MIOTILITY AND FLAGELLATION OF THE SOIL CORYNEBACTERIA

FRANCIS Il. CLARK AND P. H. CARRU. S. Departmlent of Alyriculture anrd lo?va Agricultural Experiment Station, Ames, lowal

Receive( foIr pu)lication 'March 12, 1951

Gram-variable riods showing morphological irregularities occur abundantlyin soil. For the most part, soil bacteriologists (Jensen, 1934; Topping, 1937;Clark, 1939; Lochhead, 1940) have considered these bacteria as representativesof Corynebacterium. Conn and Dimmick (1947) recently have objected to broad-ening of Corynebacteriuim to include the soil corynebacteria, and have suggestedthat these organisms be designated generically as Arthrobacter. In their descriptionof the proposed genus, motility is not mentioned. The type species Arthrobacterglobiforme wvas described as nonmotile. All doubtless soil corynebacteria wereconsidered by Jensen (1934) to be nonmotile. Bergey's Manual (Breed et al.,1948) currently places certain motile, gram-positiv-e plant pathogens in anappendix to Corynebacteriurn, at the same time admitting that the reports ofmotile species in this genus present a puzzling problem.

Topping (1937) encountered motility among the gram-positive, pleomorphicrods from soil, and Lochhead (1940) has reported motility in soil isolates whichhe designated as Bacterium globiforme. A number of Cellulomonas spp. recentlyhave been reported to showi- the pleomorphism and gram variability charac-teristic of the soil corynebacteria (Clark, 1951). Certain of these cellulolyticbacteria wvere originally described as motile, and others (e.g., Cellulomonas fima,or Corynebacterium fimi in the (6th edition of Bergey's Manual), as nonmotile.

Currently, some observations are presented concerning the occurrence ofmotility in certain soil organisms variously designated as Arthrobacter, Celllt-lomonas, or Corynebacteriium.

EXPERIMENTAL METHODS

Observations on motility were made on 42 cultures. Included in this collectionwere 10 cultures of Arthrobacter, obtained from H. J. Conn; 8 cultures of celluloly-tic bacteria, from N. R. Smith; 5 cultures of phytopathogenic corynebacteria,from R. E. Gordon; 7 of Topping's unnamed isolates from soil, 3 cultures ofJensen's soil corynebacteria, together with representative species of Agrobac-teriuim, Miycoplana, and Proteus.During the study of some cellulolytic bacteria it was noted that of 5 Celllt-

lomonas species originally described as motile, 3 appeared as motile, although2 failed to show motility, when tested routinely by a soft-agar method. Some-what later, the opportunity arose to acquire 4 cultures of Topping's "motilegroup 1". When subeultuied in 0.3 per cent agar, 2 of these cultures appearedas nonmotile. When examined thoroughly in wet mount, these 2 cultures werefound to show some actively motile cells.

I JourIInal paper no. J-1884 of the Iowa Agricultural lEx)eriment Stationi, Ames, Iowa.P'roject 965.

1

on April 5, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

FRANCIS E. CLARK AND P. H. CARR [VOL. 62

These observations suggested that for feebly motile bacteria, 0.3 per centagar was unsuitable for detecting motility. All available cultures of soil coryne-,.............

,:

Figure 1. Flagellated cells of Corynebacterium fimi no. 133

a and b, 18-hour broth cultures. X 1,500. c, 26-hour culture, X 1,500.

bacteria were re-examined for motility, by direct microscopy and by the culturalmethod, employing agar concentrations of 0.2, 0.3, 0.4, and 0.5 per cent. The

2

on April 5, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

MOTILITY OF SOIL CORYNEBACTERIA

lowest of these concentrations proved most reliable for detecting motility infeebly motile bacteria. Ambiguous, or conflicting readings between replicatetubes, were frequently obtained for such bacteria in 0.3 per cent agar, and nega-tive readings were commonly obtained in 0.4 and 0.5 per cent concentrations.In contrast, such actively motile organisms as Xanthomonas campestris, Myco-

