JOURNAL OF BACTERIOLOGY, Oct., 1965Copyright © 1965 American Society for Microbiology

Vol. 90, No. 4Printed in U.S.A.

Numerical Taxonomy of Certain CoryneformBacteria

G. A. NIGEL DA SILVA AND JOHN G. HOLTDepartment of Bacteriology, Iowa State University, Ames, Iowa

Received for publication 7 May 1965

ABSTRACTDA SILVA, G. A. NIGEL (Iowa State University, Ames), AND JOHN G. HOLT. Nu-

merical taxonomy of certain coryneform bacteria. J. Bacteriol. 20:921-927. 1965-Anelectronic computer was used to sort 32 strains of coryneforms into groups with thetree-sort program. The similarity values obtained in this procedure were then usedto construct a dendrogram depicting the. phenetic resemblance among the taxa. Theresults indicated that all the phytopathogens studied were sufficiently distinct from thetype species, Corynebacterium diphtheriae, to be excluded from the genus Corynebac-terium. The grouping of some of the phytopathogens with Microbacterium lacticumis discussed. C. fascians appeared so distinct from the other strains studied that itshould probably be excluded from the Corynebacteriaceae. The phenetic resemblance ofBrevibacterium linens and Arthrobacter globiformis was emphasized, and the newcombination, Arthrobacter linens, was proposed. In addition, because of distinct dis-similarity from the type species, it would seem desirable to exclude Arthrobactertumescens from the genus Arthrobacter. The justification for classifying Kurthia zopfiiin a family separate from the Corynebacteriaceae would appear to be open to seriousquestion. It was concluded that the present taxonomic positions of Listeria monocy-togenes, Cellulomonas biazotea, and Cellulomonas fimi are satisfactory.

"There are perhaps few groups of bacteria ofwhich the typical representatives are easier torecognize and the aberrant types more numerousand more difficult to separate than those whichoriginally constituted the genus Corynebacterium"(Jensen 1952). When first introduced by Leh-mann and Neumann (1907) to accommodate thediphtheria bacillus and certain similar parasiticorganisms, the genus was a pleasingly simpleone. Its species were characterized by gram-positive, nonsporeforming, nonmotile, nonacid-fast rods which showed a tendency to irregularstaining. During the nearly six decades since itsintroduction, however, so many different club-shaped bacteria have been included in the genus,and its outlines have become so fluid, that manyworkers have had recourse to protest againstwhat Conn and Dimmick (1947) refer to as the"misuse of the term Corynebacterium."In recent years, the word "coryneform" has

come to be used for a wide variety of gram-positive, nonsporeforming rods. Etymologically,however, the word coryneform means clublike.

I Based on a thesis submitted by the seniorauthor to the Graduate College of Iowa StateUniversity in partial fulfillment of the require-ments for the M.S. degree.

Originally, this vernacular designation was usedwith reference to those species belonging to thegenus Corynebacterium. In many instances,modern usage of this word would appear to beinconsistent with the implied meaning. For thepurpose of this paper, however, coryneform isused in the following sense: gram-positive,nonsporeforming, nonacid-fast rods showingmetachromatic granules; many are club-shapedand exhibit characteristic palisade arrangementof cells; many exhibit morphogenesis and atendency to irregular staining.The classification of these organisms has al-

ways been beset with problems. Probably themost controversial aspect, however, centersaround the inclusion of 11 plant pathogens inthe genus Corynebacterium. To the plant patholo-gist, these organisms are relatively unimportant.Yet, to the bacterial systematist, there has beena constant problem as to what genus or generathey should occupy. C. sepedonicum, for example,the potato ring rot bacterium, has been placedin six different genera since its isolation in 1913.In spite of this unsettled state of classification,few comprehensive studies have been made onthe relationship of the plant pathogens to theother coryneforms.

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The coryneform bacteria are widespread innature. Some of the soil forms have been wellstudied for their ability to degrade cellulose,others for their ability to undergo morphogenesis.Their taxonomy, however, has been sadly ne-glected. Consequently, not unlike their plantpathogenic counterparts, their generic placementhas been quite controversial.

