INTRACELLULAR STARCH FORMATION IN CORYNEBACTERIA

E. BERNARD CARRIER AND C. S. McCLESKEYDepartment of Bacteriology, Louisiana State University, Baton Rouge, Louisiana

Received for publication November 22, 1961

ABSTRACT

CARRIER, E. BERNARD (Louisiana State Uni-versity, Baton Rouge, La.) AND C. S. MCCLES-KEY. Intracellular starch formation in cor-ynebacteria. J. Bacteriol. 83:1029-1036. 1962.-Cor-ynebacterium tritici, C. striatum, C. renale, and C.pseudodiphtheriticum produce an intracellularstarch-like material when grown on nativestarches; glucose-i-phosphate, mono-, di-, andtrisaccharides do not serve as substrates for in-tracellular starch formation. C. pseudotuberculosisand C. kutscheri produce intracellular starch fromstarch substrates and glucose-i-phosphate. C.diphtheriae produces starch from glucose-i-phosphate only.

Polysaccharide synthesis bv bacteria appearsto be a common, if not universal, occurrence, butbacterial synthesis of starch-like polysaccharidesgiving a blue color with iodine is a relativelyrare phenomenon.The production of iodophilic polysaccharide

from glucose-i-phosphate has been observed inNeisseria (Hehre and Hamilton, 1946, 1948;Hehre, 1948), a mutant strain of Escherichia coli(Doudoroff et al., 1949), Corynebacterium diph-theriae and certain streptococci (Hehre, Carlson,and Neill, 1947; Carlson and Hehre, 1949), and aclostridium from pig caecum (Nasr and Baker,1949). With none of these organisms could glu-cose be substituted for glucose-i-phosphate.

Reports of the production of iodophilic mate-rial from monosaccharides by bacteria have beenmade by Svartz (1930), Spray (1948), andDoetsch et al. (1957). Bacteria capable of produc-ing starch-like material from disaccharides in-clude E. coli (Monod and Torriani, 1948), rumenstreptococci (Hobson and Mann, 1955; Crowley,1955; Crowley and Jevons, 1955), Neisseria(Hehre and Hamilton, 1946, 1948; and Hehre,1948), and a gram-negative anaerobic rod fromthe rumen of sheep (Doetsch et al., 1957).

Starches, glycogen, amylose, amylopectin, dex-

trins, maltotriose, and maltose were suitablesubstrates for the synthesis of iodophilic materialby the rumen streptococci described by Hobsonand Mann (1955), Crowley (1955), and Crowleyand Jevons (1955). Crowley and Jevons con-cluded that, since starch was hydrolized to mal-tose, the synthesis of iodophilic material wasmediated by the enzyme amylomaltase.No reports have been found of bacteria which

require, for starch synthesis, substrates exceed-ing two hexose units in length. This paper presentsevidence that certain corynebacteria produceintracellular iodophilic starch-like material onlyfrom substrates consisting of relatively longa-1,4-linked glucosidic chains.

MATERIALS AND METHODS

A total of 44 strains, representing 24 of theaerobic species of the genus Corynebacteriumdescribed in Bergey's Manual (Breed, Murray,and Smith, 1957), were initially employed in thestudy of starch utilization. The species found toproduce intracellular starch from one or moresubstrates are shown in Table 1.

Nutrient agar (Difco) was used as the basalmedium for the qualitative determination of com-pounds utilized in the formation of intracellularstarch, with the exception that the phosphoryl-ated carbohydrates were tested in filter-sterilizedyeast-extract medium (Hehre et al., 1947).

Sugars were added to nutrient agar in 1%concentrations; natural starches, Ramalin (Stein-Hall & Co., New York, N. Y.), maltoheptaose andNiigeli amylodexrins (Dexter French, Iowa StateUniversity) were added in 0.3% concentration.Sterilization was performed in an autoclave at121 C for 15 min. Superlose (Stein-Hall & Co.,New York, N. Y.), a highly purified amylose frac-tion of potato starch, and rice amylose were dis-solved in cold 0.1 N NaOH and neutralized with1 N HCI immediately before they were added tomedia; they partially retrograded when added tonutrient agar. Aqueous solutions of the Schardin-ger alpha- and beta-dextrins (Corn Products Co.,

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TABLE 1. Corynebacteria producing intracellularstarch from one or more substrates

Species Source Laboratorydesignation

C. diphtheriae type ATCC 9015 1 diulcerans

C. diphtheriae type G. T. Hauser* 1 di Ggravis

C. diphtheriae type G. T. Hauser 1 di Mmitis

C. diphtheriae type G. T. Hauser 1 di Iintermedius

C. striatum ATCC 6940 4 stC. pseudotuberculosis ATCC 8666 5 pstC. renale ATCC 10845 9 reC. kutscheri ATCC 11035 10 kuC. pseudodiphtheriticum ATCC 6981 17 psdC. tritici ATCC 11403 39 trC. tritici (CT101) M. 45 tr

P. Starrt

* Louisiana State Board of Health.t University of California.

