Flagellation and motility in Aerobacter cloacae and Escherichia coli

20

BIOCHIMICA ET BIOPHYSICA ACTA

VOL. 15

(1954)

FLAGELLATION AND MOTILITY IN A E R O B A C T E R CLOACAE A N D E S C H E R I C H I A COLI by ISABEL W. SMITH*

Bacteriology Department, University o/Edinburgh (Scotland)

The classical explanation of bacterial motility was that the bacteria moved by means of their flagella, filamentous appendages first clearly demonstrated by LOEFFLER1 in specially stained preparations. PIJPERz has expressed the opposite view, namely that bacteria move by undulating movements of the cell body and that the flagella are merely trails of extracellular slime stripped off by the movement. Most of the literature would appear to support the classical theory and this has been fully summarised by KISGMA BOLTJESa and VA~ ITERSON4. It appears, however, that no attempt has been made to examine a large range of organisms for the occurrence of motility and flagella so that their correlation might be determined. The value of such observations must depend upon the reliability of the method for observing the flagella and the thoroughness of the examinations made to establish the absence of flagella. The electron microscope now makes possible the definite identification of flagella according to their dimensions, uniform thickness and wavy form, whereas the older optical methods did not convincingly distinguish flagella from slime threads and other artefacts. METHODS Strains Observations were made with i2 strains of Aerobacter cloacae and 16 strains of Escherichia coli. The A ere. cloacae strains were isolated from ice cream by Dr. J. D. COGHLA~, and they all had the following properties: methyl-red negative, Voges-Proskauer positive, citrate u t i l i ~ t i o n positive, indol negative, Eijkman negative, liquefying gelatin, fermenting glucose, lactose, sucrose and mannitol with the production of acid and gas, and fermenting glycerol with the production of acid only (except one strain which produced both acid and gas). The E. cell strains were isolated from faeces, urine, water and milk by Dr. J. P. DUGUID, and all the strains had the following properties: methyl-red positive, Voges-Proskauer negative, citrate utilisation negative, indol positive (except one strain), not liquefying gelatin, ferment!ng glucose, lactose, mannitol and glycerol with production of acid and gas, and varying in power to ferment sucrose and dulcitol.

Cultural methods Cultures were prepared in peptone water and in a synthetic liquid medium, incubated aerobically at 35 ° C. The peptone water contained per xoo ml distilled water; "baetopeptone", x.o g and sodium chloride, 0.5 g. The synthetic medium contained per IOO ml distilled water: glucose, o.x g; ammonium sulphate, 0.5 g; phosphate (3 parts NatHPO 4 and x p a r t KHtPO4), I.o g; sodium chloride, 0.2 g; potassium sulphate, o.x g; magnesium sulphate, o.ox g; calcium chloride, o.oooI g and ferrous sulphate, o.oooi g. These media, the initial p H of which was 7-3-7-4, were sterilised by steaming for an hour at IOO° C and dispensed in test tubes in 8 ml amounts. The tube of medium was inoculated with a small amount of material from a colony and incubated aerobically at 35 ° C. * Present address: University of Aberdeen, Department of Bacteriology, Foresterhill.

Re/erences p. 24.

VOL,: 1 5 (1954)

FLAGELLATION AND MOTILITY IN .4. c l o a c a e AND E . c o l i

2I

Fig. I. E . coli s t r a i n s h o w i n g t w o flagella f r o m a dividing cell. T h e s p e c i m e n w a s c u l t u r e d in s y n t h e t i c liquid m e d i u m for 6 h o u r s a t 35 ° C. × 24,ooo.

.,. 2. E . coli s t r a i n s h o w i n g a n u m b e r of flagella. T h e s p e c i m e n w a s c u l t u r e d in p e p t o n e w a t e r

for 6 h o u r s a t 35 ° C. × 24,ooo.

'22

I, W. SMITH

VOL. 1 5

(I954)

Fig. 3. E. coli s t r a i n s h o w i n g b o t h flagella a n d pseudoflagella. T h e s p e c i m e n w a s c u l t u r e d on p e p t o n e a g a r for 16 h o u r s a t 35 ° C. x 24,000.

Observation of motility A loopful of c u l t u r e w a s e x a m i n e d w i t h t h e optical microscope b y t h e " h a n g i n g - d r o p " m e t h o d .