Certain gram-positive bacteria closely re-sembling the soil coryneforms are now classifiedin the family Brevibacteriaceae (Bergey'sManual). Some of these organisms, now con-sidered as belonging to the genus Brevibacterium,have been investigated in some detail as a resultof investigations of the microbial synthesis ofglutamic acid. Mulder and Antheunisse (1963)found that the type species, B. linens, was quitesimilar to Arthrobacter globiformis. This tends toquestion the justification for classifying these twoorganisms in two separate families.

This investigation was undertaken primarilyto determine, by use of the methods of numerical

taxonomy, the relationship of the coryneformplant pathogens to jC. diphtheriae and to the typespecies of six other genera of coryneforms.

MATERIALS AND METHODS

Organisms. A total of 32 bacterial strains wasstudied. The strain designations and sources ofthese cultures are listed in Table 1. Cultures weremaintained on a medium consisting of TrypticSoy agar (Difco) supplemented with 0.4% yeastextract.

Determination of properties. The 57 standardmorphological and physiological properties listedin Table 2 were determined for each of the or-ganisms. The procedures employed in the deter-mination of all properties, except numbers 1, 5, 6,13, 14, 18, 23, and 57, were according to standardmethods (Society of American Bacteriologists,1957). Property 1 was determined by the methodof Leifson (1960). Properties 5 and 6 were deter-mined by direct microscopic observation of thebacterial growth on an agar plate as follows. Ayoung broth culture of the species being examinedwas streaked onto an agar plate and incubated,

TABLE 1. Bacterial strains used in the computer analysis*

Family Genus Species

Corynebacteriaceae Corynebacterium C. diphtheriae (type species) ATCC 11913C. fascians ATCC 12974, 12975C. flaccumfaciens ATCC 6887, 7392C. insidiosum NCIB 83, ATCC 10253C. michiganense ATCC 4450, 10202C. poinsettiae ATCC 9069, 9682C. sepedonicum ATCC 9850C. tritici ATCC 11402, 11403

Bean wilt bacteriumt

Arthrobacter A. globiformis (type species) ATCC 8010,NCIB 8620, 8907

A. tumescem MRI 836, ATCC 6947

Cellulomonas C. biazotea (type species) ATCC 486, NCIB8077

C. fimi ATCC 484

Microbacterium M. lacticum (type species) ATCC 8180,8181

Listeria L. monocytogenes (type species) ATCC 4428,15313 (type strain)

Brevibacteriaceae Brevibacterium B. linens (type species) ATCC 9172, 9174,9175

Kurthia K. zopfii (type species) ATCC 6900, 10538

* The systematic arrangement is according to Bergey's Manual. Abbreviations: ATCC = AmericanType Culture Collection, NCIB = National Collection of Industrial Bacteria (Great Britain), MRI =Microbiology Research Institute, Ottawa, Canada.

t Acquired from J. Dunleavy, Department of Botany and Plant Pathology, Iowa State University.

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TABLE 2. Properties used in the characterization of the organisms studiedProperties

1. Motility2. Hemolysis on blood-agar3. Presence of capsule4. Metachromatic granules5. Snapping division6. Morphogenesis7. Turbidity in broth8. Sediment in broth9. Reduction of litmus milk

10. Reduction of tellurite11. Nitrates reduced12. Casein hydrolyzed13. Tributyrin hydrolyzed14. Tyrosine hydrolyzed15. Starch hydrolyzed16. Gelatin hydrolyzed17. Voges-Proskauer-positive18. Cytochrome oxidase-positive19. Catalase-positive20. Citrate-positive

21. Urea-positive22. Cellulose digestion23. Ammonia from peptone24. Methyl red-positive25. Hydrogen sulfide produced26. Resists 62 C for 10 min27. Indole produced28. Growth at 37 C29. Acid from adonitol*30. Acid from arabinose31. Acid from cellibiose32. Acid from fructose33. Acid from galactose34. Acid from glucose35. Acid from inositol36. Acid from lactose37. Acid from maltose38. Acid from mannitol39. Acid from mannose40. Acid from melibiose

