Jouwra OF BACTERIOLOGYVol. 88, No. 4, p. 838-844 October, 1964Copyright § 1964 American Society for Microbiology

Printed in U.S.A.

METABOLISM OF PENTOSES AND PENTITOLS BYAEROBACTER AEROGENES

I. DEMONSTRATION OF PENTOSE ISOMERASE, PENTULOKINASE, ANDPENTITOL DEHYDROGENASE ENZYME FAMILIES'

R. P. MORTLOCK2 AND W. A. WOODDepartment of Biochemistry, Michigan State University, East Lansing, Michigan

Received for publication 1 April 1964

ABSTRACT

MORTLOCK, R. P. (Michigan State University,East Lansing) AND W. A. WOOD. Metabolism ofpentoses and pentitols by Aerobacter aerogenes. I.Demonstration of pentose isomerase, pentuloki-nase, and pentitol dehydrogenase enzyme families.J. Bacteriol. 88:838-844. 1964.-Aerobacter aero-genes PRL-R3 is capable of utilizing as sole sub-strates for energy and growth seven of the eightaldopentoses and all of the four pentitols. Growthupon media containing either D-xylOse, L-arabi-nose, D-ribose, D-arabitol, or ribitol occurredwithin 24 hr at 26 C. When D-arabinose or L-arabi-tol were used as growth substrates, growth wascomplete within 2 days; 4 days were required forgrowth on D-lyxose or xylitol, and 3 to 4 weeks forgrowth upon L-xylose. The versatility of thisstrain of A. aerogenes is due to an ability to syn-thesize in the presence of appropriate carbohy-drates (inducers) families of enzymes which cata-lyze the metabolism of the carbohydrates (i.e.,families of pentitol dehydrogenases, aldopentoseisomerases, and pentulokinases). The specificity ofinduction for members of the enzyme families wasfound to vary, and cross induction of enzyme ac-tivity was common, especially among the pentitoldehydrogenases. Ribitol dehydrogenase activitywas detected in extracts of cells grown on all ofthe above carbohydrates with the exception ofD-xylose, L-arabinose, and D-ribose. The ribitoldehydrogenase activity of xylitol-grown cell ex-tracts was fivefold higher than the activity in ex-tracts of ribitol-grown cells.

Aerobacter aerogenes, PRL-R3, is unusualI Contribution no. 3272 of the Michigan Agri-

cultural Experiment Station. A preliminary reportof this investigation was presented at the AnnualMeeting of the American Society for Microbiologyat Kansas City, Mo., May, 1962.

2 Postdoctoral Fellow of the U.S. Public HealthService. Present address: Department of Micro-biology, University of Massachusetts, Amherst.

among bacteria in that it can grow on all eightof the aldopentoses (Simpson, unpublished data)and all four of the pentitols. This versatility ismore striking because many of these compoundsrarely, if ever, occur in nature. Fermentationstudies with specifically labeled pentoses showedthat several of the pentoses yield the same fer-mentation products and with identical labelingpatterns (Neish and Simpson, 1954; Altermatt,Simpson, and Neish, 1955). Thus, the same gen-eral metabolic routes must be involved for thefermentation of these compounds. The strategyfor pentose utilization as elucidated in manyorganisms involves isomerization of an aldopen-tose to (one of the four) ketopentoses, phos-phorylation of the ketopentoses, and epimeriza-tion of ketopentose-5-phosphates to D-xylu-lose-5-phosphate. Pentitols are utilized bydehydrogenation to the ketopentose, but theremaining steps are identical.For A. aerogenes, these processes accommodate

the whole range of C5 structures. Current infor-mation on these pathways is summarized in Fig.1. The individual reactions in the utilization ofL-arabinose, L-xylose, L-lyxose, and the pentitolswere intensively studied in this strain. As shownby Simpson, Wolin, and Wood [(1958); see alsoBurma and Horecker (1958) for similar processesin Lactobacillus plantarum], L-arabinose is isom-erized to L-ribulose which is then phosphorylatedto 4-ribulose-5-phosphate. This phosphate esteris then epimerized at carbon four to form D-xylu-lose-5-phosphate, the substrate of transketolase.Similarly, L-xylose and L-lyxose are isomerized toL-xylulose, which is then phosphorylated to L-xylulose-5-phosphate. By epimerizations at car-bons three and four, this ester is also convertedto D-xylulose-5-phosphate (Anderson and Wood,1962a). Thus, all of these isomers are convertedto D-xylulose-5-phosphate by epimerases.The oxidation of ribitol to D-ribulose and of

