JOURNAL OF BACTERIOLOGY, OCt. 1975, p. 262-268Copyright i 1975 American Society for Microbiology

Vol. 124, No. 1Printed in U.S.A.

Maltose Metabolism of Pseudomonas fluorescensARTHUR 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 toglucose via an inducible alpha-glucosidase (alpha-D-glucoside glucohydrolase,EC 3.2.1.20). No evidence for phosphorolytic cleavage or oxidation to malto-bionic acid was found in this organism. The alpha-glucosidase was totallyintracellular and was most active at pH of 7.0. Induction occurred when cellswere incubated with maltotriose or maltose. Induction was rapid and easilydetectable within the first 5 min after the addition of the inducer. Glucose and itsderivatives did not repress induction. Cells growing on DL-alanine or succinateplus 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 isoften initiated by hydrolysis of the sugar into itsmonosaccharide components. Simple hydrolysisof maltose to glucose by alpha-glucosidase oc-curs in bacteria (2, 11), but not invariably, sinceat least three other mechanisms of maltoseutilization are known (1, 2, 5, 9). Pseudomonasfluorescens W, isolated from soil and grown incomplex media, was found to secrete manyhydrolases (24, 25) into the extracellular envi-ronment. Included among the enzymes weresmall amounts of maltose-hydrolyzing activity(25). The disaccharide hydrolases of bacteriaare usually thought to be cell bound, but asurprising paucity of information about theformation and biosynthetic control of alpha-glucosidase (alpha-D-glucoside glucohydrolase,EC 3.2.1.20) of bacteria is available. It was withthese facts in mind that the present work wasundertaken.

MATERIALS AND METHODSOrganism and growth media. The bacterium

studied in this work was isolated from soil by T. L.Whiteside and identified as P. fluorescens (24). Theorganism, designated as strain W, was routinelytransferred every 2 weeks on slants of peptone-glucose-yeast extract agar (24) and, after growth for24 h at 30 C, stored at 4 C.The defined medium used in the experiments

described in this paper contained the following ingre-dients 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, wassupplied as the only required growth factor. The pH

'Present address: Department of Biochemistry, MountSinai School of Medicine of the City University of New York,New York, N.Y. 10029.

262

was 7.0 and did not change during growth. The carbonsources used in various experiments were DL-alanine,succinate, glucose, or maltose. They were autoclavedseparately (121 C for 15 min) and added aseptically tothe rest of the sterile medium, to give a final concen-tration of 14 mM maltose, 28 mM glucose, 56 mMDL-alanine, or 19 mM succinate.The organism was grown in 500-ml Erlenmeyer side

arm flasks (Belco Glass Co., Vineland, N.J.), 50 mlper flask, at 30 C on a rotary shaker. One milliliter ofa late log-phase culture was used as the inoculum.Growth was followed as increase in culture turibidityusing a Klett-Summerson photoelectric colorimeterwith a number 66 filter.

Preparation of cell extracts and other cellfractions. Cells from 50 ml of culture were harvestedby centrifugation in the cold (4 C) for 15 min at 12,000x g in a Servall model SS34 centrifuge (Sorvall Co.,Norwalk, Conn.). They were washed once with 20 mlof cold 0.1 M phosphate buffer, pH 7.0, suspended in 5ml of the same buffer, and ruptured by ultrasonicoscillation in a cuvette suspended in an ice bath for10 min at maximum intensity with an MSE ultrasonicdevice (Instrumentation Association Inc., New York,N.Y.). The suspension of ruptured cells was centri-fuged in the cold for 20 min at 27,000 x g.The supernatant fluid was carefully decanted anddesignated as cell extract.The procedure for the localization of alpha-glucosi-

dase differed from that described above. Extracellularenzyme was recovered from a late log-phase culturesupernatant fluid by precipitation with 90% satura-tion ammonium sulfate according to the method ofWhiteside and Corpe (24). The protein precipitatewas dissolved in a small volume of 0.1 M phosphatebuffer, pH 7.0. The cells, which had been sedimentedat 27,000 x g for 15 min, were washed twice in thesame buffer at 4 C, suspended in 5 ml of buffer, andbroken by ultrasonic oscillation for 15 min. They werecentrifuged at 1,085 x g for 30 min to removeunbroken cells. The supernatant fluid containing cell