Observation o[ flagella T h e c u l t u r e , a f t e r it h a d been e x a m i n e d for m o t i l i t y , w a s killed a n d fixed b y a d d i t i o n of a few d r o p s of f o r m a l i n . A drop of fixed c u l t u r e w a s placed on a collodion m e m b r a n e m o u n t . A f t e r d r y i n g in a d e s i c c a t o r t h e s p e c i m e n w a s w a s h e d w i t h distilled w a t e r a n d dried again. I t w a s t h e n s h a d o w c a s t a t a n a n g l e of 15 degrees w i t h g o l d - p a l l a d i u m alloy a n d e x a m i n e d in t h e electron micros c o p e a t 75 k v (Metropolitan-Vickers EM3). F o r e a c h s t r a i n m a n y s p e c i m e n s were e x a m i n e d a n d v a r i o u s fields p h o t o g r a p h e d .

Re]erences p. 24.

VOL. 15 (1954)

FLAGELLATION AND MOTILITY IN A . cloacae AND E. coli

23

RESULTS A ero. cloacae T h e 12 s t r a i n s of Aero. cloacae were e x a m i n e d for m o t i l i t y a n d flagellation a f t e r g r o w t h in p e p t o n e w a t e r for 16 hours a t 35 ° C. All b u t one of t h e s t r a i n s were f o u n d to e x h i b i t m o t i l i t y u n d e r these conditions. F l a g e l l a were o b s e r v e d in al~ t h e I I m o t i l e strains, b u t were n e v e r f o u n d in r e p e a t e d e x a m i n a t i o n s of t h e n o n - m o t i l e strain. T y p i c a l l y each cell bore 2 t o 4 p e r i t r i c h o u s l y s i t u a t e d flagella. TABLE I MOTILITY

AND

Strain

FLAGELLATION

Peptone wate~ 6 hour's Motility

A 5° A 51 A52 A53 A54 A55 A56 A57 A58 A93 A 96 A97 A98 A ioo A lO3 A lO4

E . dolg

IN

. + + . + + + + + . . . + . . +

STRAINS

Peptone water x6 hours

Flagel~ition Motility

.

.

+ +

.

. + + + + +

. .

. . . . .

+ +

CULTURED

Flagellation Motility

. -+ . -+ -__ +

.

. -. . +

.

+ +

.

. .

.

COND][TIONS

Pkenel-peptone water 6 holtrS

Flagellation Motility

no growth -+

Flagellation

+ m

. + + + + +

. .

DIFFERENT

Synthetic medium z6-z2 hours

-+ + + -+ -+ -+ no growth no growth

. . .

UNDER

+ +

. . .

--

+

4-

+

m

+

m

+

Note. -4- means only a few cells flagellate or motile.

Esch. coli T h e 16 s t r a i n s of E. coli were e x a m i n e d after 6 a n d 16 hours g r o w t h a t 35°C in p e p t o n e w a t e r a n d a f t e r 16-22 hours g r o w t h in s y n t h e t i c liquid m e d i u m . T h e results are s h o w n in T a b l e I. I t is seen t h a t 9 of t h e 16 s t r a i n s e x h i b i t e d m o t i l i t y a f t e r 6 h o u r s g r o w t h in p e p t o n e w a t e r . F l a g e l l a were o b s e r v e d in all of these 9 m o t i l e c u l t u r e s a n d , in spite of r e p e a t e d e x a m i n a t i o n s , in none of t h e n o n - m o t i l e ones. T h u s t h e r e was a perfect c o r r e l a t i o n b e t w e e n t h e possession of flagella a n d t h e p o w e r of a c t i v e m o t i l i t y . I n t h e I 6 - h o u r p e p t o n e w a t e r cultures, t h e presence of flagella was o b s e r v e d in all of t h e 9 s t r a i n s w h i c h h a d been f o u n d to be flagellate a n d m o t i l e a t 6 hours. T h e m o t i l i t y of t h e organisms a f t e r 16 hours g r o w t h was f o u n d to be m u c h less vigorous a n d in 5 of t h e 9 s t r a i n s it h a d c o m p l e t e l y d i s a p p e a r e d . A p p a r e n t l y t h e c o n d i t i o n s in t h e I 6 - h o u r c u l t u r e s i n h i b i t e d t h e a c t i v i t y of t h e flagella. Similarly, flagella were o b s e r v e d in t h e s y n t h e t i c m e d i u m c u l t u r e s of all t h e s t r a i n s f o u n d t o be flagellate a n d m o t i l e in p e p t o n e water, b u t o n l y 2 of these s t r a i n s e x h i b i t e d m o t i l i t y . T h e c o n d i t i o n s in t h e s y n t h e t i c med i u m t h u s s u p p o r t e d f o r m a t i o n of t h e flagella b u t d i d n o t p e r m i t t h e i r functioning. BRAUN ~ r e p o r t e d t h a t t h e a d d i t i o n of p h e n o l to t h e c u l t u r e m e d i u m caused m o t i l e organisms t o lose t h e i r m o t i l i t y . Accordingly, o b s e r v a t i o n s were m a d e on c u l t u r e s g r o w n Re/erences p. 24.