838

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

27

Dec

embe

r 20

21 b

y 22

3.17

.132

.67.

ENZYME FAMILIES

D-arabitol to D-xylulose by specific nicotinamideadenine dinucleotide (NAD)-linked dehydro-genases has been documented (Fromm, 1958;Wood, McDonough, and Jacobs, 1961; Lin, 1961),as have the oxidations of L-arabitol to L-xyluloseand xylitol to D-xylulose (Fossitt et al., 1964).For D-ribose, a difference in detail exists, in thatphosphorylation precedes isomerization.Growth of A. aerogenes on all of these five-

carbon structures implies an unusual ability tosynthesize several families of enzymes, with themembers of each famnily catalyzing the same re-

action, but each having a different specificity.Because of the value of these families in studyingdeterminants of specificity, a detailed analysis ofthis phenomenon has been undertaken.

MATERIALS AND METHODSBacteriological. A. aerogenes, PRL-R3, was

grown aerobically at 25 C on a minimal medium(Anderson and Wood, 1962a) supplemented with0.5% carbohydrate. The carbohydrate andMgSO4 were autoclaved separately and addedafter cooling. For some experiments, the carbo-hydrate was sterilized by filtration through a

type HA Millipore filter. The viable cell determi-nations were obtained by plating on the abovemedium supplemented with 1.5% Nobel Agar(Difco). Total cell counts were made with a

Petroff-Hausser counting chamber. Turbiditydeterminations were made in a Bausch & LombSpectronic-20 colorimeter, and were converted todry weight of cell material with a standard curve.

For standardized growth experiments, tubescontaining 5.5 ml of medium were inoculated togive 2 X 106 cells per ml, inclined to 450, andincubated at 26 C on a reciprocating shaker. Theinoculum was washed twice with sterile saltssolution prior to use. Cultures were consideredgrown when the turbidity at 620 m, reached an

optical density of 1.0.Analytical. Kinase activity was estimated by

use of the following coupled reaction sequencedescribed by Anderson and Wood (1962b):

(a) pentulose + adenosine triphosphate (ATP)

kinase > pentulose-5-phosphate

+ adenosine diphosphate (ADP)

(b) ADP + phosphoenolpyruvate

pyruvate kinase> pyruvate + ATP

IN A. AEROGENES !839

(c) pyruvate + reduced nicotinamide. .,{f,

adenine dinucleotide (NADIb)lactic dehydrogenae >lactate + NAD

For determination of pentitol dehydrogenaseactivity, the same reaction mixture was used,except that ATP was omitted and the rate ofpentulose reduction (NADH oxidation) at 340 m,uwas determined. To measure kinase activity inthe presence of large amounts of pentitol dehy-drogenase activity, reduced nicotinamide adeninedtnucleotide phosphate (NADPH) was substi-tuted for NADH. NADPH serves as an efficientreductant for pyruvate in the presence of excesslactic dehydrogenase, but is not utilized byNADH oxidase or the NADH-specific pentitoldehydrogenases, thereby greatly decreasing theblank rate. A unit of kinase or pentitol dehy-drogenase activity was defined as the amount ofenzyme giving an absorbancy change of 1.0 permin at 340 m,u in a reaction volume of 0.15 mland with a light path of 1 cm.

