Vol. 124, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, OCt. 1975, p. 262-268 Copyright i 1975 American Society for Microbiology
Maltose Metabolism of Pseudomonas fluorescens ARTHUR A. GUFFANTI1 AND W. A. CORPE* Department of Biological Sciences, Columbia University, New York, New York 10027
Received for publication 31 July 1975
Pseudomonas fluorescens W uses maltose exclusively by hydrolyzing it to glucose via an inducible alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20). No evidence for phosphorolytic cleavage or oxidation to maltobionic acid was found in this organism. The alpha-glucosidase was totally intracellular and was most active at pH of 7.0. Induction occurred when cells were incubated with maltotriose or maltose. Induction was rapid and easily detectable within the first 5 min after the addition of the inducer. Glucose and its derivatives did not repress induction. Cells growing on DL-alanine or succinate plus maltose exhibited lower levels of alpha-glucosidase than those grown on maltose alone or maltose plus glucose. Induction required both messenger ribonucleic acid and protein synthesis. The utilization of disaccharides by bacteria is often initiated by hydrolysis of the sugar into its monosaccharide components. Simple hydrolysis of maltose to glucose by alpha-glucosidase occurs in bacteria (2, 11), but not invariably, since at least three other mechanisms of maltose utilization are known (1, 2, 5, 9). Pseudomonas fluorescens W, isolated from soil and grown in complex media, was found to secrete many hydrolases (24, 25) into the extracellular environment. Included among the enzymes were small amounts of maltose-hydrolyzing activity (25). The disaccharide hydrolases of bacteria are usually thought to be cell bound, but a surprising paucity of information about the formation and biosynthetic control of alphaglucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) of bacteria is available. It was with these facts in mind that the present work was undertaken. MATERIALS AND METHODS Organism and growth media. The bacterium studied in this work was isolated from soil by T. L. Whiteside and identified as P. fluorescens (24). The organism, designated as strain W, was routinely transferred every 2 weeks on slants of peptoneglucose-yeast extract agar (24) and, after growth for 24 h at 30 C, stored at 4 C. The defined medium used in the experiments described in this paper contained the following ingredients in grams per liter of deionized water: (NH,)2S04, 2.0; KH2PO4, 5.3; Na2HPO4, 8.66; MgSO4 7H20, 0.2; NaCl, 0.01; FeSO4 7H20, 0.01; and MnSO4, H20, 0.01. DL-Methionine, 30 Ag/ml, was supplied as the only required growth factor. The pH 'Present address: Department of Biochemistry, Mount Sinai School of Medicine of the City University of New York, New York, N.Y. 10029.
was 7.0 and did not change during growth. The carbon sources used in various experiments were DL-alanine, succinate, glucose, or maltose. They were autoclaved separately (121 C for 15 min) and added aseptically to the rest of the sterile medium, to give a final concentration of 14 mM maltose, 28 mM glucose, 56 mM DL-alanine, or 19 mM succinate. The organism was grown in 500-ml Erlenmeyer side arm flasks (Belco Glass Co., Vineland, N.J.), 50 ml per flask, at 30 C on a rotary shaker. One milliliter of a late log-phase culture was used as the inoculum. Growth was followed as increase in culture turibidity using a Klett-Summerson photoelectric colorimeter with a number 66 filter. Preparation of cell extracts and other cell fractions. Cells from 50 ml of culture were harvested by centrifugation in the cold (4 C) for 15 min at 12,000 x g in a Servall model SS34 centrifuge (Sorvall Co., Norwalk, Conn.). They were washed once with 20 ml of cold 0.1 M phosphate buffer, pH 7.0, suspended in 5 ml of the same buffer, and ruptured by ultrasonic oscillation in a cuvette suspended in an ice bath for 10 min at maximum intensity with an MSE ultrasonic device (Instrumentation Association Inc., New York, N.Y.). The suspension of ruptured cells was centrifuged in the cold for 20 min at 27,000 x g. The supernatant fluid was carefully decanted and designated as cell extract. The procedure for the localization of alpha-glucosidase differed from that described above. Extracellular enzyme was recovered from a late log-phase culture supernatant fluid by precipitation with 90% saturation ammonium sulfate according to the method of Whiteside and Corpe (24). The protein precipitate was dissolved in a small volume of 0.1 M phosphate buffer, pH 7.0. The cells, which had been sedimented at 27,000 x g for 15 min, were washed twice in the same buffer at 4 C, suspended in 5 ml of buffer, and broken by ultrasonic oscillation for 15 min. They were centrifuged at 1,085 x g for 30 min to remove unbroken cells. The supernatant fluid containing cell
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MALTOSE METABOLISM OF P. FLUORESCENS
envelopes was then centrifuged at 27,000 x g for 30 min. The resulting supernatant fluid was termed "extract," and the sediment containing cell envelopes was suspended and washed several times. This fraction was termed "envelopes." All three fractions were assayed for alpha-glucosidase activity by hydrolysis of para-nitrophenyl-a-D-glucoside (a-PNPG). Assay for alpha-glucosidase activity. The release of glucose from maltose by cell extracts was assayed by a variation of the technique of Kusch and Wilson for beta-galactosidase (12). The reaction mixture (5 ml total volume) contained 4 mg of extract protein, 0.1 M phosphate buffer, pH 7.0, and various concentrations of maltose (10, 5, 2.5, and 1.25 mM). The reaction proceeded at 30 C and was stopped with Ba(OH)2 and ZnSO4 exactly as described by Kusch and Wilson. Glucose was assayed by the Glucostat method according to the manufacturer's instructions (Worthington Biochemical Corp., Freehold, N.J.). The Km and Vmax were determined from a Lineweaver-Burk plot. Alpha-glucosidase activity was also assayed with a-PNPG as the substrate, using a variation of the procedure of Han and Srinivasan for beta-glucosidase (6). The reaction mixture contained a sample of cell extract, envelopes, or extracellular enzyme in 2.5 ml of 0.1 M phosphate buffer, pH 7.0. After equilibration for 5 min in a 30 C water bath, 0.5 ml of 5 mM a-PNPG was added, and the reaction was allowed to proceed for 30 min. The reaction was stopped by the addition of 2 ml of 1 M Na2CO,. The yellow paranitrophenol released was read at 400 nm in a Coleman-Hitachi model 124 double-beam spectrophotometer. A unit of enzyme activity was defined as nanomoles of p-nitrophenol released per minute. A standard curve was constructed using various concentrations of p-nitrophenol. For the determination of Vmax and Km the following concentrations of a-PNPG were used: 1 mM, 500 gM, 250 ,M, 100 gM, and 50
AM.