Argo, Ill.) were filter-sterilized and added tosterile nutrient agar to give 1% concentrations.Petri plates were prepared with the variousmedia, inoculated by the streak method, and in-cubated for 5 days at 37 C. Starch formation was

tested by flooding the colonies with Gram's iodinesolution.The intracellular location of the starch-like

material was determined by microscopic examina-tion of cells suspended in Gram's iodine and bythe treatment of heated and unheated washedcells with saliva.

Nutrient broth containing 0.3% potato starchwas used as the medium for the quantitative de-termination of the utilization of this substrate.Bacteria were removed by centrifugation andthe cell-free supernatants tested for starch by themethod of McCready and Hassid (1943). Thecells were washed three or four times in distilledwater and adjusted to an optical density of 0.210(Spectronic 20, 660 m,u). To 5 ml of the adjustedcell suspension were added 0.05 ml each of Gram'siodine and 6 N HCl. Several minutes were allowedfor maximal color development, after which op-

tical densities were again determined. An in-crease in optical density (above 0.210) was con-

sidered to be due to the intracellular starch-iodinecomplex.Reducing compounds were determined on the

cell-free supernatants by the method of Somogyi(1945) and Nelson (1944). Paper partitionchromatography (MeCleskey, 1953; Townsend,1955) was used to detect carbohydrates.

Nutrient agar containing 0.3% potato starchwas used to study the effect of phosphate con-centration, pH, and growth temperature on thesynthesis of intracellular starch. Inorganic phos-phate concentration was varied from 0.5 to 3.0%,to give a tenfold ratio of phosphate to substrate.The hydrogen ion concentrations employed

varied from pH 5.5 to 8.5. Cultures were streakedon the various media and incubated at 4, 20, 30,37, and 45 C. Cultures on nutrient potato starchagar served as controls. After 5 days of incuba-tion, the plates were flooded with Gram's iodineand compared visually with the control plates.

Inorganic phosphate liberation from glucose-i-phosphate was determined by the method ofFiske and SubbaRow (1925). The medium em-ployed was that of Hehre et al. (1947). The bac-teria were removed by centrifugation, and thecell-free supernatant fluids were tested for inor-ganic phosphate.The type of intracellular iodophilic polysac-

charide produced from starch was determined bythe method of Schoch (1945).

RESULTS AND DISCUSSION

On nutrient agar containing potato starch,colonies of certain of the corynebacteria staineddark blue when flooded with Gram's iodine solu-tion (Fig. 1). A faint reddish color was usuallyobserved at the periphery of the blue colonies andextended 3 to 4 mm out into the medium. Thelight areas indicate hydrolysis of the substratestarch by contaminants which appeared 4 to 5days after the plates were inoculated. The coloniesof corynebacteria in the areas of hydrolysisstained blue when treated with iodine, suggestingthe intracellular location of the starch-like ma-terial.

Microscopic examination of cells treated withiodine showed from one to several large bluegranules per cell. The treatment of heated andunheated washed cells with saliva gave addi-tional proof that the starch-like material was lo-cated within the cells (Fig. 2). Cells which wereheated and then treated with saliva became nega-tive for starch after 25 min; unheated cells,treated in the same manner, remained positivefor starch.

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INTRACELLULAR STARCH IN CORYNEBACTERIA

FIG. 1. Colonies of Corynebacterium renale on starch agar, flooded with Gram's iodine. Light areas indi-cate hydrolysis caused by contaminants.

The substrates utilized by the corynebacteria inthe formation of intracellular starch are shownin Table 2. Glucose-i-phosphate was utilized forstarch synthesis by all strains of C. diphtheriae,as reported by Hehre et al. (1947), and also byC. pseudotuberculosis and C. kutscheri (Carrierand McCleskey, 1961).The polysaccharides produced from glucose-

1-phosphate by C. kutscheri and the intermediusstrain of C. diphtheriae gave a reddish-wine color;and those produced by C. pseudotuberculosis andmitis, gravis, and ulcerans strains of C. diph-theriae stained blue with iodine. This suggeststhat the polysaccharide of the former cultures isamylopectin or glycogen-like, similar to that pro-duced from sucrose by Neisseria sp. (Hehre,1948), and that of the latter is an amylose-typepolysaccharide.Corn starch, potato starch, rice amylose,