Isomerase activity was measured from the rateof pentulose formation by the method of Ander-son and Wood (1962a); 1 unit of isomerase in 2.0ml catalyzed the formation of 1 ,umole of pentu-lose per hr at 37 C.Oxygen utilization was determined by stand-

ard manometric methods. Protein was estimatedby the method of Lowry et al. (1951). Aldopen-toses were determined by the orcinol test (Mej-baum, 1939), with a 40-min heating time. Keto-pentose was measured by the cysteine-carbazoletest of Dische and Borenfreund (1951).

Chemical. Ribitol, D-arabitol, xylitol, D-xylose,and D-ribose were purchased from Nutritional

D-RIBOSE kinose O-RIBOSE-5-PHOSPHATE

D-ARABINOSE isomerose isomerosekinose

D-RIBULOSE D-RIBULOSE-5-PHOSPHATERIBITOL dehydrogenose L

0-XYLOSE isornerose 3pimeraseD-LYXOSE isomerose k

kinoseo-XYLULOSE 11-D-XYLULOSE-5-PH SPHAT

D-ARABITOL 4nit2roenseXYLITOL dehdromse4m

isoerneose kinose { 4-pineroseL-ARABINOSE --- L-RIBULOSE ko L-RIBULOSE-5-PHOSPHATE

L-XYLOSE isomeroseL-LYXOSE isoerose 3-epimerse

Aldopentose k/inose3-hnrsL-XYLULOSE --- L-XYLULOSE-5-PHOSPHATEL-ARABI TOL !2^dr9e~nqse A

FIG. 1. Aldopentose and pentitol metabolism byAerobacter aerogenes.

VOL'. 88, 1964

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

27

Dec

embe

r 20

21 b

y 22

3.17

.132

.67.

MORTLOCK AND WOOD

Biochemicals Corp., Cleveland, Ohio. r-Arab-inose and D-arabinose were obtained fromPfanstiehl Laboratories, Inc., Waukegan, Ill.;L-arabitol was purchased from Mann ResearchLaboratory, New York, N.Y.; D-lyxose was pur-chased from General Biochemicals, ChagrinFalls, Ohio; L-xylose, L-lyxose, and t-xylulosewere prepared as described by Anderson andWood (1962a); and L-xylose was also purchasedfrom General Biochemicals. D-Ribulose was pre-pared from the o-nitrophenylhydrazone deriva-tive as described by Muller, Montigel, and Reich-stein (1937). Recrystallization of aldopentosesand pentitols was carried out in absolute ethanol.

Enzymatic. To prepare cell-free extracts forenzyme assays, the cells were harvested by cen-trifugation, washed once in 0.3 volume of water,and broken with a 10-kc oscillator for 6 min.Whole cells and cell fragments were removed bycentrifugation.

TABLE 1. Growth of Aerobacter aerogenes PRL-R$on the various pentoses and pentitots*

Substrate used for growth of inoculum

Growth substrateD-GlU- Arab- Arab D-Lyx- L-Xy- XyIi-cose inse itol ose lose tol

D-Glucose..... 0.5t 0.5 0.5 0.5 0.5 0.5D-Xylose...... 0.5 0.5 0.5 0.5 0.5 0.5L-Xylose ...... 7 25 22 27 2 5D-Arabinose . .2 1 4 2 1.5 1L-Arabinose. . . 0.5 0.5 1 1 1 1'D-Ribose ...... 1 1 1 1 1 1D-Lyxose ...... 4 4 4 1 4 4D-Arabitol..... 0.5 0.5 1 1 0.5 1L-Arabitol..... 2 2 1 2 1 1Ribitol ...... 1 1 1 1 1 1Xylitol... 4 4 4 4 4 2

* The incubation was carried out in screw-captubes containing 5.5 ml of the salts solutionsupplemented with 0.5% of the indicated carbo-hydrate. The inoculum consisted of 2 X 106 cellsper ml, and was prepared from a culture grownon the indicated carbohydrate, harvested bycentrifugation, and washed twice with sterile saltssolution. The tubes were slanted in test tuberacks and shaken at 26 C. Turbidity determina-tions were made at time intervals, and werecorrected for carbohydrate blanks.

t Figures indicate number of days required toreach a turbidity reading of 1.0.