The optimum pH for activity was determined by adding a 0.5-ml extract sample (100 ig of protein) in 0.05 M phosphate buffer, pH 7.0, to 2 ml of each of the following buffers: 0.1 M citrate-phosphate, pH 3 to 7; 0.1 M phosphate, pH 6 to 8; 0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 8 to 9; and 0.1 M glycine-NaOH, pH 9 to 10. One-half milliliter of 5 mM a-PNPG in deionized water was added, and the enzyme was assayed as above. The pH of each reaction mixture was measured directly. Alpha-glucosidase activities in cells grown on various carbon sources. For the production of enzyme in asynchronous cultures, 14 mM maltose alone, maltose (14 mM) plus glucose (28 mM), succinate (19 mM), or DL-alanine (56 mM) was used as the carbon source. Samples (5 ml) were withdrawn at various times (early log, late log, stationary phase), and cell extracts were prepared and assayed for alpha-glucosidase activity with a-PNPG. Induction of alpha-glucosidase synthesis. Maltose or other potential inducers were added to cultures grown on glucose, succinate, or alanine as the sole carbon source, and incubation was allowed to proceed at 30 C on a rotary shaker. The cells were harvested before and after the addition of the inducer, and
263
extracts were assayed with a-PNPG as described above. For the study of induction in the first few minutes after adding maltose, 10-xnl samples were removed from a log-phase culture and placed in test tubes containing either 3 drops of toluene in a bath of ice water or 100 jg of chloramphenicol per ml to prevent any further enzyme synthesis. Cell extracts were prepared and assayed for hydrolysis of a-PNPG using the standard procedure. Other enzyme assays and chemical determinanations. The assay for glucose-i-phosphate and glucose-6-phosphate was done by the procedure of Hofnung and Schwartz (9). The assay utilized 50 IU of phosphoglucomutase to convert glucose-1-phosphate to glucose-6-phosphate. Glucose-6-phosphate dehydrogenase (30 IU) was also added to oxidize glucose6-phosphate to 6-phosphogluconic acid, and the concomitant reduction of nicotinamide adenine dinucleotide phosphate, reduced form, was followed spectrophotometrically at 340 nm. Standard curves for glucose- 1-phosphate and glucose-6-phosphate were constructed. Cell extracts were assayed at 30 C for 20 min. Maltodextrin formation was looked for by incubating either whole cells or extract with excess maltose and performing the iodine test as described by Hofnung and Schwartz (9). Inorganic phosphate was determined in appropriately diluted culture filtrate by the phosphomolybdate method described by Herbert (8). A standard curve. was prepared by plotting different concentrations of K2HPO4 against absorbance at 730 nm. Protein was measured by the method of Lowry et al. (15) with the Folin-Ciocalteau reagent, using bovine serum albumin as the protein standard. Specific activity was expressed as nanomoles of p-nitrophenol released per minute per milligram of extract protein. Manometric methods. Oxygen consumption was measured in a Warburg apparatus (American Instrument Co., Silver Spring, Md.). Washed cells in 0.1 M phosphate buffer, pH 7.0, with 5 mg (dry weight) of cells per ml were used. The Warburg flasks contained 1 ml of cells, 1 ml of the phosphate buffer, and 0.3 ml of water in the main compartment; 0.2 ml of 20% KOH in the center well; and 0.5 ml of substrate in the side arm. All experiments were run in duplicate at 37 C. Oxygen consumption and carbon dioxide production were corrected for endogenous activity. Carbon dioxide was determined by the direct method (23), using a reaction flask without KOH. The substrates tested included glucose, maltose, and maltobionate dissolved in deionized water. At the end of the reaction with maltose or glucose the contents of the vessel were centrifuged at 15,000 x g for 15 min, and the supernatant was assayed for residual sugar with the anthrone reagent (17). Polyacrylamide gel electrophoresis. Polyacrylamide gels (7.5%) in Tris-glycine buffer, pH 8.9, were prepared according to the procedure used by Winters and Corpe (25). Crude extracts (0.25 to 3.0 mg of protein) were electrophoresed at 4 mA/gel in a Buchler electrophoresis apparatus (Buchler Co., Fort Lee, N.J.). The alpha-glucosidase activity was assayed directly on the gels by incubating intact, water-rinsed gels in test tubes with 5 mM a-PNPG in
264
J. BACTERIOL.
GUFFANTI AND CORPE
0.1 M phosphate buffer, pH 7.0, at 30 C. Bands with alpha-glucosidase activity were yellow (p-nitrophenol) in color. Protein was stained on the gels with 0.25% Coomassie blue in 15% trichloroacetic acid and 25% methanol for 20 min at room temperature. 'The gels were destained with 7% acetic acid. Chemicals. Actinomycin D, rifampin, p-nitrophenol, chloramphenicol, and maltose were products of Sigma Chemical Co. (St. Louis, Mo.). Maltotriose, maltobionic acid, glucose-6-phosphate dehydrogenase (yeast), and phosphoglucomutase (rabbit muscle) were all purchased from Calbiochem (San Diego, Calif.).
RESULTS Evidence for hydrolytic cleavage of maltose. The growth of P. fluorescens W on maltose was equivalent to growth on glucose in terms of cell yield and doubling time. Washed whole cells consumed the same amount of oxygen whether incubated with 2 Mmol of glucose or 1 Amol of maltose (Table 1). For each mole of oxygen consumed, 1 mol of carbon dioxide was given off. The organism used twothirds of the amount of oxygen necessary to completely oxidize all of the sugar. The remaining third was presumably assimilated, since the supernatant showed the absence of carbohydrate. The enzyme had a greater affinity for a-PNPG than for maltose, but split maltose at a faster rate when substrate was present in excess (Fig. 1). Hydrolytic cleavage was generally confirmed when it was shown that cell extracts released more than 1 mol of glucose per mol of maltose (Table 2). In the case of small amounts of maltose almost exactly 2 mol of glucose were produced per mol of maltose. The maltose analogue, a-PNPG, was split-to glucose and p-nitrophenol. Both a-PNPG splitting activity and maltose cleaving activity were induced by growth on maltose (Table 3). Once this was established we routinely used the simple a-PNPG assay for alpha-glucosidase activity. Evidence against other mechanisms of maltose utilization. Since other means of mal-
tose utilization have been reported for bacteria, some effort was made to demonstrate their presence or
absence in P. fluorescens W. Any
type of phosphorolytic cleavage was ruled out, since sonic extracts exhaustively dialyzed against Tris-maleic acid buffer, to remove inor-
ganic phosphate, could still release glucose from maltose. Furthermore, no glucose-i-phosphate or glucose-6-phosphate was detected when extracts were incubated with maltose or dextrin. When samples of the culture supernatant were assayed at various stages of growth on an ammonium-salts medium with 0.1 M Trishydrochloride, pH 7.2, and 100 gg of K2HPO, per ml there was no improved uptake of inorganic phosphate by cells growing on maltose compared to those growing on glucose. Also, when excess maltose was added to extracts or whole cells grown on maltose no iodine-detectable polymer was found (9). Oxidation of maltose to maltobionate and subsequent cleavage to glucose and gluconate was unlikely, because whole cells (Table 1) hardly oxidized maltobionate and glucose was not released from maltobionate by extracts (Table 2). Moreover, sonically ruptured cells oxidized maltose but not maltobionate, indicating that the failure to use maltobionate was probably not due solely to its inability to penetrate the cell membrane. pH dependence, stability, and cellular location of alpha-glucosidase. A pH of 7.0 was optimum for a-PNPG hydrolysis in cell extracts (Fig. 2). The enzyme activity was stable in the extract at 4 C for at least 4 days, but lost about 80% of its activity when incubated overnight at 30 C. The P. fluorescens W alpha-glucosidase activity was shown to be almost wholly intracellular. Approximately 98% of the enzyme activity was found in cell extracts (Table 4), with very little in the cell envelopes and only traces in the culture supernatant fluid. Induction of alpha-glucosidase. Among the various sugars and sugar analogues tested as inducers of alpha-glucosidase activity only mal-
TABLE 1. Oxygen consumption and carbon dioxide production with various substrates Substratea
Maltose Glucose Maltobionate
Concn
Oxygen consumed
(pmol) (Amol1Al-mol 1 2 1
177 180 10
7.9 8.0 0.4
Carbon dioxide produced Amol Al
179 178
8.0 7.9
NDb
ND
a Substrates were incubated with washed cells, and oxygen consumption and carbon dioxide production were determined in duplicate Warburg vessels. The values shown were corrected for endogenous activity. "ND, Not determined.
