COLONIAL DIMORPHISM IN NONMOTILE SALMONELLA'

MASATOSHI ENOMOTO AND TETSUO IINONational Institute of Genetics, Mishima, Japan

Received for publication 15 April 1963

ABSTRACTENOMOTO, MASATOSHI (National Institute of

Genetics, Mishima, Japan) AND TETSUO IINO.Colonial dimorphism in nonmotile Salmonella.J. Bacteriol. 86:473-477. 1963.-Two distinctcolonial types, LP-type and SD-type, appearedamong the nonmotile mutant clones originatedfrom a strain of Salmonella typhimurium whenthey were cultivated on semisolid gelatin-agarmedium (NGA) at 37 C. These colonial typesreflect the nonmotile character of the mutantclones: a nonflagellated clone formed an LP-typecolony which was large, translucent, and paleyellowish-gray, whereas a paralyzed flagellatedclone formed an SD-type colony which was small,opaque, and dense yellowish-gray. Colonial di-morphism was also present in cells paralyzed bynonheritable change: flagellated cells, immobil-ized by treatment with homologous anti-Hserum, formed SD-type colonies, while cells whichhad lost flagella, growing in the NGA mediumcontaining 0.12% phenol, formed LP-type colo-nies. The distinction between the two colonialtypes was not impaired by addition of motilityphage or various sugars to NGA, but becameobscure when gelatin was omitted from the med-ium or the temperature of cultivation waslowered. It is concluded that the difference be-tween the two colonial types is due to the presenceor absence of flagella on the surface of the bac-terial cells. Such correlation between flagellationand colonial type was observed on nonmotilemutants of several other smooth-type Salmonellastrains and Escherichia coli but was not found ina rough-type Salmonella strain.

have been recognized by direct observation offlagella with light or electron microscopy or byagglutination tests with anti-H serum. In thepresent paper, it is reported that nonflagellatedand paralyzed clones of smooth-type Salmonellastrains form different types of colonies with eachother under proper culturing conditions.

MATERIALS AND METHODS

Organisms. Four motile Salmonella strains, S.typhimurium TM2 and SW1292, S. abortus-equiSL23 (S. abortivoequina), and S. abonySW1391, and nonmotile mutants derived fromthese strains were used for the present experi-ment. The characteristics of these strains havebeen given elsewhere (Lederberg and lino, 1956;lino, 1961a; Makelii, Lederberg, and Lederberg,1962). A motility phage, chi, which was used forthe selection of nonmotile mutants, was receivedfrom the Lister Institute of Preventive Medicine,London, England, by courtesy of B. A. D.Stocker.

Mledia. The composition of semisolid nutrientgelatin-agar medium (NGA) was as follows: 1%Kyokuto peptone, 1% Lender meat extract,0.4% agar, and 8% gelatin in distilled water, thewhole adjusted to pH 7.2. This medium is semi-solid at 37 C and solidifies at temperatures below25 C. It could, therefore, be used for testingbacterial motility or selecting motile bacteria outof a nonmotile bacterial culture, since, at 37 C,a motile clone forms a swarm while a nonmotileone forms a compact colony (Edwards and Bru-ner, 1942). In some experiments, gelatin wasomitted or anti-H serum was added.

Nonmotile mutants originated from motileSalmonella are classified into two groups: onegroup is nonflagellated and the other producesparalyzed flagella (Stocker, Zinder, and Leder-berg, 1953). Differences between these two types

l Contribution no. 477 from the National Insti-tute of Genetics, Mishima, Shizuoka-ken, Japan.

RESULTS

Detection of two colonial types among nonmotilemutants. Chi-phage attacks motile but not non-motile Salmonella cells (Meynell, 1961). Conse-quently, a medium spread with chi-phage hasbeen used for the selection of nonmotile mutantsfrom motile Salmonella strains (Stocker et al.,1953). In an experiment to isolate nonmotile

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mutants from a strain of S. typhimurium TM2,bacteria and chi-phage suspension were mixed ina tube containing the nutrient broth; the mixedsuspension was incubated for 15 hr at 37 C, thena drop of the suspension was plated on NGA.After 24 hr of cultivation at 37 C, motile cellswere mostly lysed, and temporary chi-resistantcells formed minute starlike colonies within themedium, while the nonmotile clone grew to asmooth, entire, compact colony on the medium.By detailed observation, it was found that therewere two types among the nonmotile mutantcolonies grown on the medium (Fig. 1A). One waslarge, low-convex, translucent, pale yellowish-gray (LP-type); the other was small, opaque,dense yellowish-gray (SD-type). H-antigen ofthese colonies was typed by slide agglutination.All LP-type colonies tested were H-negative,whereas all SD-type colonies carried an H-anti-gen of the parental strain, either i or 1.2. Thesecolonial and antigenic characteristics of eachclone were maintained through subsequent sub-cultures on NGA, even after the cultures hadbeen freed from chi-phage. Electron microscopicstudy of the cells of these two colonial types

showed that LP-type colonies consisted of cellswithout flagella whereas those of SD-type hadnormal but paralyzed flagella.