RESULTS

Growth characteistics. A. aerogenes, PRL-R3,utilized as a sole source of carbon and energy forgrowth seven of the eight aldopentoses and all ofthe pentitols. The times required to reach a pre-scribed growth level on these substrates areshown in Table 1. With the use of a standardinoculum of D-glucose-grown cells, growth onD-glucose, D-xylose, D-ribose, L-arabinose, ribitol,and D-arabitol was complete within 1 day. Alonger incubation time was required for the othersubstrates as follows: D-arabinose and L-arabitol,2 days; D-lyxose and xylitol, 4 days; and L-xylose,from 3 to 4 weeks. The longer growth period onthese substrates was due to a lag of approxi-mately 15 hr with D-arabinose and L-arabitol,and 3 days with D-lyxose or xylitol. When L-xylosewas used as substrate, the lag prior to growthvaried from 25 to 35 days. Growth on L-xylose,once initiated, was complete within 2 to 3 days.Varying the substrate concentrations did not sig-nificantly alter the time required for growth, butgrowth of the inoculum on a pentose or pentitolwhich afforded slow growth reduced the subse-quent growth time on the same substrate. Crossadaptation was noticed only in the case of L-xylose, xylitol, and i-arabitol; i.e., uxylose- orxylitol-grown inocula grew on L-arabitol within1 day. Extensive recrystallization of L-xylose,xylitol, D-lyxose, and D-arabinose from absoluteethanol did not decrease the time required forgrowth.

In a single experiment with lAyxose, with theuse of the standard inoculum of D-glucose-growncells, growth was complete within 2 days. n-Lyxose was not available in sufficient quantityfor further studies. Attempts to obtain growthwith L-ribose as the substrate were unsuccessful.

Carbohydrate oxidation. The ability of D-glu-cose-grown cells to acquire a system for oxida-tion of pentoses and pentitols was measuredmanometrically with washed-cell suspensions, inthe presence of salts and inducers (substrates;Fig. 2). The lag in ability to oxidize the inducerswas found to correlate with the time required toinitiate growth on the same substrates. Relativeto D-glucose, a lag of several hours was visiblefor i-arabinose, D-arabitol, D-ribose, and ribitol,presumably the time required to synthesize thenecessary enzymes. A slightly longer lag wasnormally observed with D-xylose. At 32 and 37 C,

840 J. BACTERIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

27

Dec

embe

r 20

21 b

y 22

3.17

.132

.67.

ENZYME FAMILIES IN A. AEROGENES

0

O1 2 3 4 5D-RIBOE

z L~~~~-ARABII

0

4-OURS IBITOL

z

w

._D-XYLOSE

D-LYXOSE

0L-ARABITOL

0 2 3 4 5

TIME (HOURS)

FIG. 2. Oxidation of pentoses and pentitols byglucose-grown cell suspensions at 26 C. Each flaskcontained 2.6 ml of a cell suspension (8.6 mg, dryweight, per flask), 0.8 ml of 40% KOH in the centerwell, and 0.1 ml of 1 M substrate added from the sidearm at 0 time. The cells were harvested by centrifu-gation, washed, and incubated in the salts solutionfor days prior to use. All values are corrected forendogenous activity.

the times for adaptation decreased but the re-

sults were qualitatively the same.