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envelopes was then centrifuged at 27,000 x g for 30min. The resulting supernatant fluid was termed"extract," and the sediment containing cell envelopeswas suspended and washed several times. This frac-tion was termed "envelopes." All three fractions wereassayed for alpha-glucosidase activity by hydrolysis ofpara-nitrophenyl-a-D-glucoside (a-PNPG).

Assay for alpha-glucosidase activity. The releaseof glucose from maltose by cell extracts was assayedby a variation of the technique of Kusch and Wilsonfor beta-galactosidase (12). The reaction mixture (5ml total volume) contained 4 mg of extract protein,0.1 M phosphate buffer, pH 7.0, and various concen-trations of maltose (10, 5, 2.5, and 1.25 mM). Thereaction proceeded at 30 C and was stopped withBa(OH)2 and ZnSO4 exactly as described by Kuschand Wilson. Glucose was assayed by the Glucostatmethod according to the manufacturer's instructions(Worthington Biochemical Corp., Freehold, N.J.).The Km and Vmax were determined from a Line-weaver-Burk plot.

Alpha-glucosidase activity was also assayed witha-PNPG as the substrate, using a variation of theprocedure of Han and Srinivasan for beta-glucosidase(6). The reaction mixture contained a sample of cellextract, envelopes, or extracellular enzyme in 2.5 mlof 0.1 M phosphate buffer, pH 7.0. After equilibrationfor 5 min in a 30 C water bath, 0.5 ml of 5 mMa-PNPG was added, and the reaction was allowed toproceed for 30 min. The reaction was stopped by theaddition of 2 ml of 1 M Na2CO,. The yellow para-nitrophenol released was read at 400 nm in a Cole-man-Hitachi model 124 double-beam spectrophotom-eter. A unit of enzyme activity was defined asnanomoles of p-nitrophenol released per minute. Astandard curve was constructed using various concen-trations of p-nitrophenol. For the determination ofVmax and Km the following concentrations of a-PNPGwere used: 1 mM, 500 gM, 250 ,M, 100 gM, and 50AM.The optimum pH for activity was determined by

adding a 0.5-ml extract sample (100 ig of protein) in0.05 M phosphate buffer, pH 7.0, to 2 ml of each of thefollowing buffers: 0.1 M citrate-phosphate, pH 3 to 7;0.1 M phosphate, pH 6 to 8; 0.1 M tris(hydroxy-methyl)aminomethane (Tris)-hydrochloride, pH 8 to9; and 0.1 M glycine-NaOH, pH 9 to 10. One-halfmilliliter of 5 mM a-PNPG in deionized water wasadded, and the enzyme was assayed as above. The pHof each reaction mixture was measured directly.

Alpha-glucosidase activities in cells grown onvarious carbon sources. For the production of en-zyme in asynchronous cultures, 14 mM maltose alone,maltose (14 mM) plus glucose (28 mM), succinate (19mM), or DL-alanine (56 mM) was used as the carbonsource. Samples (5 ml) were withdrawn at varioustimes (early log, late log, stationary phase), and cellextracts were prepared and assayed for alpha-glucosi-dase activity with a-PNPG.

Induction of alpha-glucosidase synthesis. Mal-tose or other potential inducers were added to culturesgrown on glucose, succinate, or alanine as the solecarbon source, and incubation was allowed to proceedat 30 C on a rotary shaker. The cells were harvestedbefore and after the addition of the inducer, and

extracts were assayed with a-PNPG as describedabove. For the study of induction in the first fewminutes after adding maltose, 10-xnl samples wereremoved from a log-phase culture and placed in testtubes containing either 3 drops of toluene in a bath ofice water or 100 jg of chloramphenicol per ml toprevent any further enzyme synthesis. Cell extractswere prepared and assayed for hydrolysis of a-PNPGusing the standard procedure.