41. Acid from raffinose42. Acid from rhamnose43. Acid from salicin44. Acid from sorbitol45. Acid from sorbose46. Acid from sucrose47. Acid from trehalose48. Acid from xylose49. Surface growth in broth50. Colony elevation51. Colony edge52. Growth form on a slant53. Surface texture of colony54. Pigmentation55. Growth in NaCl at concentra-

tionis of 2.5, 5, 7.5, 10, and12.5%

56. Gram stain57. Average length of the cell

* The basal medium employed in determining properties 29 to 48 for all species except Corynebac-terium diphtheriae was nutrient broth. Hiss serum water was used for C. diphtheriae. The method ofpreparation of the Hiss serum water was as outlined by Wilson and Miles (1964).

after which a sterile cover slip was placed on topof the growth and examined directly with a phasemicroscope. For property 5, cultures were ex-amined at intervals of 4, 6, and 10 hr. For property57, determinations were made with the aid of amicrometer on the 24-hr growth used for deter-mining the previous property. The presence ofcytochrome oxidase was determined by flooding24- to 48-hr-old agar plate cultures with a 1%solution of p-aminodimethylaniline oxalate. De-velopment of a pink colony which turned maroonand finally black was considered to be positive.Production of ammonia from peptone was deter-mined in accordance with the method outlined byDowson (1957). Hydrolysis of tyrosine and tri-butyrin were determined by use of the method ofDavis and Park (1962).

Coding offeatures for data analysis. A code sym-bol (A, B, C, D, or E) was assigned for each al-ternative state in which a property could occur.Since properties 1 to 48 existed as one of twomutually exclusive states, they were coded for thecomputer analysis as A = positive, B = negative.For properties 50 to 55, the method suggested byLockhart and Hartman (1963) was used. A dif-ferent symbol was assigned to each separate statein which a property existed.Many of the strains, particularly the soil

coryneforms, exhibited variability in their gramreaction, depending on the age of the culture atwhich the stain was made. The method employedin coding this feature is as follows. When thereaction to the Gram stain was positive at 24 and72 hr, code symbol A was assigned; when the reac-tion was gram-neia.tive at 24 hr and gram-posi-

tive at 72 hr, B was assigned; and when the reac-tion was gram-negative at 24 and 72 hr, code Cwas assigned. Property 49 was coded according tothe method of Lockhart (1964). The presence ofsurface growth was considered as a primary prop-erty; the presence of a ring or membrane, as a sec-ondary property. For property 57, all strains withan average cell length less than 3.0 ,u were assignedone code symbol; those in excess of 3.0 ,/ wereassigned an alternative symbol.

Computer operation. The machine employed inthe analysis of these data was the IBM 7074 ofthe Iowa State University Computation Center.The computer method used was one developed byLockhart, Hartman, and Smith in 1963 (unpub-lished data), and is known as the tree-sort method.In this method, the coded properties were tran-scribed onto special data cards and then suppliedto the computer. The machine searches the datafor those pairs or groups of strains which are alikein all their properties, and then prints up theseclusters of strains as forming a group. The com-puter next searches the data for those strains whichdiffer in only one property and prints up these.This procedure is repeated over and over, themachine incrementing the number of differencesby one each time until the taxa are all groupedtogether in one large group. In this way, thestrains form several smaller clusters separated atvarying difference levels which eventually jointo form one large cluster at sufficiently largelevels of difference. In the tree-sort method, thereis a direct linear relationship between the dif-ference level and the percentage similarity. [Con-version of the difference level to percentage

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similarity was obtained according to the formula:Sij = (NF - NDij)/NF, where Sij = percentagesimilarity between an ith and jth taxon, NF =total number of features represented per taxon,NDij = number of features by which the ith andjth taxon differed.] Since the latter criterion is amore convenient way of expressing phenetic re-semblance among the taxa, it was used for presen-tation of the results.

RESULTS AND DISCUSSIONThe results of the analyses were arranged in

the form of a dendrogram, which is representedin Fig. 1.