Specificity of induction-pentulokinases. Thephosphorylation of D- and L-ribulose, D- andL-xylulose, and D-ribose is an integral part ofpentose and pentitol utilization in A. aerogenes.The kinase activities for these substrates in ex-

tracts of cells grown on the various C5 substrateswere determined to establish the specificity forinduction and to obtain evidence for the numberof kinases involved. A tabulation of the specificactivities found in extracts is shown in Table 2.In the assay used, NADH oxidation resultingfrom ADP formation was automatically plottedwith high precision as a linear decrease in ab-sorbancy vs. time (Wood and Gilford, 1961).Rates taken from slopes of these lines alloweddiscernment of activity at very low levels, butestimation of kinase activity was somewhat lim-ited by the amounts of NADH oxidase and adeno-sine triphosphatase. A value of zero indicatesthat activity was not detectable or was no greater

than 1.0 unit per mg of protein. When variableresults were obtained from replicate extracts, thehighest and lowest values are given.The induction of L-ribulokinase and D-ribo-

kinase proved to be highly specific. L-Xyluloki-nase activity was found in extracts preparedfrom cells grown on L-xylose and L-arabitol;occasionally, low activities were detected in ex-tracts of xylitol-grown cells.Of the three substrates whose dissimilation is

known to involve D-xylulose as a common inter-mediate, D-xylose was the best inducer for D-xylu-lokinase, D-lyxose was the poorest, and D-arabitolwas intermediate. Xylitol, also metabolized byoxidation to D-xylulose, induced D-xylulokinaseto the same extent as did D-lyxose. Of the fivekinases, D-ribulokinase showed the least specific-ity for induction. Although this enzyme is be-

TABLE 2. Kinase activity of cells grown on variouspentoses and pentitols*

Growth D-Ribulo- D-Xylulo- L-Rib- L-Xylul D-substrate kinase kinase 0las kinasekikinase kinase

D-Glucose.. Ot 0 0 0 0D-Ribose.... 5.1-9.7 0 0 0 5.6-

6.6D-Arabinose 10-19.3 0-2.6 0 0 0D-Xylose. . .. 0-2.0 15-50 0 0 0D-Lyxose.... 0-1.0 1.4-3.3 0 0 0L-Arabinose. 1. 2-4.5 0 1.2- 0 0

8.8L-Xylose.... 1.0-5.0 0 0 1.0-8.0 0Ribitol. . 3.3-21.4 0 0 0 0D-Arabitol.. 0 9.7-11.4 0 0 0Xylitol .... 9.1-30 1.0-2.2 0 0-1.0 0L-Arabitol. 0-1.0 0 0 4.4-4.8 0

* Each cuvette contained 1.0 Mmole of MgCl2;1.5,umoles of glutathione; 8 uAmoles of tris buffer(pH 7.5); 0.5 jAmole of adenosine triphosphate;0.25,Amole of phosphoenolpyruvate; 0.1 ,umole ofNADH or NADPH; 0.05 ml of lactic dehydro-genase containing pyruvate kinase (WorthingtonBiochemical Corp., Freehold, N.J.; muscle);1.5 jumoles of pentulose; 3 to 8 units of enzyme;and water to a total volume of 0.15 ml. When arange of values was obtained with different prepa-rations, the highest and lowest values are given.A value of 0 indicates that the specific activitywas less than 1.0.

t Figures indicate specific activity in units permilligram of protein.

VOL. 88, 1964 841

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

27

Dec

embe

r 20

21 b

y 22

3.17

.132

.67.

MORTLOCK AND WOOD

lieved to be directly involved in the metabolismof only D-arabinose and ribitol, activity couldreadily be detected in extracts of D-ribose- andL-xylose-grown cells, and very high activity wasalways found in xylitol-grown cells.

Pentitol dehydrogenases. Table 3 demonstratesthe NAD-linked, pentitol dehydrogenase activi-ties in extracts. The data were calculated fromrates of NADH oxidation under conditions of thekinase assays, but without ATP added and withthe NADH oxidase rates subtracted. L-Arabitol( L-xylulose) dehydrogenase activity was nor-nmally detected only in L-arabitol-grown cells, al-though activity could be detected occasionally incells grown on other substrates. The best inducerfor D-arabitol (-- D-xylulose) dehydrogenase wasD-arabitol. Smaller amounts for this enzymewere always obtained from growth on D-xylose,D-lyxose, and xylitol. Low levels of ribitol (I D-ribulose) dehydrogenase could be detected in ex-tracts prepared from L-xylose-, D-lyxose-, D-ara-bitol-, and L-arabitol-grown cells. High activitywas present in D-arabinose- and ribitol-growncells. NADH-linked, L-ribulose reductase activ-ity was never observed.