Other enzyme assays and chemical determina-nations. The assay for glucose-i-phosphate andglucose-6-phosphate was done by the procedure ofHofnung and Schwartz (9). The assay utilized 50 IU ofphosphoglucomutase to convert glucose-1-phosphateto glucose-6-phosphate. Glucose-6-phosphate dehy-drogenase (30 IU) was also added to oxidize glucose-6-phosphate to 6-phosphogluconic acid, and the con-comitant reduction of nicotinamide adenine dinu-cleotide phosphate, reduced form, was followed spec-trophotometrically at 340 nm. Standard curves forglucose- 1-phosphate and glucose-6-phosphate wereconstructed. Cell extracts were assayed at 30 C for 20min. Maltodextrin formation was looked for by incu-bating either whole cells or extract with excess mal-tose and performing the iodine test as described byHofnung and Schwartz (9).

Inorganic phosphate was determined in appropri-ately diluted culture filtrate by the phosphomolyb-date method described by Herbert (8). A standardcurve. was prepared by plotting different concentra-tions of K2HPO4 against absorbance at 730 nm.

Protein was measured by the method of Lowry etal. (15) with the Folin-Ciocalteau reagent, usingbovine serum albumin as the protein standard. Spe-cific activity was expressed as nanomoles of p-nitro-phenol released per minute per milligram of extractprotein.Manometric methods. Oxygen consumption was

measured in a Warburg apparatus (American Instru-ment Co., Silver Spring, Md.). Washed cells in 0.1 Mphosphate buffer, pH 7.0, with 5 mg (dry weight) ofcells per ml were used. The Warburg flasks contained1 ml of cells, 1 ml of the phosphate buffer, and 0.3 mlof water in the main compartment; 0.2 ml of 20%KOH in the center well; and 0.5 ml of substrate in theside arm. All experiments were run in duplicate at 37C. Oxygen consumption and carbon dioxide produc-tion were corrected for endogenous activity. Carbondioxide was determined by the direct method (23),using a reaction flask without KOH. The substratestested included glucose, maltose, and maltobionatedissolved in deionized water. At the end of the reac-tion with maltose or glucose the contents of the vesselwere centrifuged at 15,000 x g for 15 min, and thesupernatant was assayed for residual sugar with theanthrone reagent (17).

Polyacrylamide gel electrophoresis. Polyacryl-amide gels (7.5%) in Tris-glycine buffer, pH 8.9, wereprepared according to the procedure used by Wintersand Corpe (25). Crude extracts (0.25 to 3.0 mg ofprotein) were electrophoresed at 4 mA/gel in aBuchler electrophoresis apparatus (Buchler Co., FortLee, N.J.). The alpha-glucosidase activity was as-sayed directly on the gels by incubating intact,water-rinsed gels in test tubes with 5 mM a-PNPG in

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264 GUFFANTI AND CORPE

0.1 M phosphate buffer, pH 7.0, at 30 C. Bands withalpha-glucosidase activity were yellow (p-nitro-phenol) in color.

Protein was stained on the gels with 0.25% Coomas-sie blue in 15% trichloroacetic acid and 25% methanolfor 20 min at room temperature. 'The gels were

destained with 7% acetic acid.Chemicals. Actinomycin D, rifampin, p-nitro-

phenol, chloramphenicol, and maltose were productsof 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.).

