VARIATION IN SHAPE AND ARRANGEMENT OF BACTERIAL FLAGELLA EINAR LEIFSON AND RUDOLPH HUGH' Department of Microbiology, Stritch School of Medicine and Graduiate School, Loyola University, Chicago, Illinois Received for publication July 23, 1952 Few studies have been reported in the litera- ture on variation in shape and arrangement of the flagella on bacteria. Leifson (1951) reported a shape variation of the flagella of Salmonella wichita. The variant showed twice the humber of curves per unit length compared to the parent strain. In the same publication Leifson also re- ported a monotrichous organism which produced filamentous variants showing lateral flagella. The lateral flagella were interpreted to be es- sentially polar. Gray and Thornton (1928) de- scribed two organisms which they placed in a new genus Mycoplana. The one organism was de- scribed as having "polar, peritrichous" and the other as having "polar or peritrichous" flagella. Unfortunately, their illustrations are only draw- ings, and the flagellation shown is difficult or impossible to evaluate. METHODS AND MATERIALS Flagella staining. The technique used for staining flagella is that of Leifson (1951). For thorough cleaning of slides it was found neces- sary to use hot (70 C) cleaning solution for about 10 hours. The SO2 vapor from the hot cleaning solution has a very deleterious effect on the stain- ing process, and the solution should be kept under a hood or in a separate room. Media. Unless otherwise stated liquid media were used. The usual composition was peptone, 0.5 per cent; yeast extract, 0.2 per cent; sodium phosphates, 0.3 per cent; pH of about 7.1. The nature of the peptone and the yeast extract had no appreciable effect on the results obtained. Cultures. The data presented are based mainlv on the work with three types of organisms which may be described briefly as follows: (1) Aeromonas (Pseudomonas) sp. These or- ganisms are classified in Bergey's Manual of Determinative Bacteriology, 6th ed., under the genus Pseudomonas, but since they are carbo- I Acknowledgment is made to the Standard Oil Company of Indiana for support of this work in the form of a fellowship to the junior author. hydrate fermenters the genus Aeromonas, first proposed by Kluyver and van Niel (1936), seems preferable. The validity of this genus has been emphasized by Miles and Miles (1951) and by Stanier and Adams (1944). Many species of the genus were studied and several showed the flagellar variation described. (2) Organisms K-517 and K-493 were obtained from Robert Keller, Academy of Natural Sciences of Philadelphia, who isolated them from fresh water streams. Both organisms produced a yellow nonwater soluble pigment and were apparently identical both physiologically and antigenically. The organisms were gram negative rods, about 0.7 , by 3 ,A, actively motile with predominantly a single polar flagellum and/or several peritri- chous flagella. They grew readily on simple pep- tone media. Detectable acid was not produced from glucose nor from any other of the common carbohydrates. Gelatin was liquefied, but nitrate, indole, urea, and H2S reactions were negative. These organisms could be classified equally well as Xanthomonas or Flavobacterium. (3) Organism H-247 was obtained from the American Type Culture Collection as number 8461 and originally labeled Vibrio percolans by Mludd and Warren (1923). It corresponded physi- ologically to the original description with the exception of nitrate which we found to be reduced to nitrite. The organism is a small (about 0.5 ,u by 2 ,), gram negative rod which at times may show a slight curvature. It is actively motile, and the original culture showed typically 2 to 4 polar lophotrichous flagella and/or several peritrichous flagella. Serology. The antigenic constitution of the variants was determined in the usual manner by cross agglutination and agglutinin adsorption. Rabbits were used for preparing the antisera. "O" antigens were prepared by placing saline suspensions of the organisms in boiling water for 2j1 hours. Flagella could not be demon- strated on the heated organisms. The "H" anti- gens were 0.5 per cent formalin-saline suspen- 263 on June 23, 2020 by guest http://jb.asm.org/ Downloaded from
Transcript
VARIATION IN SHAPE AND ARRANGEMENT OF BACTERIAL FLAGELLA
EINAR LEIFSON AND RUDOLPH HUGH'Department of Microbiology, Stritch School of Medicine and Graduiate School, Loyola University,
Chicago, Illinois
Received for publication July 23, 1952
Few studies have been reported in the litera-ture on variation in shape and arrangement ofthe flagella on bacteria. Leifson (1951) reporteda shape variation of the flagella of Salmonellawichita. The variant showed twice the humberof curves per unit length compared to the parentstrain. In the same publication Leifson also re-ported a monotrichous organism which producedfilamentous variants showing lateral flagella.The lateral flagella were interpreted to be es-sentially polar. Gray and Thornton (1928) de-scribed two organisms which they placed in anew genus Mycoplana. The one organism was de-scribed as having "polar, peritrichous" and theother as having "polar or peritrichous" flagella.Unfortunately, their illustrations are only draw-ings, and the flagellation shown is difficult orimpossible to evaluate.