TABLE 3. Pentitol dehydrogenase activities of cellsgrown on various pentoses and pentitols*

Growth substrate

D-Glucose ....

D-Ribose ....D-Arabinose.D-Xylose. ....

D-Lyxose .....

L-Arabinose.L-Xylose ..

Ribitol .......

D-Arabitol....Xylitol .......

L-Arabitol

Dehydrogenase activities(units/mg of protein)

Ribitoldehydro-genase

0

0

38-1450

1.1-5.10

1.0-4.346-1542.1-2.7125-8003.7-16

D-Arabitoldehydro-genase

0

0

0-2.41.0-4.13.1-17

0

0

0-2.347-177

7.3-9.30-1.8

L-Arabitoldehydro-genase

0

0

0

0

0-1.70

0

0-2.50

0-3.40-5.4

L-Ribulosereductase

0

0

0

0

0

0

0

0

0

0

0

* The assay system was identical to that inTable 2 except that the adenosine triphosphatewas omitted. The values given are for pentulosereduction. When different activities were obtainedwith different preparations, the highest and lowestvalues are given. A value of 0 indicates activitywas not detectable (less than 1.0).

TABLE 4. Isomerase activities of cells grown onvarious pentoses and pentitols*

Growth substrate

D-Glucose....D-Ribose ......D-Arabinose...D-Xylose.D-Lyxose.L-Arabinose...L-Xylose ...

Ribitol. ..D-Arabitol ...Xylitol .......L-Arabitol. ..

Specific activity (units per mg of protein)

D-Arab-inose

isomerase

01-2

17-39000

239-5840-10

0-0.20

D-Xyloseisom-erase

000

3-101-40000

0-0.50

L-Xyloseisom-erase

00

0.5-1000

8-150000

L-Arab-inoseisom-erase

00000

8-50000000

D-Lyxoseisom-erase

0000

2-3000000

* The assay system consisted of 130 inmoles ofcacodylate buffer (pH 7.0), 10 ,umoles of metal(MnCl2, for D- and L-arabinose isomerases,MgCl2 for D-xylose and D-lyxose isomerases, andCoCl2 for L-xylose isomerase), 100 umoles of sub-strate, and water to a total volume of 2.0 ml.Tubes were incubated at 37 C, and samples wereremoved at time intervals for analysis. A value of0 indicates a specific activity of less than 0.2.

Pentose isomerases. Table 4 demonstrates thespecificity of induction of the aldopentose isom-erases. High blank values in the eysteine-car-bazole test, due to the acid-catalyzed conversionof residual aldopentose to ketopentose, the in-herently low activity of the isomerases in crudeextracts, and the possibility of further isomeriza-tion of the ketopentose to the epimeric aldopen-tose, decreased the sensitivity of the assay. Theinduction of L-arabinose (-k L-ribulose) isomer-ase and D-lyxose (-* D-xylulose) isomerase ac-tivities in detectable amounts occurred only withL-arabinose and D-lyxose as inducers, respec-tively. In contrast, D-xylose (I D-xylulose)isomerase activity was detected in cells grown oneither D-xylose or D-lyxose. Activity for D-arabi-nose isomerase and L-xylose (I L-xylulose) isom-erase was detected in cells grown on eitherD-arabinose or L-xylose. The isomerizations ofD-arabinose and L-xylose are believed to be dueto a single enzyme. Trace amounts of D-arabinoseisomerase activity could also be detected in ex-tracts of cells grown on D-ribose and ribitol.

842 J. BACTERIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

27

Dec

embe

r 20

21 b

y 22

3.17

.132

.67.