MALTOSE METABOLISM OF P. FLUORESCENS
VOL. 124, 1975
265
x
1 .O-
20-
0>
C
E
E
CL2
101
CD
Ia.1
MALTOSE (mM) ALPHA-PNPG (10-2 mM) FIG. 1. Kinetics of maltose and a-PNPG hydrolysis. Cells were grown on maltose as the sole carbon source,
and extracts were prepared. Various concentrations of maltose were added to a reaction mixture, and the amount of glucose released was measured. Various amounts of a-PNPG were added to another reaction mixture, and the amount of p-nitrophenol released was measured. Activities are expressed per milligram of extract protein.
tose and maltotriose were satisfactory (Table 3). When grown on succinate or DL-alanine none of the following repressed the induction by maltose: 2-deoxyglucose, glucose, sorbose, glucose1-phosphate, glucose-6-phosphate, gluconate, alpha-methylglucoside, alpha-phenylglucoside, or a mixture of gluconate and glucose. Chloramphenicol inhibited synthesis of alpha-glucosidase above the noninduced level (Table 5). Essentially the same was true of actinomycin D, an inhibitor of messenger ribonucleic acid synthesis, but it only inhibited when cells were pretreated with ethylenediaminetetraacetic acid to alter the permeability of the outer wall (13). Ethylenediaminetetraacetic acid alone did not inhibit alpha-glucosidase induction. Rifampin, another transcriptional inhibitor, allowed some induction (Table 5). This was probably due to its slow penetration into this organism (Sheela Amrute, personal communication). Induction was very rapid in growing cells and was detectable within the first 5 min after adding maltose. Production of alpha-glucosidase during growth on various carbon sources. The highest specific activity of a-glucosidase occurred in
TABLE 2. Release of glucose from maltose by cell extracts
Substrate0 Maltose
Maltobionate
Concn (nmol/ml)
Glucose liberated (nmol/ml)
550 275 140 70 275 550
610 330 250 140 0 0
aOne milliliter of extract (0.6 mg of protein) from maltose-grown cells was incubated for 30 min at 30 C in a reaction mixture containing 0.1 M phosphate buffer, pH 7.0, and the indicated substrate concentration in a final volume of 3 ml.
the late log phase of growth on all four carbon sources tested (Fig. 3). The activity with maltose as the sole carbon source or with maltose plus glucose was very similar. When succinate or DL-alanine was present with maltose in the medium, the specific activities were lower at comparable stages of growth and never reached the level attained in cultures with maltose alone
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J. BACTERIOL.
GUFFANTI AND CORPE
TABLE 3. Induction of alpha-glucosidase by several sugars Sp act Inducera
a-PNPG assay (nmol/ min/mg)
Glucose (nmoVl min/mg)
None Maltose Maltose + glucose (10 mM) Maltotriose a-PNPG a-Phenylglucoside a-Methylglucoside Glucose Cellobiose Isomaltose Lactose Maltobionate Sucrose Maltose grownc
0.25 3.75 3.71 3.46 0.75 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 25.0
0.35 3.37 NDb ND ND ND ND ND ND ND ND ND ND ND
a Cultures were grown on the regular synthetic medium with 19 mM succinate as the sole carbon source. A final concentration of 1 mM inducer was added to a mid-log-phase culture, and incubation was continued for 1 h at 30 C. The cells were harvested, extracted, and assayed with a-PNPG or maltose (glucose release) in the usual way. IND, Not determined. cAlpha-glucosidase was assayed in a mid-log-phase culture growing on 28 mM maltose as the sole carbon source.
0.7
or maltose plus glucose. In each case the inducer, maltose, was present in excess. No efflux of a-glucosidase activity into the culture medium was found at any stage of growth. No suggestion of diauxic growth was observed when succinate-grown cells were inoculated into a medium containing 5 mM succinate and 10 mM maltose. The same could be said for cells transferred from DL-alanine to DL-alanine (5 mM) and maltose (10 mM), except that growth was a little slower than on maltose alone. The doubling times on glucose, maltose, or succinate were essentially the same (6 h), but cells growing on DL-alanine doubled in turbidity every 12 h.
Polyacrylamide gel electrophoresis of crude cell extracts. Only one band of enzyme activity was detected on 7.5% polyacrylamide gels by hydrolysis of a-PNPG, when up to 3 mg TABLE 4. Localization of alpha-glucosidase in maltose-grown P. fluorescens W Source of enzyme
Culture supernatant Cell envelopes Cell extract
Alpha. glucosidase) (U)
toa
0.33 0.33 30.0
1 1 98
%o
%of
a Cells were grown on maltose as the sole carbon source and units of a-glucosidase activity are expressed as nanomoles of p-nitrophenol released per minute by 10 ml of cell culture material.
0.6
TABLE 5. Effect of translational and transcriptional inhibitors on the induction of alpha-glucosidase Inhibitor
Concn (gg/ml)
min/mg)
0.4-
'~0.3-
0.2-
FIG. 2. pH dependence of alpha-PNPG hydrolysis. Cells were grown on 14 mM maltose as the sole carbon source and broken by ultrasonic oscillation, and extracts were obtained. The reaction mixture contained 0.1 mg of extract protein; alpha-glucosidase was expressed in nanomoles of p-nitrophenol released per minute per milliliter of extract.
Nonea Actinomycin D None"
Rifampin Nonec Chloramphenicol No maltose
2.13
80 10 100
0.39 6.39 0.79 1.59 0.25 0.29
a A mid-log-phase culture growing on glucose was divided in half, and 1 mM EDTA and 1 mM maltose were added to each portion. Incubation continued on a rotary shaker at 30 C for 30 min, and then the culture was assayed for alpha-glucosidase in the usual way. 'Mid-log-phase cells were grown on glucose and divided in half, and 1 mM maltose was added to each portion. Incubation was as above, but for 2 h. c Mid-log cultures were grown on alanine as carbon source and divided in thirds, and 1 mM maltose was added to only two cultures. Incubation and assay were as in footnote a.
VOL. 124, 1975
MALTOSE METABOLISM OF P. FLUORESCENS
267
24
500 21
400
613, r-
300
1--
18
a
15
F"
20 CA 1-
=
m-
f^
100
F"
12
80
60 9
el)
31, .1
40 6
z
201
S
10
15
20
2 5
30
3
5
T IM"E ( HO0U RS)
FIG. 3. Specific activities of a-glucosidase during growtih on different carbon sources. In each case the inoculum was a late log-phase culture which had been grown in the absence of maltose on the carbon source to be tested. The concentrations of carbon sources were as follows: maltose (14 mM), glucose (28 mM), succinate (19 mM), and DL-alanine (56 mM). (0) represents growth on glucose plus maltose or succinate plus maltose; (x) is growth on alanine plus maltose. Specific activities are expressed in nanomoles of p-nitrophenol released per minute per milligram of extract protein in cultures growing on glucose and maltose (0), succinate and maltose (A), or alanine and maltose (a).