Superlose, Ramalin, and, to a limited extent,dextrin were utilized by C. striatum, C. pseudo-tuberculosis, C. renale, C. kutscheri, C. tritici, andC. pseudediphtheriticum in the formation of

intracellular starch. In all these species, theintracellular polysaccharide produced from sub-strate starch was of the amylose type.Only those corynebacteria producing iodophilic

polysaccharide from glucose-i-phosphate releaseda significant amount of inorganic phosphate fromthat substrate (Fig. 3). C. pseudotuberculosis andC. diphtheriae gravis produced a blue-stainingpolysaccharide and released 0.0685 and 0.0705g/100 ml, respectively, of inorganic phosphateafter 117 hr. C. kutscheri produced a wine-redstaining polysaccharide and released 0.0308g/100 ml of inorganic phosphate. The resultsindicate that those species which do not producestarch from glucose-i-phosphate lack a trans-port mechanism for this compound, or else donot possess phosphorylase.Hehre (1948) showed that by increasing the

molecular concentration of inorganic phosphateto sixfold that of the substrate concentration,the action of phosphorylase obtained from aNeisseria sp. was inhibited. A tenfold ratio ofinorganic phosphate to substrate starch failed to

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FIG. 2. Effect of saliva on heated and unheatedstarch-grown washed cells of corynebacteria. Iodinpwas added after exposure to saliva for 25 min. Tube1, unheated cells; tube 2, unheated cells plus saliva;tube 8, heated cells plus saliva; tube 4, heated cells;tube 5, starch plus saliva.

inhibit the formation of intracellular starch fromsubstrate starch by the corynebacteria tested.Although C. pseudotuberculosis and C. kutschericontain phosphorylase, the failure of high con-

centrations of inorganic phosphate to inhibitstarch synthesis from a starch substrate suggeststhat this is not the primary means by which theseorganisms synthesize intracellular starch fromstarch. Further evidence is suggested by the be-havior of C. kutscheri in forming amylose fromstarch, but amylopectin-like (red-staining) ma-

terial from glucose-l-phosphate.Green and Stumpf (1947) noted that the

Schardinger dextrins inhibited potato phos-phorylase and they concluded that the dextrinswere competing with starch for the same activesite on the enzyme. Schardinger alpha- andbeta-dextrin failed to inhibit intracellular starchsynthesis by the corynebacteria, but the alpha-dextrin increased the amount of iodine requiredfor formation of the blue starch-iodine complex,probably by sequestration of iodine (French, per-

sonal communication; Schoch, personal communi-cation).The initial pH of the growth medium and

TABLE 2. Synthesis of intracellular starch-like polysaccharide from various substrates by corynebacteria*

44 0 0

Culture oS0 .o3

0 04 .0C

C. diphtheriae(gravis) + - - - - - - -

C. diphtheriae (mitis) + - - - - - - -C. diphtheriae +

(intermedius)C. diphtheriae + - - - - - - -

(ulcerans)C. kutscheri + - + + + + + 4 - -

C. pseudotuberculosis + - + + + + + 4 - -

C. pseudo- - - + + + + + 41 - -

diphtheriticumC. renale - - + + + + + 4 - -

C. striatum - - + + + + + 4 - -

C. tritici - - + + + + + 4 - -

* All cultures were negative on glucose, fructose, maltose, sucrose, lactose, cellobiose, galactose,raffinose, melezitose, inulin, Schardinger alpha- and beta-dextrin, dextran, beta-limit dextrin, malto-heptaose, and Nageli amylodextrin. Symbols: + indicates production of intracellular starch; indi-cates slight or doubtful production of intracellular starch; - indicates no production of intracellularstarch.

t Amylose from potato.t Amylopectin from potato.

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1962]~INTRACELLULAR STARCH IN CORYNEBACTERIA 13

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Culture ~~~~~pH Temperature (C)

5.5 6.0 6.5 7.5 8.0 8.5 4 20 30 37 45

4 st +* + + + + + + + + (+5 pst () + + + + + + + + (+9 re (4±) + + + + + + + + (+lOku (4-) + + (+ (+ (+ + + + NG17 psd NG NG (+ + + (+ + + + NG39 tr (4-) (±4) ±4 + 41 (4±) + + + NG NG45 tr (4-) (4d) d4 + 4d (±4) + + + NG NG

* Parentheses indicate moderate growth inhibition; NG indicates no growth; + indicates produc-tion of intracellular starch; ±i indicates slight or doubtful production of intracelluilar starch.

growth temperature had little effect on synthesisof intracellular starch; starch was formed at alltemperatures and at all hydrogen ion concentra-tions allowing good growth of the organisms(Table 3).