ENZYME FAMILIES IN A. AEROGENES

DISCUSSION

The data indicate that the strain of A. aero-genes is capable of utilizing all of the eight aldo-pentoses except L-ribose and all four of the penti-tols as substrates for growth. [The inability toobserve growth on L-ribose as had been observedby Simpson (unpublished data) has not been re-solved.] With D-lyxose, xylitol, and L-xylose,where a long lag occurs prior to growth, the possi-bility exists that mutation and selection ratherthan utilization of pre-existing genetic informa-tion is involved. Standard plating experimentshave proved inconclusive in answering this ques-tion, because growth occurred on salt-agar platesin the absence of added carbohydrate. Growth onL-xylose is of special interest because of the verylong lag. In a number of fluctuation tests per-formed with L-xylose, and with a population of6.6 X 105 viable cells per tube, growth was notvisable in any tube before 20 days. Thereafter,growth occurred in all tubes in the next 45 daysor less. Thus, this experiment produced no definiteevidence for spontaneous mutation to L-xyloseutilization. However, L-xylose-grown cells retainthe ability of rapid growth on L-xylose even aftersubsequent transfer on D-glucose, indicating apersistence of information and suggesting a muta-tion to L-xylose utilization. It should be notedthat, with the possible exception of a specificpermease and L-xVlulose-5-phosphate 3-epimer-ase, all of the enzymes required for L-xylosemetabolism can be induced within 2 days bygrowth on either L-arabitol or D-arabinose. Thatis, L-xylulokinase and L-ribulose- 5-phosphate4-epimerase were demonstrated in extracts of cellsgrown on L-arabitol (Fossitt et al., 1964), andL-xylose isomerase is believed to be identical withD-arabinose isomerase which is induced bV D-arabinose (Anderson and Wood, 1962a).With respect to the families of kinases, pentitol

dehydrogenases, and isomerases, it is apparentthat synthesis of at least four types each of pentu-lokinase, pentitol dehydrogenase, and pentoseisomerase are possible. It is recognized, however,that purification and careful characterization arerequired before the final number can be estab-lished. Lack of specificity may reduce the numberof distinct enzymes in each family; or, in contrast,more members of each family may exist since dif-ferent proteins, with common enzymatic activi-ties, may be synthesized in response to separate

inducers, as in the case of the aslpartokinases(Stadtman et al., 1961).

D-Ribulokinase and ribitol dehydrogenase ap-peared not to be under as strict control as werethe other enzymes studied, since nonspecific in-duction of these enzymes was commonly observed.The D-ribulokinase activity observed in extractsof L-arabinose-grown cells may be explained bythe nonspecificity of L-ribulokinase. This enzyme,purified from L-arabinose-grown cells, phos-phorylates D-ribulose, L-arabitol, and ribitol(Simpson and Wood, 1958). The L-xylulokinasefrom L-xylose cells and the D-xylutlokinase fromD-xylose cells were purified and were shown to bespecific (Anderson and Wood, 1962b; Bhuyanand Simpson, 1962). The ribitol dehydrogenaseactivities observed after growth on D-lyxose andD-arabitol might be explained by the formation ofsmall amounts of D-ribulose in metabolism and thesubsequent induction of low levels of ribitol de-hydrogenase, as observed with A. aerogenes, 1033,by Hulley, Jorgensen, and Lin (1963). Perhapsthe most interesting example of cross induction isthe presence in xvlitol-grown cells of higher levelsof D-ribulokinase and ribitol dehydrogenase thanwere obtained by growth on any other inducer,including D-arabinose and ribitol.

ACKNOWLEDGMENTS

The authors wish to thank Amy Rogers forvaluable technical assistance.

This investigation was stupported by a grant-in-aid from the National Science Foundation.

LITERATURE CITED

ALTERMATT, H. A., F. J. SIMPSON, AND A. C.NEISH. 1955. The anaerobic dissimilation ofD-ribose-1-C"4, D-xylose-2-C14, and D-xylose-5-C14, by Aerobacter aerogenes. Can. J. Biochem.Physiol.33:615-621.