Figur e 2. Flagellated cells of A rthrobacter si'm plumrl and of Cellulomtionas gelidaa. A. simpliin D K17, 22-hour broth culture, X 7,500. b. A. sit plllm 1 T-11, 22-hours,

X 7,500. c. C. yelida, A.T.C.C. no. 488, 22-hours, X 7,500.

plana bullata, and Agrobacteriuni radiobacter producedl diffuse clouding apartfrom the line of inoculation not only in 0.3 per cent agar, but also in 0.4 and0.5 per cent agar.By cultures in 0.2 per cent agar and by direct microscopic studies, active

motility has been noted for certain bacteria hitherto described as nonmotile.Among 10 cultures labeled Arthrobacter when received, 2 cultures of A. siimplum(Conn's D 117 and L T-JI) were found motile. Coryn.cbacteriflm simpleX, culture

1951] 3

on April 5, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

FRANCIS E. CLARItK AND P. H. CAR[Ol6

no. 140 of N. R. Smith, was also found motile. Among the cellulolytic bacteriaavailable for study, the motility of Cellutlomonas biazotea, C. cellasca, C. gelida,and C. sutbalbas (A.T.C.C. no. 486 to 489, respectively) has been confirmed, andin addition, Corynebactcriun fimi (Smith's culture 133), and Cellulomonas liquata(A.T.C.C. no. 485), both previously known only as nonmotile, have beenfound motile.

Of the phytopathogenic corynebacteria, C. flaccumfaciens and C. poin-settiae, two species usually described as motile, showed motility in 0.2 and 0.3per cent agar, but not in 0.4 per cent agar. C. michiganense, a species usuallydescribed as nonmotile, showed motility in 0.2 per cent agar, but not in 0.3per cent.

Flagella staining, following the piocedures recommended by Conn and Wolfe(1938), was undertaken on those bacteria formerly named whose motility pre-viously has not been reported. Individual cells of Arthrobacter simplum, Coryne-bacteriium simplex, C. Jimi, and Ccllidoinonas liquata were found that showedfrom one to four flagella. The flagella were randomly located, that is, theyoccurred either in polar or lateral positions, but most commonly, polar.

Flagella staining was also undertaken on C. flaccutmfaciens and C. poinsettiae.Single cells of these species wvere found to possess from one to several flagella.In cultures 24 hours old, flagella usually appeared single and polar; in 12-hourcultures, cells with 2 or 3 flagella were, collectively, roughly as numerous asthose with but a single flagellum. For singly flagellated cells, the flagellum wasnot alwX-ays in a polar position. When several flagella were present on a singlecell, one or more of them might occur in a lateral position.The cells of Arthrobacter simplum, Corynebacterium fimi, C. simplex, C. michi-

ganense, and Celltilomonas gelida were shadow-cast with gold and were examinedwith an electron microscope. Representative micrographs are shown in figures1 and 2.

DISCUSSION

Qirskov expressed the opinion that motility is by no means a rare property inthe angular rods and small mycelial fungi, and Topping believed it impossibleto distinguish eertain motile and nonmotile pleomorphic organisms from soilby any characteristic other than motility, and concluded that it seemed reason-able to classify the motile organisms along with "their nonmotile relations"(Topping, 1937). Lochhead (1940) has reported the occurrence of occasionalmotile strains in the "Bacterium globiforme" group.Dowson (1949), and others have placed certain gram-positive plant pathogens

in Corynebacteriium. Some of these phytopathogenic bacteria show motility,others fail to do so. Dowson has noted that C. flaccutmfaciens is reported asmotile by American workers, whereas an Australian strain is ieported as non-motile. Discrepancy in the motility responses of C. michiganense was encounteredby Conn, Wolfe, and Ford (1940): "Of three strains of P. michiganensis, oneappears to be nonmotile, wvhile the other two have the same type of flagellationas the above" (described as degenerate peritrichous flagellation) and . . . "the

