UDPGlcNAc-ORase) activities which convert UDPGlcNAc to the N-acetylquinovosamine and N-acetylfucosamine precursor, UPD4-keto-QuiNAc (i.e., uridine 5'-diphosphate-4keto-2-N-acetyl-2,6-dideoxyglucose), and an additional unidentified uridine 5'-diphosphateketo-2-N-acetyl-2,6-dideoxyhexose was demonstrated by Daniel et al. (1), indicating the possibility of two enzyme activities. Both UDP-4keto products are measurable as 335-nm absorbing compounds in 0.1 N alkali (1). Production of both products can be shown to require nicotinamide adenine dinucleotide (A. Daniel and R. W. Wheat, unpublished data). A lyophilized, washed residue of acetone-powdered whole cells obtained from overnight cultures was used for most of these prior studies. The acetone-dried cell enzyme preparation was stable and could be stored with no loss of activity for several years (1), although once solubilized, 50% or more loss of activity overnight was not unusual. In addition preliminary attempts to solubilize and purify the enzyme from acetonedried cells indicated the presence of two forms of the enzyme, one being easily water extractable and the other particulate. The particulate activity, which varied from 30 to 50% of the total activity, was slowly released in soluble form with extended times of extraction and could be maximally demonstrated only after sonic disruption. Even after sonic oscillation a portion of the enzyme activity sometimes appeared to be particulate, indicating again the possibility of two forms of the enzyme. The present work was therefore undertaken to determine the cellular location of the ' Present address: Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria. 2 Present address: Wadley Institutes of Molecular Medicine, Dallas, Tex. 75235.

UDPGlcNAc-ORase activity or activities in this bacterium. C. freundii ATCC 10053, maintained on nutrient agar, was grown on nutrient broth (Difco Laboratories) with 1% glucose overnight (16 h). Sterile nutrient broth (400 ml) containing 10% glucose (added aseptically) in a 2-liter Erlenmeyer flask was inoculated with 5 ml of an overnight culture of C. freundii ATCC 10053 and incubated at 37°C with vigorous shaking. For preparation of spheroplasts, cells were harvested in log phase growth after 4 to 5 h of incubation, i.e., when absorbance reached 0.4 to 0.60 at 600 nm (Spectronic 20). Logarithmic growth was completed by 12 h, and the stationary phase cells were therefore harvested after overnight growth at about 16 to 18 h. Cells were harvested by centrifugation at 10,000 x g for 5 min at 0 to 4°C, and 800 mg (wet weight) was suspended in 20 ml of the required diluent (40 mg/ml). Different cell batches of both log and stationary phase cells were used for all experiments listed in Table 1. In experiments indicated as A and B, equal amounts (800 mg each) of the same cell batch of either log or stationary phase cells were used for more direct comparative purposes. Osmotic shock experiments were carried out according to conditions worked out for C. freundii by Neu and Chou (4). Spheroplasts were prepared by the procedure of Osborn et al. (5). Conversion of log phase cells to spheroplast (>98%), monitored by phase-contrast microscopy, was usually complete within 5 to 30 min, whereas stationary phase cells required up to 90 min for partial conversion to spheroplasts. Outer and cytoplasmic membranes were separated by the procedure of Schnaitman (7) as modified by Koplow and Goldfine (3). Total membranes (e.g., 10.4 mg of protein) were suspended in cold 25% sucrose containing 5 mM ethylenediaminetetraacetate (EDTA), pH 7.5, and layered onto a discontinuous gradient




VOL. 127, 1976

TABLE 1. Solubilization of C. freundii ATCC 10053 UDPGlcNAc-ORase activity UDPGlcNAc-ORase (total act) Conditions and treatment




Cells were suspended in 0.01 M Tris-hydrochloride (pH 7.8) and centrifugeda before sonic treatment Residue' Supernatant



Stationary phase cells

141.5 5.6

135.5 10.8

2.4 306.0

74.0 152.0

Cells as above, were sonically treated before centrifugationa

Residueb Supernatant 3

Log phase cells

Cells were osmotically shocked (+0.5 mM MgCl2), centrifugeda A. No MgCl2 Residue Residue after freeze-thawing residue Supernatant (periplasm) B. With MgCl2 Residue Residue after freeze-thawing residue

40.1 190.0 5.6 8.0 30.0

Cells were converted to spheroplasts and centrifugeda Residue (spheroplasts, sonically treated) Supernatant (periplasm)

296.0 6.0

Spheroplasts A. Osmotically shocked, centrifugeda Residue' Supernatant B. Sonically treated, centrifuged"