ANDERSON, R. L., AND W. A. WOOD. 1962a. Path-way of L-xylose and L-lyxose degradation inAerobacter aerogenes. J. Biol. Chem. 237:296-303.

ANDERSON, R. L., AND W. A. WOOD. 1962b. Purifi-cation and properties of L-xylulokinase. J.Biol. Chem. 237:1029-1052.

BHUYAN, B. K., AND F. J. SIMPSON. 1962. Someproperties of the D-xylulokinase of Aerobacteraerogenes. Can. J. Microbiol. 8:737-745.

BURMA, D. P., AND B. L. HORECKER. 1958. Pentosefermentation by Lactobacillus plantarum. IV.

VO L. 88, 1964 843

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

27

Dec

embe

r 20

21 b

y 22

3.17

.132

.67.

844 MORTLOCK

L-Ribulose-5-phosphate 4-epimerase. J. Biol.Chem. 231:1053-1064.

DISCHE, Z., AND E. BORENFREUND. 1951. A newspectrophotometric method for the detectionand determination of keto sugars and trioses.J. Biol. Chem. 192:583-587.

FOSSITT, D., R. P. MORTLOCK, R. L. ANDERSON,AND W. A. WOOD. 1964. Pathways of L-arabitoland xylitol metabolism in Aerobacter aerogenes.J. Biol. Chem. 239: 2110-2115.

FROMM, H. J. 1958. Ribitol dehydrogenase. I.Purification and properties of the enzyme. J.Biol. Chem. 233:1049-1052.

HULLEY, S. B., S. B. JORGENSEN, AND E. C. C.LIN. 1963. Ribitol dehydrogenase in Aerobac-ter aerogenes 1033. Biochim. Biophys. Acta 67:219-225.

LIN, E. C. C. 1961. An inducible D-arabitol dehy-drogenase from Aerobacter aerogenes. J. Biol.Chem. 236:31-36.

LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measure-ment with the Folin phenol reagent. J. Biol.Chem. 193:265-275.

MEJBAUM, W. 1939. Estimation of small amountsof pentose especially in derivatives of adenylicacid. Z. Physiol. Chem. 258:117-120.

MULLER, H., C. MONTIGEL, AND T. REICHSTEIN.

AND WOOD J. BACTERIOL.

1937. Reine L-erythrulose (L-2-keto-tetrose).Helv. Chim. Acta 20:1468-1473.

NEISH, A. C., AND F. J. SIMPSON. 1954. Anaerobicdissimilation of D-glucose-l-C14, D-arabinose-1-C'4, and L-arabinose-1-C'4 by Aerobacteraerogenes. Can. J. Biochem. Physiol. 32:147-153.

SIMPSON, F. J., M. J. WOLIN, AND W. A. WOOD.1958. Degradation of L-arabinose by Aerobac-ter aerogenes. I. A pathway involving phos-phorylated intermediates. J. Biol. Chem. 230:457-472.

SIMPSON, F. J., AND W. A. WOOD. 1958. Degrada-tion of L-arabinose by Aerobacter aerogenes.II. Purification and properties of L-ribuloki-nase. J. Biol. Chem. 230:473-486.

STADTMAN, E. R., G. N. COHEN, G. LEBRAS, ANDH. DE ROBICHON-SZULMAJSTER. 1961. Feed-back inhibition and repression of aspartoki-nase activity in Echerichia coli and Saccharo-myces cerevisiae. J. Biol. Chem. 236:2033-2038.

WOOD, W. A., AND S. R. GILFORD. 1961. A systemfor automatic recording of absorbancy and itsapplication to enzyme-catalyzed reactions.Anal. Biochem. 2:589-600.

WOOD, W. A., M. J. MCDONOUGH, AND L. B.JACOBS. 1961. Ribitol and D-arabitol utiliza-tion by Aerobacter aerogenes. J. Biol. Chem.236:2190-2195.

S . _ _IL ).

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

27

Dec

embe

r 20

21 b

y 22

3.17

.132

.67.