PERCENTAGE SIMI LARITY70 8060

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From the dendrogram it is evident that atleast four distinct clusters emerge. The largestof these clusters is delimited by a phenon level of82%, and contains five of the phytopathogenicspecies, C. poinsettiae, C. tritici, C. sepedonicum,C. insidiosum, and C. michiganense. Clusterformation revolves around M. lacticum. Thephenetic resemblance among this cluster appearsto be reasonably convincing. However, it ispossible that this resemblance is not entirelycorrect and may have resulted from the fact thatMicrobacterium lacticum was incompletely com-pared with the phytopathogens. One aspect of

GROUPS90 100

- ARTHROBACTER TUMESCENS

- --C KURTHIA ZOPFII

CORYNEBACTERIUM DIPHTHERtAE

CORYNEBACTERIUM FLACCUMFACIENS

.I { -

II LI

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BEAN WILT BACTERIUM

CELLULOMONAS BIAZOTEACELWLOMONAS FIMI

CORYNEBACTERIUM POINSETTIAE

OORYNEBACTERIUM MICHIGANENSEATCC 10202

MICROBACTERIUM LACTICUM

CORYNEBACTERIUM INSIDIOSUM

CORYNEBACTERIUM TRITICI

CORYNEBACTERIUM SEPEDONICUM

CORYNEBACTERIUM MICHIGANENSEATCC 4450

-f.Z. LISTERIA MONOCYTOGENES

I}2

-3

4

5

-6

NCIB 8907I ARTHROBACTER GLOBIFORMIS ATCC 8010

NCI B 8602- 7

BREVIBACTERIUM LINENSATCC 9174ATCC 9172ATCC 9175

fE111171111 CORYNEBACTERIUM FASCIANS

I

FIG. 1. Dendrogram depicting the phenetic resemblance among the species studied. The horizontal linesrepresent the taxa and the levels at which they unite to form groups. The broken vertical lines indicate thephenon levels.

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this incomplete comparison involves the path-ways for glucose metabolism. Most of the coryne-form phytopathogens appear to metabolizeglucose oxidatively (Katznelson, 1958; Starr,1959). On the other hand, M. lacticum appearsto use the Embden-Meyerhof pathway. Surelysuch differences in enzymatic constitution shouldbe taken into consideration when computingphenetic resemblance. However, it is difficult toassess the extent to which this property should beweighted. Consequently, it was not used in deter-mining these similarity values.The second largest cluster is made up of strains

of A. globiformis and B. linens. The cluster isdelimited by a phenon level of approximately83%. It appears as the most distinct cluster ofthe organisms studied, and, in spite of the factthat the two constituent species each belong todifferent families, the grouping is not altogethersurprising. Other workers have also reportedthat these two species appear to be pheneticallysimilar (Mulder and Antheunisse, 1963). It issurprising to find, however, that A. tumescensis so distinct from its type species, A. globiformis.It can be seen that this species has a phenonlevel, in relation to the other species, of just over75%.

Several species in the genus Arthrobacter ap-pear to be out of place. Cummins (1962) andCummins and Harris (1958, 1959) pointed outthat major discrepancies exist between the cell-wall constituents of A. globiformis and A. tumes-cens. Zagallo and Wang (1962) presented evi-dence of significant metabolic dissimilarity amongthe species in this genus. They found, for example,that the type species used mainly the Embden-Meyerhof pathway for the metabolism of glu-cose, but that other species-A. simplex, A. pas-cens, and A. atrocyaneus-relied primarily on theintermediary formation of gluconate.The tendency in the past few years has been

to assign species to the genus Arthrobacter mainlyon the basis of morphology, often despite distinctdifferences in nutritional requirements (Zagalloand Wang, 1962). This practice is understandablein view of the fact that all too often it is difficultto obtain significant physiological response fromthese organisms. This has led to the questionableinclusion of many species in this genus. It wouldseem that our results cast further doubt on theorganization of this genus as it stands in Bergey'sManual. Clearly, on the basis of the comparativerelationships presented in Fig. 1, A. tumescensno more belongs in the genus Arthrobacter thandoes Listeria monocytogenes or, for that matter,C. diphtheriae.The genus Brevtibacterium has similarly be-

come the repository for a variety of gram-posi-tive bacteria. The species in this genus range