[VOL. 624

on April 5, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

MOTILITY OF SOIL CORYNEBACTERIA

very sluggish motility of the motile organisms of this group and the difficultyof staining their flagella make it at least doubtful as to whether it may not bepossible in the future to demonstrate flagella on some of those which are stillthought to be nonmotile."The similarity of flagellation reported by various workers for corynebacteria

of soil and plant origin is worthy of comment. Kellerman et al. (1913) reportedfrom 1 to 3 peritrichous flagella for Cellulomonas biazotea, C. cellasea, and C.gelida; for other cellulolytic species, one or several polar flagella were reported.The motile, gram-positive phytopathogenic bacteria assigned to Corynebacteriumare commonly described as possessing single polar flagella. Careful inspection ofyoung cultures of C. flawcumfaciens and C. poinsettiae has shown that more thanone, and laterally placed, flagella are not uncommon. Starr and Pirone (1942)in their initial description of C. poinsettiae state: "Motile by one, rarely two,polar or lateral flagella." A "degenerate peritrichous flagellation" has beennoted for cultures of C. michiganense by Conn, Wolfe, and Ford (1940). Topping(1937) reported from 1 to 4 flagella for isolates of noncellulolytic globiformebacteria from soil.More extensive studies will be required in order to define the systematic

relationships of these organisms to each other and to the genus Corynebacterium,but the posibility is raised that certain cellulolytic, some phytopathogenic, andA. globiforme bacteria of soil origin should not be widely separated in taxonomicschemes.

SUMMARY

Motility is reported for cultures of Corynebacterium simplex, C. fimi, Cellulo-monas liquata, and Arthrobacter simplum, all of which previously have beenknown as nonmotile species. Motility was also encountered in Corynebacteriummichiganense, commonly known as nonmotile.

Flagella were demonstrated both by light and electron microscopy. Thebacteria in question were found to possess from one to four flagella, in polaror lateral positions. This type of flagellation previously has been reported forspecies of Cellulomotans and for unnamed, noncellulolytic bacteria resemblingArthrobacter globiforme isolated from soil.

REFERENCES

BREED, R. S., MURRAY, E. G. D., AND HITCHENS, A. P. 1948 Bergey's Manual of De-terminative Bacteriology. 6th ed. Williams & Wilkins, Baltimore.

CLARK, FRANCIs E. 1939 Notes on types of bacteria associated with plant roots. Trans.Kansas Acad. Sci., 43, 75-84.

CLARK, FRANCIS E. 1951 The generic classification of certain cellulolytic bacteria.Soil Sci. Soc. America Proc., 15 (in press).

CONN, H. J., AND DIMMIcK, I. 1947 Soil bacteria similar in morphology to Mycobacteriumand Corynebacterium. J. Bact., 54, 291-303.

CONN, H. J., AND WOLFE, G. E. 1938 Flagella staining as a routine test for bacteria.J. Bact., 36, 517-520.

CONN, H. J., WOLFE, G. E., AND FORD, M. 1940 Taxonomic relationships of Alcaligene8spp. to certain soil saprophytes and plant parasites. J. Bact., 39, 207-226.

1951] 5

on April 5, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

6 FRANCIs E. CLARK AND P. H. CARR [VOL. 62

DOWBON, W. J. 1949 Manual of Bacterial Plant Diseases. Adam and Chas. Black,London.

JENSEN, H. L. 1934 Studies on saprophytic mycobacteria and corynebacteria. Proc.Linnean Soc., N. W. Wales, 59, 19-62.

KELLERMAN, K. F., MCBETH, I. G., SCAIES, F. M., AND SMITH, N. R. 1913 Identificationand classification of cellulose-dissolving bacteria. Centralbl. f. Bakt. etc., II Abt.,39, 502-522.

LOCHHEAD, A. G. 1940 Qualitative studies of soil micro-organisms. III. Influence ofplant growth on the character of the bacterial flora. Can. J. Research, Sec. C, 18,42-53.

STARR, M. P., and PIRONE, P. P. 1942 Phytomonas poinsettiae n. -p., the cause of a bac-terial disease of poinsettia. Phytopath. 32, 1076-1081.

TOPPING, L. E. 1937 The predominant micro-organisms in soils. I. Description andclassification of the organisms. Centralbl. f. Bakt. etc., II Abt., 97, 289-304.

on April 5, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/