RESULTSEvidence for hydrolytic cleavage of

maltose. The growth of P. fluorescens W on

maltose was equivalent to growth on glucose interms of cell yield and doubling time. Washedwhole cells consumed the same amount ofoxygen whether incubated with 2 Mmol of glu-cose or 1 Amol of maltose (Table 1). For eachmole of oxygen consumed, 1 mol of carbondioxide was given off. The organism used two-thirds of the amount of oxygen necessary tocompletely oxidize all of the sugar. The remain-ing third was presumably assimilated, since thesupernatant showed the absence of carbohy-drate. The enzyme had a greater affinity fora-PNPG than for maltose, but split maltose at afaster rate when substrate was present in excess

(Fig. 1). Hydrolytic cleavage was generallyconfirmed when it was shown that cell extractsreleased more than 1 mol of glucose per mol ofmaltose (Table 2). In the case of small amountsof 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 split-ting activity and maltose cleaving activity were

induced by growth on maltose (Table 3). Oncethis was established we routinely used thesimple a-PNPG assay for alpha-glucosidaseactivity.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 theirpresence or absence in P. fluorescens W. Anytype of phosphorolytic cleavage was ruled out,since sonic extracts exhaustively dialyzedagainst Tris-maleic acid buffer, to remove inor-ganic phosphate, could still release glucose frommaltose. Furthermore, no glucose-i-phosphateor glucose-6-phosphate was detected when ex-

tracts 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 Tris-hydrochloride, pH 7.2, and 100 gg of K2HPO,per ml there was no improved uptake ofinorganic phosphate by cells growing on maltosecompared to those growing on glucose. Also,when excess maltose was added to extracts or

whole cells grown on maltose no iodine-detecta-ble polymer was found (9). Oxidation of maltoseto maltobionate and subsequent cleavage toglucose and gluconate was unlikely, becausewhole cells (Table 1) hardly oxidized malto-bionate and glucose was not released frommaltobionate by extracts (Table 2). Moreover,sonically ruptured cells oxidized maltose butnot maltobionate, indicating that the failure touse maltobionate was probably not due solely toits inability to penetrate the cell membrane.pH dependence, stability, and cellular loc-

ation 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 theextract at 4 C for at least 4 days, but lost about80% of its activity when incubated overnight at30 C.The P. fluorescens W alpha-glucosidase ac-

tivity was shown to be almost wholly intracellu-lar. Approximately 98% of the enzyme activitywas found in cell extracts (Table 4), with very

little in the cell envelopes and only traces in theculture supernatant fluid.

Induction of alpha-glucosidase. Among thevarious sugars and sugar analogues tested as

inducers of alpha-glucosidase activity only mal-

TABLE 1. Oxygen consumption and carbon dioxide production with various substrates

Concn Oxygen consumed Carbon dioxide producedSubstratea (pmol)(Amol1Al-mol Al Amol

Maltose 1 177 7.9 179 8.0Glucose 2 180 8.0 178 7.9Maltobionate 1 10 0.4 NDb ND

a Substrates were incubated with washed cells, and oxygen consumption and carbon dioxide production weredetermined in duplicate Warburg vessels. The values shown were corrected for endogenous activity."ND, Not determined.

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MALTOSE METABOLISM OF P. FLUORESCENS 265

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 theamount of glucose released was measured. Various amounts of a-PNPG were added to another reactionmixture, and the amount of p-nitrophenol released was measured. Activities are expressed per milligram ofextract protein.

tose and maltotriose were satisfactory (Table 3).When grown on succinate or DL-alanine none ofthe following repressed the induction by mal-tose: 2-deoxyglucose, glucose, sorbose, glucose-1-phosphate, glucose-6-phosphate, gluconate,alpha-methylglucoside, alpha-phenylglucoside,or a mixture of gluconate and glucose.