maltose. Moreover, cell extracts did not release any glucose from maltobionate. The intracellular location of alpha-glucosidase in P. fluorescens W was consistent with DISCUSSION that reported for other bacterial carbohydrases The evidence is clear that P. fluorescens W found in gram-negative bacteria (4, 16). Such a utilizes maltose by hydrolizing it to glucose location would necessitate a maltose-concenalone by means of an inducible alpha-glucosi- trating system probably similar to other bactedase. If glucose plus some other derivative of rial permease systems. The maltose permease in glucose were the product of this reaction one P. fluorescens W is currently under investigawould expect to find no more than 1 mol of tion. Since only maltose and maltotriose were glucose released from 1 mol of maltose. There good inducers of a-glucosidase we believe that the inducer binding site is quite specific for an was no indication that inorganic phosphate was al-4 glucoside. The aryl analogue, a-PNPG, is necessary for maltose cleavage, nor were glucose-i-phosphate or glucose-6-phosphate de- an excellent substrate but a very poor inducer tected as products of maltose metabolism as has for this enzyme. This could also be due to its been reported for Escherichia coli (9) and Neis- inability to readily enter the cell. Like that of other bacterial systems (18) seria (5). The Warburg respirometry showed that mal- induction of the Pseudomonas alpha-glucositobionate was not oxidized either by whole cells dase was accomplished within only a few minutes of the addition of the inducer. This indior sonically ruptured cells. If this pseudomonad could oxidize maltose to maltobionate and then cated rapid entry of the inducer and attachment split it in a manner common to other pseudomo- to the inducer binding site. Both de novo nands and Alcaligenes (2), then maltobionate synthesis of messenger ribonucleic acid and should have been oxidized to the same extent as protein were necessary for the induction of the
of extract protein was loaded onto the gel. This indicated the presence of only one species of alpha-glucosidase in this organism.
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J. BACTERIOL.
GUFFANTI AND CORPE
enzyme. The same holds true for other inducible bacterial enzymes (16). The catabolite and or transient repression characteristic of such inducible systems as betagalactosidase in E. coli (14, 16, 22) was not detected in this organism. Alpha-glucosidase yield on maltose medium was not affected when glucose was included simultaneously. The apparent repressive effect of succinate was not too surprising in a strict aerobe such as Pseudomonas. Pseudomonads lack a constitutive phosphoenolpyruvate-glucose transport system (19, 20) and a functional Embden-Meyerhof pathway (21). Unlike E. coli, the enzymes for glucose transport and catabolism are inducible in these strict aerobes (3, 10). Tiwari and Campbell (21) showed that succinate-grown Pseudomonas aeruginosa, when placed on glucose-succinate medium, exhibited diauxic growth. The tricarboxylic acid cycle enzymes were constitutive, but the inducible EntnerDoudoroff enzymes were present in low levels until the succinate was used up. Diauxic growth on succinate and maltose was not observed in P. fluorescens W, but some repression of alpha-glucosidase synthesis was detected. This may indicate a less stringent control over inducible enzyme systems by tricarboxylic acid cycle intermediates in this organism than in P. aeruginosa. As far as alanine repression is concerned, pseudomonads have been shown to utilize amino acids in preference to sugars (7), but the mechanism for this is not really known. The results of the electrophoresis studies of the cell extract indicated the presence of a single alpha-glucosidase in P. fluorescens W. If the enzyme had subunit structure and was able to form multiple complexes of the subunits in a manner similar to E. coli beta-galactosidase (4), then more than one band of alpha-glucosidase activity would have been detected on the gels. LITERATURE CITED 1. Bentley, R., and L. Slechta. 1960. Oxidation of monoand disaccharides to aldonic acids by Pseudomonas species. J. Bacteriol. 79:346-354. 2. Bernaerts, M. J., and J. DeLey. 1960. Microbiological formation and preparation of 3-ketoglycosides from disaccharides. J. Gen. Microbiol. 22:129-136. 3. Eisenberg, R. C., S. J. Butters, S. C. Quay, and S. B. Friedman. 1974. Glucose uptake and phosphorylation in Pseudomonas fluorescens. J. Bacteriol. 120:147-153. 4. Erickson, R. P., and E. Steers. 1970. Comparative study of isoenzyme formation of bacterial beta-galactosidase. J. Bacteriol. 102:79-84. 5. Fitting, C., and H. W. Scherp. 1952. Observations on a strain of Neisseria meningitidis in the presence of glucose and maltose. J. Bacteriol. 64:287-298.
6. Han, Y. W., and V. R. Srinivasan. 1969. Purification and characterization of beta-glucosidase of Alcaligenes faecalis. J. Bacteriol. 100:1355-1363. 7. Hamilton, P. B., and G. Shelley. 1971. Chemotactic response to amino acids by Pseudomonas aeruginosa in semisolid nitrate medium. J. Bacteriol. 108:596-598. 8. Herbert, D., P. J. Phipps, and R. E. Strange. 1971. Chemical analysis of microbiol cells, p. 228. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 5B. Academic Press Inc., New York. 9. Hofnung, M., and M. Schwartz. 1967. La maltodextrine phosphorlase d'Escherichia coli. Eur. J. Biochem. 2:132-145. 10. Hylemon, P. B., and P. V. Phibbs. 1972. Independent regulation of hexose catabolizing enzymes and glucose transport activity in Pseudomonas aeruginosa. Biochem. Biophys. Res. Commun. 48:1041-1050. 11. Katznelson, H., and S. W. Tanenbaum. 1954. Observations on maltose oxidation by Acetobacter melanogenum. J. Bacteriol. 68:368-372. 12. Kusch, M., and T. H. Wilson. 1973. Defective lactose utilization by a mutant of Escherichia coli energyuncoupled for lactose transport: the advantages of active transport versus facilitated diffusion. Biochim. Biophys. Acta 311:109-122. 13. Leive, L. 1965. RNA degradation and the assembly of ribosomes in actinomycin-treated Escherichia coli. J. Mol. Biol. 13:862-865. 14. Loomis, W. F., and B. Magasanik. 1966. Nature of the effector of catabolite repression of beta-galactosidase in Escherichia coli. J. Bacteriol. 92:170-177. 15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 16. Nakada, D., and B. Magasanik. 1962. Catabolite repression and the induction of beta-galactosidase. Biochim. Biophys. Acta 61:835-837. 17. Neish, A. C. 1952. Analytical methods for bacterial fermentations. Report 46-8-3. National Research Council of Canada, Saskatoon, Saskatchewan, Canada. 18. Pardee, A. B., and L. S. Prestridge. 1961. Initial kinetics of enzyme induction. Biochim. Biophys. Acta 49:77-88. 19. Phibbs, P. V., and R. G. Eagon. 1970. Transport and phosphorylation of glucose, fructose, and mannitol by Pseudomonas aeruginosa. Arch. Biochem. Biophys.
138:470-482. 20. Romano, A. H., S. J. Eberhard, S. L. Dingle, and T. D. McDowell. 1970. Distribution of the phosphoenolpyruvate:glucose phosphotransferase system in bacteria. J. Bacteriol. 104:808-813. 21. Tiwari, N. P., and J. J. R. Campbell. 1969. Enzymatic control of the metabolic activity of Pseudomonas aeruginosa grown in glucose or succinate medium. Biochim. Biophys. Acta 192:395-401. 22. Tyler, B., W. F. Loomis, and B. Magasanik. 1967. Transient repression of the lac operon. J. Bacteriol. 94:2001-2011. 23. Umbreit, W., R. H. Burris, and J. F. Stauffer (ed.). 1957. Manometric techniques, 3rd ed. Burgess Publishing
Co., Minneapolis. 24. Whiteside, T. L., and W. A. Corpe. 1969. Extracellular enzymes produced by Pseudomonas sp. and their effect on cell envelopes of Chromobacterium violaceum. Can. J. Microbiol. 15:81-92. 25. Winters, H., and W. A. Corpe. 1971. Polyacrylamide gel electrophoresis of exoenzymes produced by Pseudomonas fluorescens strain W. Can. J. Microbiol. 17:241-248.