from physiologically inert to active fermentativeand oxidative organisms. Indeed, the only thingdefinite about the generic description in Bergey'sManual is that the organisms are gram-positive,nonsporeforming, unbranched rods. It has beenpointed out previously that B. linens possessesmany of the characteristics peculiar to coryne-forms. In addition, Su and Yamada (1960) havecalled attention to the fact that this species isquite different physiologically from most of theother species in the genus. In view of this evi-dence, and because our analysis clearly shows thecloseness of B. linens to A. globiformis, we proposethat B. linens be transferred to the genus Arthro-bacter and the new combination, Arthrobacterlinens (Wolff) da Silva and Holt, be created.It will be necessary to study the other speciesin the genus Brevibacterium to determine theirtaxonomic position. Such studies are in progressat this time, and it is hoped that the reallocationof the remaining species will soon be forthcoming.The next largest clusters consist of three plant

pathogens and three cellulolytic coryneforms.These phytopathogens, which are represented byC. flaccumfaciens and an unnamed strain causingsoybean wilt, appear to be distinct from the groupof phytopathogens previously discussed. Theycould probably be considered as belonging to aseparate genus. The Cellulomonas strains appearto be distinct. This is a welcome finding, sincemore often than not the sole criterion used inassigning organisms to this genus is their abilityto attack cellulose. It would thus appear that, asfar as these species of Cellulomonas are concerned,the ability to degrade cellulose is sufficientlyhighly correlated with other features to make it areliable diagnostic feature with which to separatethese organisms from the other coryneforms.The species Corynebacterium fascians was the

most distinct of all the species studied. It had aphenon level of less than 70%, indicating markeddifference from the other coryneforms. Severalworkers (Starr et al., 1943; Conn and Dimmick,1947) have expressed the opinion that it does notbelong in the genus Corynebacterium. In fact,Conn and Dimmick considered it to be closelyrelated to Nocardia, which would place it in anentirely different order. Cummins (1962) statedthat, on the basis of its cell-wall constituents, itmore closely resembles Mycobacterium smeg-matis. Further, Lacey (1955) observed that, bygrowing C. fascians strains on a synthetic mediumlow in nitrogen, filamentous growth could beobtained which resembled that produced byNocardia species. In addition, under such condi-tions, most of the strains she studied were acid-fast. On the other hand, evidence in support ofits present taxonomic position is not entirelylacking. It has been found that the guanine plus

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cytosine ratio is approximately the same in C.fascians as it is in C. diphtheriae (Marmur,Falkow, and Mandel, 1963). Nevertheless, mostevidence points to the fact that there is justifica-tion for considering C. fascians as being suffi-ciently distinct to question the desirability ofits inclusion in any genus of the Corynebacteri-aceae. Hence, in line with previous workers, weare of the opinion that it should be excludedfrom the genus Corynebacterium. However, weare reluctant to propose that it be assigned to aparticular taxon, since the appropriate compara-tive studies are as yet incomplete.From the analyses, it is evident that the phyto-

pathogens differ significantly from the type ofthe genus in which they are presently classified.These findings confirm the doubts expressed byprevious workers as to the correctness of includ-ing these organisms in the genus Corynebacterium.Under the circumstances, it would seem desirablethat these species be excluded from this genusand allocated to other genera. The disposition oftwo of these phytopathogenic species has alreadybeen discussed. It has been pointed out that, inthe case of the third group, additional study maybe necessary to confirm the validity of the cluster.However, for the purposes of allocation of thespecies of this cluster to a different taxon, twotype species are available, M. lacticum and C.insidiosum. The latter name was used bySavulescu (1947) for the type of the genus Burk-holderiella. Since the name is both validly pub-lished and legitimate, it would seem to be ap-propriate. However, the name M. lacticum haspriority over B. insidiosum by virtue of beingproposed earlier. Hence, under the circumstances,the generic name Burkholderiella cannot be usedfor this group of organisms as long as M. lacticumis included. On the other hand, for reasons dis-cussed previously, M. lacticum would also beundesirable.

In order not to complicate matters further, weare reluctant at this time to suggest that thefive species of plant pathogens be placed in aseparate genus. However, we wish to emphasizethe need for further study aimed at removingthese species from the genus Corynebacteriumand placing them in appropriate new taxa.The present taxonomic rank assigned to

Kurthia and Listeria (Bergey's Manual), asreflected by the strains of these genera studied,would appear to be satisfactory. Their taxonomicposition in relation to each other appears lesssatisfactory. This is evidenced by the fact thatboth K. zopfii and L. monocytogenes have thesame phenon level in relation to the other strains,and yet they are classified in separate families.