24

I . w . SMITH

VOL. 15 (1954)

6 h o u r s a t 35°C in p e p t o n e w a t e r c o n t a i n i n g o.I % (w/v) p h e n o l . Of 6 s t r a i n s f o u n d flage l l a t e a n d m o t i l e in t h e a b s e n c e of p h e n o l , o n l y 3 s t r a i n s s h o w e d f l a g e l l a a n d n o n e exh i b i t e d m o t i l i t y in t h e p r e s e n c e of p h e n o l . T h e a c t i o n of t h e p h e n o l m a y h a v e b e e n to i n h i b i t p r o d u c t i o n of t h e flagella. L i k e t h e Aero. cloacae s t r a i n s , t h e E . coli s t r a i n s t y p i c a l l y e x h i b i t e d 2 - 4 p e r i t r i c h o u s l y s i t u a t e d flagella p e r cell (Figs. I a n d 2). I n a d d i t i o n to t h e t r u e flagella, a n o t h e r k i n d of f i l a m e n t o u s a p p e n d a g e was s o m e t i m e s seen. E l e c t r o n m i c r o g r a p h s of c e r t a i n m o t i l e a n d n o n - m o t i l e s t r a i n s s h o w e d t h e p r e s e n c e of p e r f f r i c h o u s l y s i t u a t e d f i l a m e n t s w h i c h w e r e m o r e n u m e r o u s , s h o r t e r , t h i n n e r a n d m o r e i r r e g u l a r l y b e n t t h a n t r u e flagella (Fig. 3). T h e s e " p s e u d o f l a g e l l a " w e r e e a s i l y d i s t i n g u i s h e d f r o m t h e t r u e flagella. ACKOWLEDGEMENTS T h e a u t h o r w i s h e s t o e x p r e s s h e r t h a n k s t o D r . J . P. DUGUID a n d P r o f e s s o r T. J . MACKIE for t h e i r i n t e r e s t a n d e n c o u r a g e m e n t , a n d t o t h e U n i v e r s i t y of E d i n b u r g h for t w o P o s t g r a d u a t e S t u d e n t s h i p s for t h e y e a r s 1951-1953. SUMMARY By the use of the electron microscope, flagella were shown to be present in all of the I i motile strains of Aerobacter cloacae and 9 motile strains of Escherichia coli, and to be absent from a nonmotile strain of Aerobacter cloacae and from all of 7 non-motile strains of Escherichia coll. These findings support the traditional view that flagella are organs of bacterial locomotion. Growth of Escherichia coli strains in synthetic liquid media resulted in the production of immobile flagella, whereas the addition of phenol to the medium resulted in a loss of motility due to the inhibition of the production of the flagella. RI~SUM]~ L'examen au microscope dlectronique a permis de mettre en dvidence l'existence de flagelles chez i i souches mobiles d'Aerobacter cloacae et 9 souches mobiles d'Escherichia coli, et leur absence chez une souche non mobile d'Aerobacter cloacae et chez 7 souches non mobiles d'Escherichia coll. Ces rdsultats sont en accord a v e c l a conception traditionnelle qui voit dans les flagelles les organes de la locomotion des bactdries. La culture d'Escherichia coli dans des milieux'synthdtiques liquides entraine l'apparition de flagelles immobiles, tandis que l'addition de phdnol au milieu provoque une perte de la mobilitd lide ~t l'inhibition de la production des flagelles. ZUSAMMENFASSUNG Es wurde mit Hilfe dos Elektronenmikroskopes gezeigt, dass alle II beweglichen Stgmme yon Aerobacter cloacae und die 9 beweglichen St~imme yon Escherichia coli Flagellen besitzen, wiihrend sie bei den nicht beweglicben St~kmmen yon Aerobacter cloacae und bei den 7 nicht bewegliehen Stiimmen yon Escherichia coli fehlen. Diese Befunde stiitzen die herkbmmliche Ansicht, dass die Flagellen Organe der Bakterienbewegung sind. Wachsen Escherichia coli St~mme in synthetisehem

fliissigem Medium, so bilden sich unbewegliche Flagellen. Zusatz yon Phenol zum Medium fiihrt zum Verlust der Beweglichkeit durch die Hemmung der Flagellenbildung. REFERENCES

x F. LOEFFLER, Z . B . P . Orig., 6 (1889) 209. A. PIJPER, J. Path. Bact., 58 (x946) 325 . 3 T. J. KINGMA BOLTJES, J. Path. Bact., 60 (1948) 275. 4 "~V.VAN ITERSON, Vlth. International Congress of Microbiology, Bacterial Cytology (I953) P- 24. H. BRAUN, Bet. Klin. Wschr., 55 (I918) 637. R e c e i v e d M a r c h 3 I s t , 195 4