JOURNAL OF BACTERIOLOGY, OCt. 1975, p. 262-268 Copyright i 1975 American Society for Microbiology
Maltose Metabolism of Pseudomonas fluorescens ARTHUR A. GUFFANTI1 AND W. A. CORPE* Department of Biological Sciences, Columbia University, New York, New York 10027
Received for publication 31 July 1975
Pseudomonas fluorescens W uses maltose exclusively by hydrolyzing it to glucose via an inducible alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20). No evidence for phosphorolytic cleavage or oxidation to maltobionic acid was found in this organism. The alpha-glucosidase was totally intracellular and was most active at pH of 7.0. Induction occurred when cells were incubated with maltotriose or maltose. Induction was rapid and easily detectable within the first 5 min after the addition of the inducer. Glucose and its derivatives did not repress induction. Cells growing on DL-alanine or succinate plus maltose exhibited lower levels of alpha-glucosidase than those grown on maltose alone or maltose plus glucose. Induction required both messenger ribonucleic acid and protein synthesis. The utilization of disaccharides by bacteria is often initiated by hydrolysis of the sugar into its monosaccharide components. Simple hydrolysis of maltose to glucose by alpha-glucosidase occurs in bacteria (2, 11), but not invariably, since at least three other mechanisms of maltose utilization are known (1, 2, 5, 9). Pseudomonas fluorescens W, isolated from soil and grown in complex media, was found to secrete many hydrolases (24, 25) into the extracellular environment. Included among the enzymes were small amounts of maltose-hydrolyzing activity (25). The disaccharide hydrolases of bacteria are usually thought to be cell bound, but a surprising paucity of information about the formation and biosynthetic control of alphaglucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) of bacteria is available. It was with these facts in mind that the present work was undertaken. MATERIALS AND METHODS Organism and growth media. The bacterium studied in this work was isolated from soil by T. L. Whiteside and identified as P. fluorescens (24). The organism, designated as strain W, was routinely transferred every 2 weeks on slants of peptoneglucose-yeast extract agar (24) and, after growth for 24 h at 30 C, stored at 4 C. The defined medium used in the experiments described in this paper contained the following ingredients in grams per liter of deionized water: (NH,)2S04, 2.0; KH2PO4, 5.3; Na2HPO4, 8.66; MgSO4 7H20, 0.2; NaCl, 0.01; FeSO4 7H20, 0.01; and MnSO4, H20, 0.01. DL-Methionine, 30 Ag/ml, was supplied as the only required growth factor. The pH 'Present address: Department of Biochemistry, Mount Sinai School of Medicine of the City University of New York, New York, N.Y. 10029.
was 7.0 and did not change during growth. The carbon sources used in various experiments were DL-alanine, succinate, glucose, or maltose. They were autoclaved separately (121 C for 15 min) and added aseptically to the rest of the sterile medium, to give a final concentration of 14 mM maltose, 28 mM glucose, 56 mM DL-alanine, or 19 mM succinate. The organism was grown in 500-ml Erlenmeyer side arm flasks (Belco Glass Co., Vineland, N.J.), 50 ml per flask, at 30 C on a rotary shaker. One milliliter of a late log-phase culture was used as the inoculum. Growth was followed as increase in culture turibidity using a Klett-Summerson photoelectric colorimeter with a number 66 filter. Preparation of cell extracts and other cell fractions. Cells from 50 ml of culture were harvested by centrifugation in the cold (4 C) for 15 min at 12,000 x g in a Servall model SS34 centrifuge (Sorvall Co., Norwalk, Conn.). They were washed once with 20 ml of cold 0.1 M phosphate buffer, pH 7.0, suspended in 5 ml of the same buffer, and ruptured by ultrasonic oscillation in a cuvette suspended in an ice bath for 10 min at maximum intensity with an MSE ultrasonic device (Instrumentation Association Inc., New York, N.Y.). The suspension of ruptured cells was centrifuged in the cold for 20 min at 27,000 x g. The supernatant fluid was carefully decanted and designated as cell extract. The procedure for the localization of alpha-glucosidase differed from that described above. Extracellular enzyme was recovered from a late log-phase culture supernatant fluid by precipitation with 90% saturation ammonium sulfate according to the method of Whiteside and Corpe (24). The protein precipitate was dissolved in a small volume of 0.1 M phosphate buffer, pH 7.0. The cells, which had been sedimented at 27,000 x g for 15 min, were washed twice in the same buffer at 4 C, suspended in 5 ml of buffer, and broken by ultrasonic oscillation for 15 min. They were centrifuged at 1,085 x g for 30 min to remove unbroken cells. The supernatant fluid containing cell
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MALTOSE METABOLISM OF P. FLUORESCENS
envelopes was then centrifuged at 27,000 x g for 30 min. The resulting supernatant fluid was termed "extract," and the sediment containing cell envelopes was suspended and washed several times. This fraction was termed "envelopes." All three fractions were assayed for alpha-glucosidase activity by hydrolysis of para-nitrophenyl-a-D-glucoside (a-PNPG). Assay for alpha-glucosidase activity. The release of glucose from maltose by cell extracts was assayed by a variation of the technique of Kusch and Wilson for beta-galactosidase (12). The reaction mixture (5 ml total volume) contained 4 mg of extract protein, 0.1 M phosphate buffer, pH 7.0, and various concentrations of maltose (10, 5, 2.5, and 1.25 mM). The reaction proceeded at 30 C and was stopped with Ba(OH)2 and ZnSO4 exactly as described by Kusch and Wilson. Glucose was assayed by the Glucostat method according to the manufacturer's instructions (Worthington Biochemical Corp., Freehold, N.J.). The Km and Vmax were determined from a Lineweaver-Burk plot. Alpha-glucosidase activity was also assayed with a-PNPG as the substrate, using a variation of the procedure of Han and Srinivasan for beta-glucosidase (6). The reaction mixture contained a sample of cell extract, envelopes, or extracellular enzyme in 2.5 ml of 0.1 M phosphate buffer, pH 7.0. After equilibration for 5 min in a 30 C water bath, 0.5 ml of 5 mM a-PNPG was added, and the reaction was allowed to proceed for 30 min. The reaction was stopped by the addition of 2 ml of 1 M Na2CO,. The yellow paranitrophenol released was read at 400 nm in a Coleman-Hitachi model 124 double-beam spectrophotometer. A unit of enzyme activity was defined as nanomoles of p-nitrophenol released per minute. A standard curve was constructed using various concentrations of p-nitrophenol. For the determination of Vmax and Km the following concentrations of a-PNPG were used: 1 mM, 500 gM, 250 ,M, 100 gM, and 50
AM.