On the basis of our findings, there seems to belittle justification for placing the genus Kurthiain a family separate from the Corynebacteriaceae.

ACKNOWLEDGMENTWe wish to thank R. E. Buchanan for the com-

ments and advice which he so generously offeredduring the course of this investigation.

LITERATURE CITEDCONN, H. J., AND I. DIMMICK. 1947. Soil bacteria

similar in morphology to Mycobacterium andCorynebacterium. J. Bacteriol. 54:291-303.

CUMMINS, C. S. 1962. Chemical composition andantigenic structure of cell walls of Corynebac-terium, Mycobacterium, Nocardia, Actinomycesand Arthrobacter. J. Gen. Microbiol. 28:35-50.

CUMMINS, C. S., AND H. HARRIS. 1958. Studies onthe cell wall composition and taxonomy ofActinomycetales and related groups. J. Gen.Microbiol. 18:173-189.

CUMMINS, C. S., AND H. HARRIS. 1959. Taxonomicposition of Arthrobacter. Nature 184:831-832.

DAVIS, G. H. G., AND R. W. A. PARK. 1962. Ataxonomic study of certain bacteria currentlyclassified as Vibrio species. J. Gen. Microbiol.27:101-119.

DOWSON, J. W. 1957. Plant diseases due to bac-teria, 2nd ed. Cambridge Univ. Press, Cam-bridge, England.

JENSEN, H. L. 1952. The coryneform bacteria.Ann. Rev. Microbiol. 6:77-90.

KATZNELSON, H. 1958. Metabolism of phyto-pathogenic bacteria. II. Metabolism of carbo-hydrates by cell-free extracts. J. Bacteriol.75:540-543.

LACEY, M. S. 1955. The cytology and relationshipsof C. fascians. Brit. Mycol. Soc. Trans. 38:49-58.

LEHMANN, K. B., AND R. 0. NEUMANN. 1907.Atlas und Grundriss der BakteriologischenDiagnostik. J. F. Lehmann, Munich.

LEIFSON, E. 1960. Atlas of bacterial flagellation.Academic Press, Inc., New York.

LOCKHART, W. R. 1964. Scoring data and group-formation in quantitative taxonomy. Develop.Ind. Microbiol. 5:162-158.

LOCKHART, W. R., AND P. A. HARTMAN. 1963.Formation of monothetic groups in quantita-tive bacterial taxonomy. J. Bacteriol. 85:68-77.

MARMUR, J., S. FALKOW, AND M. MANDEL. 1963.New approaches to bacterial taxonomy. Ann.Rev. Microbiol. 17:329-372.

MULDER, E. G., AND J. ANTHEUNISSE. 1963.Morphologie physiologie et ecologie des Arthro-bacter. Ann. Inst. Pasteur 105:46-74.

SAVULESCU, T. 1947. Contribution a la classifica-tion des bacteriacees phytopathogenes. AnaleleAcad. Romana Mem., Ser. III 22(4) :1-26.

SOCIETY OF AMERICAN BACTERIOLOGISTS. 1957.Manual of microbiological methods. McGraw-Hill Book Co., Inc., New York.

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STARR, M. P. 1959. Bacteria as plant pathogenes.Ann. Rev. Microbiol. 13:211-238.

STARR, M. P., J. E. WEISS, H. P. KLEIN, ANDC. B. SISSELMAN. 1943. Growth of phyto-pathogenic bacteria in a synthetic asparaginmedium. Phytopathology 33:314-318.

SU, Y., AND K. YAMADA. 1960. Studies on L-glu-tamic acid fermentation. I. Isolation of aL-glutamic acid producing strain and its taxo-

nomical studies. Bull. Agr. Chem. Soc. Japan240:69-74.

WILSON, G. S., AND A. A. MILES. 1964. Topleyand Wilson's principles of bacteriology andimmunity, 5th ed. The Williams & Wilkins Co.,Baltimore.

ZAGALLO, A. C., AND C. H. WANG. 1962. Compara-tive carbohydrate metabolism in Arthrobacter.J. Gen. Microbiol. 29:389-401.

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