Chloramphenicol inhibited synthesis of al-pha-glucosidase above the noninduced level(Table 5). Essentially the same was true ofactinomycin D, an inhibitor of messenger ribo-nucleic acid synthesis, but it only inhibitedwhen cells were pretreated with ethylenedia-minetetraacetic acid to alter the permeabilityof the outer wall (13). Ethylenediaminetetra-acetic acid alone did not inhibit alpha-glucosi-dase induction. Rifampin, another transcrip-tional inhibitor, allowed some induction (Table5). This was probably due to its slow penetra-tion into this organism (Sheela Amrute, per-sonal communication). Induction was very rapidin growing cells and was detectable within thefirst 5 min after adding maltose.Production of alpha-glucosidase during

growth on various carbon sources. The high-est specific activity of a-glucosidase occurred in

TABLE 2. Release of glucose from maltose by cellextracts

GlucoseSubstrate0 Concn liberated(nmol/ml) (nmol/ml)

Maltose 550 610275 330140 25070 140

Maltobionate 275 0550 0

aOne milliliter of extract (0.6 mg of protein) frommaltose-grown cells was incubated for 30 min at 30 Cin a reaction mixture containing 0.1 M phosphatebuffer, pH 7.0, and the indicated substrate concentra-tion in a final volume of 3 ml.

the late log phase of growth on all four carbonsources tested (Fig. 3). The activity with mal-tose as the sole carbon source or with maltoseplus glucose was very similar. When succinateor DL-alanine was present with maltose in themedium, the specific activities were lower atcomparable stages of growth and never reachedthe level attained in cultures with maltose alone

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266 GUFFANTI AND CORPE

TABLE 3. Induction of alpha-glucosidase by severalsugars

Sp act

Inducera a-PNPG assay Glucose(nmol/ (nmoVlmin/mg) min/mg)

None 0.25 0.35Maltose 3.75 3.37Maltose + glucose (10 mM) 3.71 NDbMaltotriose 3.46 NDa-PNPG 0.75 NDa-Phenylglucoside 0.25 NDa-Methylglucoside 0.25 NDGlucose 0.25 NDCellobiose 0.25 NDIsomaltose 0.25 NDLactose 0.25 NDMaltobionate 0.25 NDSucrose 0.25 NDMaltose grownc 25.0 ND

a Cultures were grown on the regular synthetic mediumwith 19 mM succinate as the sole carbon source. A finalconcentration of 1 mM inducer was added to a mid-log-phaseculture, and incubation was continued for 1 h at 30 C. Thecells were harvested, extracted, and assayed with a-PNPG ormaltose (glucose release) in the usual way.

IND, Not determined.cAlpha-glucosidase was assayed in a mid-log-phase cul-

ture growing on 28 mM maltose as the sole carbon source.

0.7

0.6

0.4-

'~0.3-

0.2-

FIG. 2. pH dependence of alpha-PNPG hydrolysis.Cells were grown on 14 mM maltose as the sole carbonsource and broken by ultrasonic oscillation, andextracts were obtained. The reaction mixture con-tained 0.1 mg of extract protein; alpha-glucosidasewas expressed in nanomoles of p-nitrophenol releasedper minute per milliliter of extract.

or maltose plus glucose. In each case the in-ducer, maltose, was present in excess. No effluxof a-glucosidase activity into the culture me-dium was found at any stage of growth. Nosuggestion of diauxic growth was observed whensuccinate-grown cells were inoculated into amedium containing 5 mM succinate and 10mMmaltose. The same could be said for cellstransferred from DL-alanine to DL-alanine (5mM) and maltose (10 mM), except that growthwas a little slower than on maltose alone. Thedoubling times on glucose, maltose, or succinatewere essentially the same (6 h), but cells grow-ing on DL-alanine doubled in turbidity every12 h.Polyacrylamide gel electrophoresis of

crude cell extracts. Only one band of enzymeactivity was detected on 7.5% polyacrylamidegels by hydrolysis of a-PNPG, when up to 3 mg

TABLE 4. Localization of alpha-glucosidase inmaltose-grown P. fluorescens W

Alpha. %oSource of enzyme glucosidase) %of(U) toa

Culture supernatant 0.33 1Cell envelopes 0.33 1Cell extract 30.0 98

a Cells were grown on maltose as the sole carbonsource and units of a-glucosidase activity are ex-pressed as nanomoles of p-nitrophenol released perminute by 10 ml of cell culture material.