The optimum pH for activity was determined by adding a 0.5-ml extract sample (100 ig of protein) in 0.05 M phosphate buffer, pH 7.0, to 2 ml of each of the following buffers: 0.1 M citrate-phosphate, pH 3 to 7; 0.1 M phosphate, pH 6 to 8; 0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 8 to 9; and 0.1 M glycine-NaOH, pH 9 to 10. One-half milliliter of 5 mM a-PNPG in deionized water was added, and the enzyme was assayed as above. The pH of each reaction mixture was measured directly. Alpha-glucosidase activities in cells grown on various carbon sources. For the production of enzyme in asynchronous cultures, 14 mM maltose alone, maltose (14 mM) plus glucose (28 mM), succinate (19 mM), or DL-alanine (56 mM) was used as the carbon source. Samples (5 ml) were withdrawn at various times (early log, late log, stationary phase), and cell extracts were prepared and assayed for alpha-glucosidase activity with a-PNPG. Induction of alpha-glucosidase synthesis. Maltose or other potential inducers were added to cultures grown on glucose, succinate, or alanine as the sole carbon source, and incubation was allowed to proceed at 30 C on a rotary shaker. The cells were harvested before and after the addition of the inducer, and
263
extracts were assayed with a-PNPG as described above. For the study of induction in the first few minutes after adding maltose, 10-xnl samples were removed from a log-phase culture and placed in test tubes containing either 3 drops of toluene in a bath of ice water or 100 jg of chloramphenicol per ml to prevent any further enzyme synthesis. Cell extracts were prepared and assayed for hydrolysis of a-PNPG using the standard procedure. Other enzyme assays and chemical determinanations. The assay for glucose-i-phosphate and glucose-6-phosphate was done by the procedure of Hofnung and Schwartz (9). The assay utilized 50 IU of phosphoglucomutase to convert glucose-1-phosphate to glucose-6-phosphate. Glucose-6-phosphate dehydrogenase (30 IU) was also added to oxidize glucose6-phosphate to 6-phosphogluconic acid, and the concomitant reduction of nicotinamide adenine dinucleotide phosphate, reduced form, was followed spectrophotometrically at 340 nm. Standard curves for glucose- 1-phosphate and glucose-6-phosphate were constructed. Cell extracts were assayed at 30 C for 20 min. Maltodextrin formation was looked for by incubating either whole cells or extract with excess maltose and performing the iodine test as described by Hofnung and Schwartz (9). Inorganic phosphate was determined in appropriately diluted culture filtrate by the phosphomolybdate method described by Herbert (8). A standard curve. was prepared by plotting different concentrations of K2HPO4 against absorbance at 730 nm. Protein was measured by the method of Lowry et al. (15) with the Folin-Ciocalteau reagent, using bovine serum albumin as the protein standard. Specific activity was expressed as nanomoles of p-nitrophenol released per minute per milligram of extract protein. Manometric methods. Oxygen consumption was measured in a Warburg apparatus (American Instrument Co., Silver Spring, Md.). Washed cells in 0.1 M phosphate buffer, pH 7.0, with 5 mg (dry weight) of cells per ml were used. The Warburg flasks contained 1 ml of cells, 1 ml of the phosphate buffer, and 0.3 ml of water in the main compartment; 0.2 ml of 20% KOH in the center well; and 0.5 ml of substrate in the side arm. All experiments were run in duplicate at 37 C. Oxygen consumption and carbon dioxide production were corrected for endogenous activity. Carbon dioxide was determined by the direct method (23), using a reaction flask without KOH. The substrates tested included glucose, maltose, and maltobionate dissolved in deionized water. At the end of the reaction with maltose or glucose the contents of the vessel were centrifuged at 15,000 x g for 15 min, and the supernatant was assayed for residual sugar with the anthrone reagent (17). Polyacrylamide gel electrophoresis. Polyacrylamide gels (7.5%) in Tris-glycine buffer, pH 8.9, were prepared according to the procedure used by Winters and Corpe (25). Crude extracts (0.25 to 3.0 mg of protein) were electrophoresed at 4 mA/gel in a Buchler electrophoresis apparatus (Buchler Co., Fort Lee, N.J.). The alpha-glucosidase activity was assayed directly on the gels by incubating intact, water-rinsed gels in test tubes with 5 mM a-PNPG in
264
J. BACTERIOL.
GUFFANTI AND CORPE
0.1 M phosphate buffer, pH 7.0, at 30 C. Bands with alpha-glucosidase activity were yellow (p-nitrophenol) in color. Protein was stained on the gels with 0.25% Coomassie blue in 15% trichloroacetic acid and 25% methanol for 20 min at room temperature. 'The gels were destained with 7% acetic acid. Chemicals. Actinomycin D, rifampin, p-nitrophenol, chloramphenicol, and maltose were products of Sigma Chemical Co. (St. Louis, Mo.). Maltotriose, maltobionic acid, glucose-6-phosphate dehydrogenase (yeast), and phosphoglucomutase (rabbit muscle) were all purchased from Calbiochem (San Diego, Calif.).
RESULTS Evidence for hydrolytic cleavage of maltose. The growth of P. fluorescens W on maltose was equivalent to growth on glucose in terms of cell yield and doubling time. Washed whole cells consumed the same amount of oxygen whether incubated with 2 Mmol of glucose or 1 Amol of maltose (Table 1). For each mole of oxygen consumed, 1 mol of carbon dioxide was given off. The organism used twothirds of the amount of oxygen necessary to completely oxidize all of the sugar. The remaining third was presumably assimilated, since the supernatant showed the absence of carbohydrate. The enzyme had a greater affinity for a-PNPG than for maltose, but split maltose at a faster rate when substrate was present in excess (Fig. 1). Hydrolytic cleavage was generally confirmed when it was shown that cell extracts released more than 1 mol of glucose per mol of maltose (Table 2). In the case of small amounts of maltose almost exactly 2 mol of glucose were produced per mol of maltose. The maltose analogue, a-PNPG, was split-to glucose and p-nitrophenol. Both a-PNPG splitting activity and maltose cleaving activity were induced by growth on maltose (Table 3). Once this was established we routinely used the simple a-PNPG assay for alpha-glucosidase activity. Evidence against other mechanisms of maltose utilization. Since other means of mal-
tose utilization have been reported for bacteria, some effort was made to demonstrate their presence or
absence in P. fluorescens W. Any
type of phosphorolytic cleavage was ruled out, since sonic extracts exhaustively dialyzed against Tris-maleic acid buffer, to remove inor-
ganic phosphate, could still release glucose from maltose. Furthermore, no glucose-i-phosphate or glucose-6-phosphate was detected when extracts were incubated with maltose or dextrin. When samples of the culture supernatant were assayed at various stages of growth on an ammonium-salts medium with 0.1 M Trishydrochloride, pH 7.2, and 100 gg of K2HPO, per ml there was no improved uptake of inorganic phosphate by cells growing on maltose compared to those growing on glucose. Also, when excess maltose was added to extracts or whole cells grown on maltose no iodine-detectable polymer was found (9). Oxidation of maltose to maltobionate and subsequent cleavage to glucose and gluconate was unlikely, because whole cells (Table 1) hardly oxidized maltobionate and glucose was not released from maltobionate by extracts (Table 2). Moreover, sonically ruptured cells oxidized maltose but not maltobionate, indicating that the failure to use maltobionate was probably not due solely to its inability to penetrate the cell membrane. pH dependence, stability, and cellular location of alpha-glucosidase. A pH of 7.0 was optimum for a-PNPG hydrolysis in cell extracts (Fig. 2). The enzyme activity was stable in the extract at 4 C for at least 4 days, but lost about 80% of its activity when incubated overnight at 30 C. The P. fluorescens W alpha-glucosidase activity was shown to be almost wholly intracellular. Approximately 98% of the enzyme activity was found in cell extracts (Table 4), with very little in the cell envelopes and only traces in the culture supernatant fluid. Induction of alpha-glucosidase. Among the various sugars and sugar analogues tested as inducers of alpha-glucosidase activity only mal-
TABLE 1. Oxygen consumption and carbon dioxide production with various substrates Substratea
Maltose Glucose Maltobionate
Concn
Oxygen consumed
(pmol) (Amol1Al-mol 1 2 1
177 180 10
7.9 8.0 0.4
Carbon dioxide produced Amol Al
179 178
8.0 7.9
NDb
ND
a Substrates were incubated with washed cells, and oxygen consumption and carbon dioxide production were determined in duplicate Warburg vessels. The values shown were corrected for endogenous activity. "ND, Not determined.