Effect of medium or temperature. On NGA, thedistinction between LP- and SD-type becameless clear as the temperature of cultivation waslowered. At 25 C, both nonflagellated and para-lyzed clones formed SD-type colonies (Table 1,column 4). The colonial dimorphism was notapparent on a nutrient agar plate containing 1.5%agar in the nutrient broth. Omission of gelatinfrom NGA also resulted in the loss of colonialdimorphism. On such medium, irrespective of thetemperature, all nonmotile clones formed LP-typecolonies (Table 1, column 5). Addition of varioussugars (galactose, arabinose, xylose, rhamnose,glucose, or lactose) to NGA in a concentration of1.0% did not alter the two colonial types (Table1, column 6).

Colonial types of clones paralyzed by anti-Hserum. By addition of anti-H serum to NGA,motile Salmonella cells in contact with homolo-gous H-antigen are temporarily paralyzed andform compact colonies instead of swarms (Leder-berg and lino, 1956). A diphasic strain of S.

S F~~~~~~~~~~~~~~~~~~~~~............

.._SX .w~~EI

CFIG. 1. Colonial types of nonmotile Salmonella mutants. Originated from (A) S. typhimurium TM2, (B)

S. typhimurium SW1292, (C) S. abortus-equi SL23. Large colonies are LP-type, formed by nonflagellatedmutants. Small colonies are SD-type formed by paralyzed mutants. Cultured in NGA at 37 C for 24 hr.

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VOL. 86, 1963 COLONIAL DIMORPHISM IN NONMOTILE SALMONELLA

typhimurium, TM2 (i:1.2), and its monophasicmutant strain, SW1061 (-:1.2), were grown on

NGA supplemented with corresponding anti-Hserum, and the colonial types of the cells tem-porarily paralyzed by anti-H serum were exam-

ined. SW1061 is a mutant which is nonflagellatedin antigenic phase 1 and produces normal 1.2-type flagella in phase 2 (Iino, 1961b). The resultsare summarized in Table 2. On NGA to whichanti-phase 1 (i-type) serum was added, a phase 2clone of either TM2 or SW1061 formed a swarm.

In phase 1, TM2 was paralyzed and producedSD-type colonies in the presence of anti-phase 1serum. On NGA to which anti-phase 2 (1.2-type)serum was added, phase 2 clones of both strainswere paralyzed and grew to SD-type colonies,whereas a phase 1 clone of TM2 spread as a

swarm. Nonflagellated phase 1 clones of SW1061grew to LP-type colonies on both media as wellas plain NGA. Thus, it was indicated that theflagellated clone temporarily paralyzed by anti-Hserum formed an SD-type colony on NGA,whereas a nonflagellated clone formed an LP-type colony regardless of presence or absence ofanti-H serum in NGA.

Colonial type of clones deflagellated by phenol. Anappropriate concentration of phenol in a mediumprevents the synthesis of flagella (reviewed byLacey, 1961). When the cells of TM2 or SW1061were cultivated overnight in a broth containing0.12% phenol and plated on NGA containing thesame concentration of phenol, the colonies com-

posed of deflagellated cells became visible after48 hr at 37 C. Their colonial characters were thesame as those of LP-type nonflagellated mutants(Table 2).

Generality of colonial dimorphism in nonmotileSalmonella and Escherichia coli. In order to ex-

amine the generality of correlation betweencolonial type and flagellation in nonmotile mutantclones, of either nonflagellated or paralyzed type,further examination was made of mutants iso-

lated from S. typhimurium TM2 on NGA me-dium. Among the mutants examined, 18 strainswere nonflagellated types, of which 1 was ob-tained by ultraviolet-light treatment, 4 werespontaneous mutants isolated from NGA cul-tures, and the others were selected by chi-phage.All formed LP-type colonies on NGA at 37 C.The remaining 65 strains were paralyzed mutantsof spontaneous origin isolated with or withoutchi-phage selection. These paralyzed mutants allformed SD-type colonies. The differences in thecolonial type between nonflagellated and para-lyzed clones were also confirmed for the non-motile mutants derived from S. typhimuriumSW1292, S. abortus-equi SL23 (Table 1 and Fig.1B, C), and three Escherichia strains, W3094,

TABLE 1. Colonial type of nonmotile Salmonellamutants grown on NGA media

Colonial type on NGA

Original motile Nonmotilestrain mutant* ¢ ¢5) S v

C4be

S. typhimurium SJ78 (mot-) SD SD LP SDTM2 SL478 (mot- SD SD LP SD

ath- phe-)SJ374 (fla-) LP SD LP LPSL480 (fla- LP SD LP LP

ade- pro-)

S. typhimurium SJ498 (mot-) SD SD LP SDSW1292 SJ398 (fla-) LP SD LP LP

S. abortus-equi SJ186 (mot-) SD SD LP SDSL23 SJ149 (fla-) LP SD LP LP

* Symbols: mot-, paralyzed mutant; fla-, non-flagellated mutant; ath-, phe-, ade-, pro-: mu-

tants requiring adenine-thiamine, phenylalanine,adenine, or proline, respectively.

t Galactose, arabinose, xylose, rhamnose, glu-cose, or lactose was added in 1% concentration.