7.2 264.0

65.0 150.8




Supernatant, recentrifugeda 3.8 4.3 Residue 167.8 264.0 Supernatant a Centrifugation at 30,000 x g/60 min per 4°C. b Subjected to sonic treatment for 4 min at 0 to 10°C in 1-min bursts in a Branson model S110 Sonifier and recentrifuged at 30,000 x g for 1 h at 0 to 4°C the second 30,000 x g supernatant solution was tested for UDPGlcNAc-ORase activity. c Centrifugation at 3,000 x g/30 min. d Negligible 3,000 x g residue.

which contained 4.8 ml of 2.02 M, 6.8 ml of 1.44 M, and 12 ml of 0.77 M sucrose solutions, each containing 5 mM EDTA (pH 7.5). Gradients were centrifuged at 26,000 rpm in a Spinco SW27 rotor for 15 h. The two membrane fractions were visually apparent after centrifugation. Fractions of approximately 1 ml were collected by removal of material from the bottom of the gradient tube with a coarse needle and peristaltic pump. The absorbance of each fraction was read at 280 and 260 nm. Peak fractions were pooled and diluted fourfold with Tris-hydrochloride buffer and centrifuged at 270,000 x g for 2 h in a fixed angle rotor. The pellets were then suspended in 10 mM Tris-hydrochloride

buffer (pH 7.8) containing 0.5 mM each EDTA and diethiothreitol, and suitable portions were assayed for UDPGlcNAc-ORase as described below and for reduced nicotinamide adenine dinucleotide oxidase as a marker for inner membranes as described by Osborn et al. (5). Calibration of gradients and initial locations of outer-membrane fractions were determined by use of a ['4C]galactose-labeled lipopolysaccharide outer-membrane marker prepared from a uridine 5'-diphosphate-galactose-4-epimeraseless mutant, G-30, of Salmonella typhimurium LT-2, graciously donated by Osborn and Rothfield (5), and later, by visual observation and by assay of periodate-thiobarbituric acid reactive




material as described by Osborn et al. (5), as a marker for outer-membrane lipopolysaccharide of C. freundii ATCC 10053. UDPGlcNAc-ORase activity was assayed in reaction mixtures of 0.4-ml total volume (containing 0.2 ml of enzyme fraction or dilution, 5 mM UDPGlcNAc, 100 mM Tris-hydrochloride [pH 7.8], and 0.5 mM each nicotinamide adenine dinucleotide, EDTA, and dithiothreitol) which were incubated for 1 h at 37°C (compare with reference 1). The mixture was then heated for 30 s in a boiling-water bath and centrifuged at 0 to 4°C at 15,000 x g for 30 min, and 0.1 ml of the supernatant was mixed with 0.9 ml of 0.11 M NaOH. Absorbance at 335 nm was recorded 2 min after mixing. UPDGlcNAc-ORase activity units are reported as product formed per hour at 370C, measured as optical density units at 335 nm in 0.1 N NaOH. The UDPGlcNAc-ORase activity of C. freundii ATCC 10053 can easily be sedimented by centrifugation and remains particulate and nondemonstrable until the cells are lysed by one of several methods (Table 1). A periplasmic (e.g., compare with reference 4) or peripheral cytoplasmic membrane (e.g., compare with references 2, 4, and 6) location of the UDPGlcNAcORase was ruled out by experiments based on the work of Neu and Chou, who previously demonstrated that periplasmic hydrolases can be extracted from C. freundii cells by osmotic shock (4). Upon repeating Neu and Chou's experiments with C. freundii ATCC 10053, we were able to confirm that 5-nucleotidase activity was released by osmotic shock (not shown), whereas very little UDPGlcNAc-ORase activity (Table 1, experiment 3A) was released until after freeze-thaw (or sonic) treatment of the osmotically shocked cell residue. Inclusion of 0.5 mM MgCl., in the sucrose-Tris-hydrochloride-EDTA preshock fluid (to stabilize the cytoplasmic membrane [compare with reference 4]) markedly increased the difficulty of solubilizing UDPGlcNAc-ORase activity by freezethaw of the osmotically shocked cell residue (Table 1, experiment 3B). The results from experiments 3A and 3B indicated a cytoplasmic or inner (i.e., cytoplasmic)-membrane association or location of the enzyme. Localization of activity in the cytoplasm was indicated in experiments 4 and 5 of Table 1, in which more than 95% of the enzyme activity was observed in the soluble fraction of lysed spheroplasts. Also indicated in Table 1 is that lysis by sonic and lysozyme-EDTA-osmotic shock treatments yielded very similar results which depended upon the age of the cells used. That is, cells harvested in approximate mid-log