MALTOSE METABOLISM OF P. FLUORESCENS
VOL. 124, 1975
265
x
1 .O-
20-
0>
C
E
E
CL2
101
CD
Ia.1
MALTOSE (mM) ALPHA-PNPG (10-2 mM) FIG. 1. Kinetics of maltose and a-PNPG hydrolysis. Cells were grown on maltose as the sole carbon source,
and extracts were prepared. Various concentrations of maltose were added to a reaction mixture, and the amount of glucose released was measured. Various amounts of a-PNPG were added to another reaction mixture, and the amount of p-nitrophenol released was measured. Activities are expressed per milligram of extract protein.
tose and maltotriose were satisfactory (Table 3). When grown on succinate or DL-alanine none of the following repressed the induction by maltose: 2-deoxyglucose, glucose, sorbose, glucose1-phosphate, glucose-6-phosphate, gluconate, alpha-methylglucoside, alpha-phenylglucoside, or a mixture of gluconate and glucose. Chloramphenicol inhibited synthesis of alpha-glucosidase above the noninduced level (Table 5). Essentially the same was true of actinomycin D, an inhibitor of messenger ribonucleic acid synthesis, but it only inhibited when cells were pretreated with ethylenediaminetetraacetic acid to alter the permeability of the outer wall (13). Ethylenediaminetetraacetic acid alone did not inhibit alpha-glucosidase induction. Rifampin, another transcriptional inhibitor, allowed some induction (Table 5). This was probably due to its slow penetration into this organism (Sheela Amrute, personal communication). Induction was very rapid in growing cells and was detectable within the first 5 min after adding maltose. Production of alpha-glucosidase during growth on various carbon sources. The highest specific activity of a-glucosidase occurred in
TABLE 2. Release of glucose from maltose by cell extracts
Substrate0 Maltose
Maltobionate
Concn (nmol/ml)
Glucose liberated (nmol/ml)
550 275 140 70 275 550
610 330 250 140 0 0
aOne milliliter of extract (0.6 mg of protein) from maltose-grown cells was incubated for 30 min at 30 C in a reaction mixture containing 0.1 M phosphate buffer, pH 7.0, and the indicated substrate concentration in a final volume of 3 ml.
the late log phase of growth on all four carbon sources tested (Fig. 3). The activity with maltose as the sole carbon source or with maltose plus glucose was very similar. When succinate or DL-alanine was present with maltose in the medium, the specific activities were lower at comparable stages of growth and never reached the level attained in cultures with maltose alone
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J. BACTERIOL.
GUFFANTI AND CORPE
TABLE 3. Induction of alpha-glucosidase by several sugars Sp act Inducera
a-PNPG assay (nmol/ min/mg)
Glucose (nmoVl min/mg)
None Maltose Maltose + glucose (10 mM) Maltotriose a-PNPG a-Phenylglucoside a-Methylglucoside Glucose Cellobiose Isomaltose Lactose Maltobionate Sucrose Maltose grownc
0.25 3.75 3.71 3.46 0.75 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 25.0
0.35 3.37 NDb ND ND ND ND ND ND ND ND ND ND ND
a Cultures were grown on the regular synthetic medium with 19 mM succinate as the sole carbon source. A final concentration of 1 mM inducer was added to a mid-log-phase culture, and incubation was continued for 1 h at 30 C. The cells were harvested, extracted, and assayed with a-PNPG or maltose (glucose release) in the usual way. IND, Not determined. cAlpha-glucosidase was assayed in a mid-log-phase culture growing on 28 mM maltose as the sole carbon source.
0.7
or maltose plus glucose. In each case the inducer, maltose, was present in excess. No efflux of a-glucosidase activity into the culture medium was found at any stage of growth. No suggestion of diauxic growth was observed when succinate-grown cells were inoculated into a medium containing 5 mM succinate and 10 mM maltose. The same could be said for cells transferred from DL-alanine to DL-alanine (5 mM) and maltose (10 mM), except that growth was a little slower than on maltose alone. The doubling times on glucose, maltose, or succinate were essentially the same (6 h), but cells growing on DL-alanine doubled in turbidity every 12 h.
Polyacrylamide gel electrophoresis of crude cell extracts. Only one band of enzyme activity was detected on 7.5% polyacrylamide gels by hydrolysis of a-PNPG, when up to 3 mg TABLE 4. Localization of alpha-glucosidase in maltose-grown P. fluorescens W Source of enzyme
Culture supernatant Cell envelopes Cell extract
Alpha. glucosidase) (U)
toa
0.33 0.33 30.0
1 1 98
%o
%of
a Cells were grown on maltose as the sole carbon source and units of a-glucosidase activity are expressed as nanomoles of p-nitrophenol released per minute by 10 ml of cell culture material.
0.6
TABLE 5. Effect of translational and transcriptional inhibitors on the induction of alpha-glucosidase Inhibitor
Concn (gg/ml)
min/mg)
0.4-
'~0.3-
0.2-
FIG. 2. pH dependence of alpha-PNPG hydrolysis. Cells were grown on 14 mM maltose as the sole carbon source and broken by ultrasonic oscillation, and extracts were obtained. The reaction mixture contained 0.1 mg of extract protein; alpha-glucosidase was expressed in nanomoles of p-nitrophenol released per minute per milliliter of extract.
Nonea Actinomycin D None"
Rifampin Nonec Chloramphenicol No maltose
2.13
80 10 100
0.39 6.39 0.79 1.59 0.25 0.29
a A mid-log-phase culture growing on glucose was divided in half, and 1 mM EDTA and 1 mM maltose were added to each portion. Incubation continued on a rotary shaker at 30 C for 30 min, and then the culture was assayed for alpha-glucosidase in the usual way. 'Mid-log-phase cells were grown on glucose and divided in half, and 1 mM maltose was added to each portion. Incubation was as above, but for 2 h. c Mid-log cultures were grown on alanine as carbon source and divided in thirds, and 1 mM maltose was added to only two cultures. Incubation and assay were as in footnote a.
VOL. 124, 1975
MALTOSE METABOLISM OF P. FLUORESCENS
267
24
500 21
400
613, r-
300
1--
18
a
15
F"
20 CA 1-
=
m-
f^
100
F"
12
80
60 9
el)
31, .1
40 6
z
201
S
10
15
20
2 5
30
3
5
T IM"E ( HO0U RS)
FIG. 3. Specific activities of a-glucosidase during growtih on different carbon sources. In each case the inoculum was a late log-phase culture which had been grown in the absence of maltose on the carbon source to be tested. The concentrations of carbon sources were as follows: maltose (14 mM), glucose (28 mM), succinate (19 mM), and DL-alanine (56 mM). (0) represents growth on glucose plus maltose or succinate plus maltose; (x) is growth on alanine plus maltose. Specific activities are expressed in nanomoles of p-nitrophenol released per minute per milligram of extract protein in cultures growing on glucose and maltose (0), succinate and maltose (A), or alanine and maltose (a).