TABLE 2. Colonial type of Salmonella clones paralyzed by anti-H serum or deflagellated by phenolColonial type with

Strain H-antigen Anti-i Anti-1.2 Phenol-NGA serum-NGA serum-NGA NGA

S. typhirnurium TM2 Phase 1 i Swarm SD Swarm LPPhase 2 1.2 Swarm Swarm SD LP

S. typhimurium SW1061 Phase 1 - LP LP LP LPPhase 2 1.2 Swarm Swarm SD LP

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W3096, and W3106, derived form E. coli K-12.An exception was a strain of S. abony SW1391.This strain forms rough colonies, and the dis-tinction of colonial type between nonflagellatedand paralyzed mutants was not clear: bothmutant types formed colonies similar to SD-typeon NGA.

DIscussIoN

There are many instances in which colonialcharacteristics are attributed to certain mor-phological, physiological, or serological featuresof the bacterial cells composing a colony. Espe-cially, a change in bacterial cell surface antigenis often noticed as a change in colonial type. InSalmonella, the colonies which contain Vi-positivecells are more opaque than those composed fromVi-negative cells (Craigie and Brandon, 1936),and the loss of 0 antigen accompanies the changein colonial type from smooth to rough (Ark-wright, 1921). The relationship of another bac-terial surface antigen, flagellar antigen, to colonialtype has been noted in connection with locomo-tive function. A clone of motile flagellated bac-teria forms a swarm, while a nonmotile bacterialclone forms a compact colony on a semisolidmedium (Edwards and Bruner, 1942). The pres-ent study demonstrated that a semisolid nutrientagar medium containing gelatin (NGA) differ-entiates not only a colonial type of motile fromnonmotile cell but, among the latter, distinguishestwo types in smooth Salmonella strains. The twotypes are composed of nonflagellated and para-lyzed clones and form different colonial types,referred to in this paper as LP- and SD-types,respectively. The colonial type on NGA is con-stant, regardless of whether the nonmotilecharacter is caused by mutation or by an environ-mental factor such as anti-H serum or phenol.Presence or absence of flagella on nonmotile cellsalso determines these colonial types. An exceptionwas observed on a rough-type strain of S. abonySW1391. The colonial type of this strain may beaffected strongly by other factors than flagella.The cells of this strain are filamentous and clingtogether. Therefore, it is possible that the clingingtogether of the filamentous cells produces a con-dition similar to that of paralyzed flagellated cells.

It is likely that the difference in color betweenthe two colonial types is due to the density of thecomponent cells of the colonies, since SD-typecolonies are smaller than those of LP-type though

there is no significant difference between non-flagellated and paralyzed clones in rate of celldivision or size of a cell. The intertwisting of theflagella of the cells may restrict mechanically thespreading on the medium, or the electric chargeof flagellar protein may interfere with the mode ofaggregation of the cells in a colony. It is worthnoting that the colonies of paralyzed mutantsgrown on semisolid medium without gelatin showthe LP-type which is characteristic of nonflagel-lated clones grown on NGA.NGA has been used for detection of motile

mutants from nonmotile bacteria (Stocker et al.,1953). On this medium, progenies of a motilemutant cell among more than 108 nonmotile cellscan be detected as a swarm spreading out from acompact mass of nonmotile cells. For the isolationof a nonmotile clone from motile bacterial culture,NGA is not suitable. For this purpose, motilityphage has been used as the most effective selectiveagent. For example, chi-phage plated with Salmo-nella cells on a cultural medium kills only motilecells and allows the nonmotile cells to grow toform colonies. As reported in the present paper,chi-phage does not affect the distinctive colonialcharacteristics of both nonflagellated and para-lyzed clones on NGA. Therefore, by using NGAand chi-phage combined, it is now possible toisolate either nonflagellated or paralyzed mutantsin Salmonella with high efficiency.Whether colonial morphology is useful for

identification of nonflagellated and paralyzed bac-teria other than Salmonella is an interestingquestion. The finding that a colonial dimorphismas in Salmonella is also present in nonmotilemutants originated from three motile clones of E.coli K-12 suggests the generality of the phenome-non.

ACKNOWLEDGMENT

This investigation was supported by researchgrant AI-02872 from the National Institute ofAllergy and Infectious Diseases, U.S. PublicHealth Service.

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EDWARDS, P. R., AND D. W. BRUNER. 1942. Sero-

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