TABLE 5. Effect of translational and transcriptionalinhibitors on the induction of alpha-glucosidase

Inhibitor Concn (gg/ml) min/mg)

Nonea 2.13Actinomycin D 80 0.39None" 6.39Rifampin 10 0.79Nonec 1.59Chloramphenicol 100 0.25No maltose 0.29

a A mid-log-phase culture growing on glucose wasdivided in half, and 1 mM EDTA and 1 mM maltosewere added to each portion. Incubation continued ona rotary shaker at 30 C for 30 min, and then theculture was assayed for alpha-glucosidase in the usualway.

'Mid-log-phase cells were grown on glucose anddivided in half, and 1 mM maltose was added to eachportion. Incubation was as above, but for 2 h.

c Mid-log cultures were grown on alanine as carbonsource and divided in thirds, and 1 mM maltose wasadded to only two cultures. Incubation and assay wereas in footnote a.

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500400

300

20

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=

m-100

80

60

40

24

21

18

15

12

9

6

S 10 1 5 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 theinoculum was a late log-phase culture which had been grown in the absence of maltose on the carbon source tobe 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 releasedper minute per milligram of extract protein in cultures growing on glucose and maltose (0), succinate andmaltose (A), or alanine and maltose (a).

of extract protein was loaded onto the gel. Thisindicated the presence of only one species ofalpha-glucosidase in this organism.

DISCUSSIONThe evidence is clear that P. fluorescens W

utilizes maltose by hydrolizing it to glucosealone by means of an inducible alpha-glucosi-dase. If glucose plus some other derivative ofglucose were the product of this reaction one

would expect to find no more than 1 mol ofglucose released from 1 mol of maltose. Therewas no indication that inorganic phosphate wasnecessary for maltose cleavage, nor were glu-cose-i-phosphate or glucose-6-phosphate de-tected as products of maltose metabolism as hasbeen reported for Escherichia coli (9) and Neis-seria (5).The Warburg respirometry showed that mal-

tobionate was not oxidized either by whole cellsor sonically ruptured cells. If this pseudomonadcould oxidize maltose to maltobionate and thensplit it in a manner common to other pseudomo-nands and Alcaligenes (2), then maltobionateshould have been oxidized to the same extent as

maltose. Moreover, cell extracts did not releaseany glucose from maltobionate.The intracellular location of alpha-glucosi-

dase in P. fluorescens W was consistent withthat reported for other bacterial carbohydrasesfound in gram-negative bacteria (4, 16). Such alocation would necessitate a maltose-concen-trating system probably similar to other bacte-rial permease systems. The maltose permease inP. fluorescens W is currently under investiga-tion. Since only maltose and maltotriose were

good inducers of a-glucosidase we believe thatthe inducer binding site is quite specific for an

al-4 glucoside. The aryl analogue, a-PNPG, isan excellent substrate but a very poor inducerfor this enzyme. This could also be due to itsinability to readily enter the cell.Like that of other bacterial systems (18)

induction of the Pseudomonas alpha-glucosi-dase was accomplished within only a few min-utes of the addition of the inducer. This indi-cated rapid entry of the inducer and attachmentto the inducer binding site. Both de novo

synthesis of messenger ribonucleic acid andprotein were necessary for the induction of the

201

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268 GUFFANTI AND CORPE

enzyme. The same holds true for other induciblebacterial enzymes (16).The catabolite and or transient repression