Earlier reports of the synthesis of iodophilicpolysaccharides by bacteria listed sugar phos-phates, maltose, sucrose, and pentoses as satis-factory substrates. None of these seems to be a

primary intermediate in the formation of intra-cellular starch by the corynebacteria grown inthe presence of starch (Table 2). To test the pos-sibility that degradation products of starch,other than those included in Table 2, may beintermediates in the formation of intracellularstarch, chromatographic examinations, deter-minations of reducing compounds, and measure-ments of substrate and intracellular starch were

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CARRIER AND McCLESKEY

60 80 100 120

TIME IN HOURS

FIG. 4. Reducing compounds produced in potatostarch broth by corynebacteria. di G, Corynebacte-rium diphtheriae; 4 st, C. striatum; 5 pst, C. pseudo-tuberculosis. None of the corynebacteria which pro-

duced intracellular starch from starch substrateproduced reducing compounds from starch.

made on the culture fluids at intervals throughoutthe incubation period. Since C. diphtheriae gravispossesses an amylase and does not form intra-cellular starch from potato starch, it was includedin these studies as a control. C. tritici was notincluded since it produced intracellular starchonly feebly from substrate starch.The chromatograms revealed no carbohydrates

in the mobile phase, except with C. diphtheriae.The results of tests for reducing substances are

presented in Fig. 4. None of the species produc-ing intracellular starch from the starch substrateshowed significant increases in reducing sub-stances.The changes in substrate starch, intracellular

starch, and reducing compounds effected by fourspecies of corynebacteria which produce intra-cellular starch are shown in Fig. 5. In each in-stance, the decrease in substrate starch was ac-

companied by an increase in intracellular starch,without significant changes in the reducing valueof the medium.Though tests with culture supernatants and

concentrated cell suspensions revealed no evi-dence of amylase activity, either by increase inreducing substances or by chromatography, thesupernatants of sonic-treated cultures activelydegraded starch. Intracellular starch usually de-creased in cultures after depletion of the starchsubstrate.

The disappearance of substrate starch and theappearance of intracellular starch, without theoccurrence of reducing sugars, suggests that pos-sibly glucose or other fragments are transferredfrom the ends of the linear components of starchinto the cell where new starch molecules areformed, or else the intact amylose molecule istransported directly into the cell. If the formerhypothesis is true, the mechanism of intracellularstarch synthesis appears to be by a "transglycos-ylase" (Hehre, 1951), unique in that it requiresas substrate relatively long chains of a-1,4-linked glucosidic units. The failure to utilize glu-cose, glucose-containing di- and trisaccharides,beta-limit dextrin, phytoglycogen, glycogen,hepataose, and Nageli amylodextrin suggests a re-quirement for chains longer than 20 glucose units.Since the only satisfactory substrate for starchsynthesis was one giving blue color with iodine,and the full blue color depends on a chain lengtlhof 30 to 35 glucose units (Swanson, 1948), per-haps the chains must be at least 30 glucose unitsin length.Our reluctance to accept the hypothesis that

amylose molecules may be transported throughthe membrane of the bacterial cell led us (Car-rier and McCleskey, 1961) to tentatively acceptthe first hypothesis, that the synthesis is mediatedby a "transglycosylase" (Hehre, 1951). Usingthe precedent of nomenclature for amylosucrase(Hehre, Hamilton, and Carlson, 1949) andamylomaltase (Monod and Torriani, 1948), thedescriptive term "amyloamylase" was applied tothis enzyme which requires lolng chains ofa-1 ,4-linked glucosidic units as substrate for theformation of intracellular starch.The possibility that amylose may be trans-

ported directly into the cell has not been dis-proved, and the following observations appear tosupport this view. All the cultures which produceintracellular starch from substrate starch pro-duce amylose; no amylopectin has been detected.Only those substrates which stain blue with io-dine result in the formation of blue-staining intra-cellular polysaccharide. The reddish zone ob-served around colonies on starch agar may bedue to the taking up of the amylose fraction ofstarch, leaving the red-staining amylopectin. Theformation of intracellular amylose from Ramalin(a commercial amylopectin) may be due to thepresence of a small amount (about 5%) ofamylose in that product. Whether these organ-

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INTRACELLULAR STARCH IN CORYNEBACTERIA

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CARRIER AND MIcCLESKEY

isms synthesize starch with a transglycosylase or

accumulate it by means of a permease, theyappear to possess characteristics unusual among

the bacteria.

ACKNOWLEDGMENT

This study was supported in part by researchgrant E-2924 from the National Institute ofAllergy and Infectious Diseases, U. S. PublicHealth Service.

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HEHRE, E. J., D. M. HAMILTON, AND A. S. CARLSON.1949. Synthesis of a polysaccharide of thestareh-glycogen class from sucrose by a cell-free, bacterial enzyme system (amylosucrase).J. Biol. Chem. 177:267-279.

HOBSON, P. N., AND S. 0. MANN. 1955. Some fac-tors affecting the formation of iodophilicpolysaccharide in group D streptococei fromthe rumen. J. Gen. Microbiol. 13:420-435.

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