phase (4 to 5 h of growth) were susceptible to lysis by both lysozyme-EDTA-osmotic shock and brief sonic treatment, and enzyme activity was predominantly located in the 30,000 x g supernatant solution. However, cells in stationary phase from overnight cultures (16-h growth) were found to be less susceptible under the same conditions of sonic or lysozymeEDTA-osmotic shock treatment. Extended sonic treatment was necessary to demonstrate that some 30% or more of the UDPGlcNAcORase activity was invariably present in the 30,000 x g residue of 16 h or older cells. This sonic treatment-resistant activity could also be sedimented by interposing a 3,000 x g centrifugation of 20 to 60 min before sedimentation at 30,000 x g. The 3,000 x g residue was found by microscopic examination to contain mostly unbroken cells. The 3,000 x g residue "resistant" cells were subsequently observed in overnight or older cultures but not in 4-h cultures (e.g., Table 1, experiment 5B). Further examination of the 16-h, 3,000 x g sedimentable whole-cell fraction revealed that these cells slowly formed spheroplasts with additional lysozyme treatment (e.g., up to 90 min). Lysis of these "late" spheroplasts released UDPGlcNAc-ORase activity which was soluble after centrifugation for 2 h at 270,000 x g. Brief sonic treatment of the 4-h cell culture spheroplasts, followed by centrifugation at 270,000 x g for 2 h to sediment membranes, yielded UDPGlcNAc-ORase activity almost quantitatively (>98%) located in the 270,000 x g soluble fraction (Table 2). An expected observation was that UDPGlcNAc-ORase activity in solutions prepared from spheroplasts was more stable during storage than were soluble prepaTABLE 2. Distribution of UDPGkcNAc-ORase activity in C. freundii ATCC 10053" Fractions

UDPGlc- NADH ORase oxidase (sp act) ( act)

1. Cells (sonically treated) 2. Spheroplasts (sonically treated) a. 270,000 x g supernatant b. 270,000 x g residue



2.5 0.0

0.0015 0.270

3. Membranes (from 2b) separated on sucrose density gradient a. Inner (cytoplasmic membrane) b. Outer membrane

0.0 0.0

0.36 0.027

a UDPGlcNAc-ORase specific activity: Micromoles per minute per milligram of protein; assayed as described by Osborn et al. (5). Nicotinamide adenine dinucleotide (NADH) oxidase specific activity: Optical density units, at 335 nm, per hour per milligram of protein; assayed as described in the text.


VOL. 127, 1976

rations made from whole acetone-dried or lyophilized cells. And finally, neither the envelope fraction (270,000 x g residue) nor the outer- or inner-membrane fractions prepared from the washed 270,000 x g pellet by discontinuous sucrose density gradient centrifugations according to the procedure of Schnaitman (7), as modified by Koplow and Goldfine (3), retained significant UDPGlcNAc-ORase activity (Table 2). It appears, therefore, that UDPGlcNAc-ORase activity of C. freundii ATCC 10053 is cytoplasmic and is not intrinsically part of the periplasm or the outer- or inner-membrane complexes.



4. 5.

This research was supported by Public Health Service grant AI-01659 from the National Institute of Allergy and Infectious Diseases.


LITERATURE CITED 1. Daniel, A., R. A. Raff, and W. R. Wheat. 1972. Identifi-



cation of products of the uridinediphospho-N-acetylD-glucosamine oxidoreductase system from Citrobacter freundii ATCC 10053. J. Bacteriol. 110:110-116. Davies, J. E., and R. E. Benveniste. 1974. Enzymes that inactivate antibiotics in transit to their targets. Ann. N.Y. Acad. Sci. 235:130-136. Koplow, J., and H. Goldfine. 1974. Alterations in the outer membrane of the cell envelope of heptose-deficient mutants of Escherichia coli. J. Bacteriol. 117:527-543. Neu, H. C., and J. Chou. 1967. Release of surface enzymes inEnterobacteriaceae by osmotic shock. J. Bacteriol. 94:1934-1945. Osborn, M. J., J. E. Gander, E. Parisi, and J. Carson. 1972. Mechanism of assembly of the outer membrane ofSalmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J. Biol. Chem. 247:3962-3972. Richmond, M. H., and N. A. C. Curtis. 1974. The interplay of 13-lactamases and intrinsic factors in the resistance of gram-negative bacteria to penicillins and cephalosporins. Ann. N.Y. Acad. Sci. 235:553-567. Schnaitman, C. A. 1970. Protein composition of the cell wall and cytoplasmic membrane of Escherichia coli. J. Bacteriol. 104:890-901.

Cellular localization of uridine 5'-diphosphate-N-acetyl-D-glucosamine oxidoreductase activity in Citrobacter freundii.

JOURNAL OF BACTERIOLOGY, Aug. 1976, p. 1032-1035 Copyright ©D 1976 American Society for Microbiology Vol. 127, No. 2 Printed in U.S.A. Cellular Loca...
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