characteristic of such inducible systems as beta-galactosidase in E. coli (14, 16, 22) was notdetected in this organism. Alpha-glucosidaseyield on maltose medium was not affected whenglucose was included simultaneously. The ap-parent repressive effect of succinate was not toosurprising in a strict aerobe such as Pseudo-monas. Pseudomonads lack a constitutive phos-phoenolpyruvate-glucose transport system (19,20) and a functional Embden-Meyerhof path-way (21). Unlike E. coli, the enzymes forglucose transport and catabolism are induciblein these strict aerobes (3, 10). Tiwari andCampbell (21) showed that succinate-grownPseudomonas aeruginosa, when placed on glu-cose-succinate medium, exhibited diauxicgrowth. The tricarboxylic acid cycle enzymeswere constitutive, but the inducible Entner-Doudoroff enzymes were present in low levelsuntil the succinate was used up. Diauxicgrowth on succinate and maltose was not ob-served in P. fluorescens W, but some repressionof alpha-glucosidase synthesis was detected.This may indicate a less stringent control overinducible enzyme systems by tricarboxylic acidcycle intermediates in this organism than in P.aeruginosa. As far as alanine repression isconcerned, pseudomonads have been shown toutilize 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 asingle alpha-glucosidase in P. fluorescens W. Ifthe enzyme had subunit structure and was ableto form multiple complexes of the subunits in amanner similar to E. coli beta-galactosidase (4),then more than one band of alpha-glucosidaseactivity would have been detected on the gels.

LITERATURE CITED1. Bentley, R., and L. Slechta. 1960. Oxidation of mono-

and disaccharides to aldonic acids by Pseudomonasspecies. J. Bacteriol. 79:346-354.

2. Bernaerts, M. J., and J. DeLey. 1960. Microbiologicalformation and preparation of 3-ketoglycosides fromdisaccharides. J. Gen. Microbiol. 22:129-136.

3. Eisenberg, R. C., S. J. Butters, S. C. Quay, and S. B.Friedman. 1974. Glucose uptake and phosphorylationin Pseudomonas fluorescens. J. Bacteriol. 120:147-153.

4. Erickson, R. P., and E. Steers. 1970. Comparative studyof isoenzyme formation of bacterial beta-galactosidase.J. Bacteriol. 102:79-84.

5. Fitting, C., and H. W. Scherp. 1952. Observations on astrain of Neisseria meningitidis in the presence ofglucose and maltose. J. Bacteriol. 64:287-298.

6. Han, Y. W., and V. R. Srinivasan. 1969. Purification andcharacterization of beta-glucosidase of Alcaligenesfaecalis. J. Bacteriol. 100:1355-1363.

7. Hamilton, P. B., and G. Shelley. 1971. Chemotacticresponse to amino acids by Pseudomonas aeruginosa insemisolid 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 microbiol-ogy, vol. 5B. Academic Press Inc., New York.

9. Hofnung, M., and M. Schwartz. 1967. La maltodextrinephosphorlase d'Escherichia coli. Eur. J. Biochem.2:132-145.

10. Hylemon, P. B., and P. V. Phibbs. 1972. Independentregulation of hexose catabolizing enzymes and glucosetransport activity in Pseudomonas aeruginosa. Bio-chem. Biophys. Res. Commun. 48:1041-1050.

11. Katznelson, H., and S. W. Tanenbaum. 1954. Observa-tions on maltose oxidation by Acetobactermelanogenum. J. Bacteriol. 68:368-372.

12. Kusch, M., and T. H. Wilson. 1973. Defective lactoseutilization by a mutant of Escherichia coli energy-uncoupled for lactose transport: the advantages ofactive transport versus facilitated diffusion. Biochim.Biophys. Acta 311:109-122.

13. Leive, L. 1965. RNA degradation and the assembly ofribosomes in actinomycin-treated Escherichia coli. J.Mol. Biol. 13:862-865.

14. Loomis, W. F., and B. Magasanik. 1966. Nature of theeffector of catabolite repression of beta-galactosidase inEscherichia 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 Folinphenol reagent. J. Biol. Chem. 193:265-275.

16. Nakada, D., and B. Magasanik. 1962. Catabolite repres-sion and the induction of beta-galactosidase. Biochim.Biophys. Acta 61:835-837.

17. Neish, A. C. 1952. Analytical methods for bacterialfermentations. Report 46-8-3. National Research Coun-cil of Canada, Saskatoon, Saskatchewan, Canada.

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