maltose. Moreover, cell extracts did not release any glucose from maltobionate. The intracellular location of alpha-glucosidase in P. fluorescens W was consistent with DISCUSSION that reported for other bacterial carbohydrases The evidence is clear that P. fluorescens W found in gram-negative bacteria (4, 16). Such a utilizes maltose by hydrolizing it to glucose location would necessitate a maltose-concenalone by means of an inducible alpha-glucosi- trating system probably similar to other bactedase. If glucose plus some other derivative of rial permease systems. The maltose permease in glucose were the product of this reaction one P. fluorescens W is currently under investigawould expect to find no more than 1 mol of tion. Since only maltose and maltotriose were glucose released from 1 mol of maltose. There good inducers of a-glucosidase we believe that the inducer binding site is quite specific for an was no indication that inorganic phosphate was al-4 glucoside. The aryl analogue, a-PNPG, is necessary for maltose cleavage, nor were glucose-i-phosphate or glucose-6-phosphate de- an excellent substrate but a very poor inducer tected as products of maltose metabolism as has for this enzyme. This could also be due to its been reported for Escherichia coli (9) and Neis- inability to readily enter the cell. Like that of other bacterial systems (18) seria (5). The Warburg respirometry showed that mal- induction of the Pseudomonas alpha-glucositobionate was not oxidized either by whole cells dase was accomplished within only a few minutes of the addition of the inducer. This indior sonically ruptured cells. If this pseudomonad could oxidize maltose to maltobionate and then cated rapid entry of the inducer and attachment split it in a manner common to other pseudomo- to the inducer binding site. Both de novo nands and Alcaligenes (2), then maltobionate synthesis of messenger ribonucleic acid and should have been oxidized to the same extent as protein were necessary for the induction of the
of extract protein was loaded onto the gel. This indicated the presence of only one species of alpha-glucosidase in this organism.
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J. BACTERIOL.
GUFFANTI AND CORPE
enzyme. The same holds true for other inducible bacterial enzymes (16). The catabolite and or transient repression characteristic of such inducible systems as betagalactosidase in E. coli (14, 16, 22) was not detected in this organism. Alpha-glucosidase yield on maltose medium was not affected when glucose was included simultaneously. The apparent repressive effect of succinate was not too surprising in a strict aerobe such as Pseudomonas. Pseudomonads lack a constitutive phosphoenolpyruvate-glucose transport system (19, 20) and a functional Embden-Meyerhof pathway (21). Unlike E. coli, the enzymes for glucose transport and catabolism are inducible in these strict aerobes (3, 10). Tiwari and Campbell (21) showed that succinate-grown Pseudomonas aeruginosa, when placed on glucose-succinate medium, exhibited diauxic growth. The tricarboxylic acid cycle enzymes were constitutive, but the inducible EntnerDoudoroff enzymes were present in low levels until the succinate was used up. Diauxic growth on succinate and maltose was not observed in P. fluorescens W, but some repression of alpha-glucosidase synthesis was detected. This may indicate a less stringent control over inducible enzyme systems by tricarboxylic acid cycle intermediates in this organism than in P. aeruginosa. As far as alanine repression is concerned, pseudomonads have been shown to utilize amino acids in preference to sugars (7), but the mechanism for this is not really known. The results of the electrophoresis studies of the cell extract indicated the presence of a single alpha-glucosidase in P. fluorescens W. If the enzyme had subunit structure and was able to form multiple complexes of the subunits in a manner similar to E. coli beta-galactosidase (4), then more than one band of alpha-glucosidase activity would have been detected on the gels. LITERATURE CITED 1. Bentley, R., and L. Slechta. 1960. Oxidation of monoand disaccharides to aldonic acids by Pseudomonas species. J. Bacteriol. 79:346-354. 2. Bernaerts, M. J., and J. DeLey. 1960. Microbiological formation and preparation of 3-ketoglycosides from disaccharides. J. Gen. Microbiol. 22:129-136. 3. Eisenberg, R. C., S. J. Butters, S. C. Quay, and S. B. Friedman. 1974. Glucose uptake and phosphorylation in Pseudomonas fluorescens. J. Bacteriol. 120:147-153. 4. Erickson, R. P., and E. Steers. 1970. Comparative study of isoenzyme formation of bacterial beta-galactosidase. J. Bacteriol. 102:79-84. 5. Fitting, C., and H. W. Scherp. 1952. Observations on a strain of Neisseria meningitidis in the presence of glucose and maltose. J. Bacteriol. 64:287-298.
6. Han, Y. W., and V. R. Srinivasan. 1969. Purification and characterization of beta-glucosidase of Alcaligenes faecalis. J. Bacteriol. 100:1355-1363. 7. Hamilton, P. B., and G. Shelley. 1971. Chemotactic response to amino acids by Pseudomonas aeruginosa in semisolid nitrate medium. J. Bacteriol. 108:596-598. 8. Herbert, D., P. J. Phipps, and R. E. Strange. 1971. Chemical analysis of microbiol cells, p. 228. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 5B. Academic Press Inc., New York. 9. Hofnung, M., and M. Schwartz. 1967. La maltodextrine phosphorlase d'Escherichia coli. Eur. J. Biochem. 2:132-145. 10. Hylemon, P. B., and P. V. Phibbs. 1972. Independent regulation of hexose catabolizing enzymes and glucose transport activity in Pseudomonas aeruginosa. Biochem. Biophys. Res. Commun. 48:1041-1050. 11. Katznelson, H., and S. W. Tanenbaum. 1954. Observations on maltose oxidation by Acetobacter melanogenum. J. Bacteriol. 68:368-372. 12. Kusch, M., and T. H. Wilson. 1973. Defective lactose utilization by a mutant of Escherichia coli energyuncoupled for lactose transport: the advantages of active transport versus facilitated diffusion. Biochim. Biophys. Acta 311:109-122. 13. Leive, L. 1965. RNA degradation and the assembly of ribosomes in actinomycin-treated Escherichia coli. J. Mol. Biol. 13:862-865. 14. Loomis, W. F., and B. Magasanik. 1966. Nature of the effector of catabolite repression of beta-galactosidase in Escherichia coli. J. Bacteriol. 92:170-177. 15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 16. Nakada, D., and B. Magasanik. 1962. Catabolite repression and the induction of beta-galactosidase. Biochim. Biophys. Acta 61:835-837. 17. Neish, A. C. 1952. Analytical methods for bacterial fermentations. Report 46-8-3. National Research Council of Canada, Saskatoon, Saskatchewan, Canada. 18. Pardee, A. B., and L. S. Prestridge. 1961. Initial kinetics of enzyme induction. Biochim. Biophys. Acta 49:77-88. 19. Phibbs, P. V., and R. G. Eagon. 1970. Transport and phosphorylation of glucose, fructose, and mannitol by Pseudomonas aeruginosa. Arch. Biochem. Biophys.
138:470-482. 20. Romano, A. H., S. J. Eberhard, S. L. Dingle, and T. D. McDowell. 1970. Distribution of the phosphoenolpyruvate:glucose phosphotransferase system in bacteria. J. Bacteriol. 104:808-813. 21. Tiwari, N. P., and J. J. R. Campbell. 1969. Enzymatic control of the metabolic activity of Pseudomonas aeruginosa grown in glucose or succinate medium. Biochim. Biophys. Acta 192:395-401. 22. Tyler, B., W. F. Loomis, and B. Magasanik. 1967. Transient repression of the lac operon. J. Bacteriol. 94:2001-2011. 23. Umbreit, W., R. H. Burris, and J. F. Stauffer (ed.). 1957. Manometric techniques, 3rd ed. Burgess Publishing
Co., Minneapolis. 24. Whiteside, T. L., and W. A. Corpe. 1969. Extracellular enzymes produced by Pseudomonas sp. and their effect on cell envelopes of Chromobacterium violaceum. Can. J. Microbiol. 15:81-92. 25. Winters, H., and W. A. Corpe. 1971. Polyacrylamide gel electrophoresis of exoenzymes produced by Pseudomonas fluorescens strain W. Can. J. Microbiol. 17:241-248.