METHODS AND MATERIALS
Flagella staining. The technique used forstaining flagella is that of Leifson (1951). Forthorough cleaning of slides it was found neces-sary to use hot (70 C) cleaning solution for about10 hours. The SO2 vapor from the hot cleaningsolution has a very deleterious effect on the stain-ing process, and the solution should be kept undera hood or in a separate room.
Media. Unless otherwise stated liquid mediawere used. The usual composition was peptone,0.5 per cent; yeast extract, 0.2 per cent; sodiumphosphates, 0.3 per cent; pH of about 7.1. Thenature of the peptone and the yeast extract hadno appreciable effect on the results obtained.
Cultures. The data presented are based mainlvon the work with three types of organisms whichmay be described briefly as follows:
(1) Aeromonas (Pseudomonas) sp. These or-ganisms are classified in Bergey's Manual ofDeterminative Bacteriology, 6th ed., under thegenus Pseudomonas, but since they are carbo-
I Acknowledgment is made to the Standard OilCompany of Indiana for support of this work inthe form of a fellowship to the junior author.
hydrate fermenters the genus Aeromonas, firstproposed by Kluyver and van Niel (1936), seemspreferable. The validity of this genus has beenemphasized by Miles and Miles (1951) and byStanier and Adams (1944). Many species of thegenus were studied and several showed theflagellar variation described.
(2) Organisms K-517 and K-493 were obtainedfrom Robert Keller, Academy of Natural Sciencesof Philadelphia, who isolated them from freshwater streams. Both organisms produced a yellownonwater soluble pigment and were apparentlyidentical both physiologically and antigenically.The organisms were gram negative rods, about0.7 , by 3 ,A, actively motile with predominantlya single polar flagellum and/or several peritri-chous flagella. They grew readily on simple pep-tone media. Detectable acid was not producedfrom glucose nor from any other of the commoncarbohydrates. Gelatin was liquefied, but nitrate,indole, urea, and H2S reactions were negative.These organisms could be classified equally wellas Xanthomonas or Flavobacterium.
(3) Organism H-247 was obtained from theAmerican Type Culture Collection as number8461 and originally labeled Vibrio percolans byMludd and Warren (1923). It corresponded physi-ologically to the original description with theexception of nitrate which we found to be reducedto nitrite. The organism is a small (about 0.5 ,uby 2 ,), gram negative rod which at times mayshow a slight curvature. It is actively motile,and the original culture showed typically 2 to 4polar lophotrichous flagella and/or severalperitrichous flagella.
Serology. The antigenic constitution of thevariants was determined in the usual manner bycross agglutination and agglutinin adsorption.Rabbits were used for preparing the antisera."O" antigens were prepared by placing salinesuspensions of the organisms in boiling waterfor 2j1 hours. Flagella could not be demon-strated on the heated organisms. The "H" anti-gens were 0.5 per cent formalin-saline suspen-
Figures 1-7Figuire 1. Aerotiuonas liquefaciens (Kluyver L417) showing a polar flagellum characteristic in the later
stages of the growth cycle. Photomicrograph X 4,000.Figure 2. Same organism as shown in figure 1. Peritrichous flagella characteristic in the early stages
of the growth cycle. Photomicrograph X 4,000.Figure 3. Aeromonas hydrophila (ATCC 7965) showing a polar flagellum characteristic in the later
stages of the growth cycle. Photomicrograph X 3,000.
sions carefully checked for tvpical flagellation.The "H" agglutinations were read after fourhours and the "O" agglutinations after 20hours. Agglutinin adsorptions were made byadding 9 parts of bacterial suspension to 1 partof serum, incubating for 2 hours at 37 C followedby centrifugation. A second adsorption was madeby suspending packed organisms in the super-natant serum from the first adsorption and pro-ceeding as before. The serum from the secondadsorption was checked for the presence ofantibodies for the homologous antigen, and ifany were demonstrable in dilution of 1:40further adsorption was done.
EXPERIMENTAL RESULTS
Experiments with Aeromonas. Several speciesof Aeromonas when incubated in broth at 20 Cfor about 16 hours showed some individuals witha single polar flagellum, some with a polarflagellum and one or more lateral flagella, andsome with several lateral flagella only. When theincubation time was extended beyond 24 hours,only polar monotrichous individuals could befound. At higher incubation temperatures, suchas 35 C, lateral flagella could be demonstratedonly during the first 5 hours or so of incubation.At longer incubation times only polar monotri-chous individuals could be found. In figures 1,2, 3, 4, 5, and 6 is illustrated the flagellar varia-tion of two Aeromonas species. It may be notedthat the curvature of the polar and lateralflagella is alike.
Experiments with media of various composi-tions showed a tendency for the developmentof a greater proportion of peritrichously flagel-lated individuals in a medium containing bothpeptone and yeast extract as compared to onewith peptone only. However, in all the mediatested, the relationship of flagellation to growthphase remained the same.From repeated experiments it became ap-
parent that the organisms produced lateralflagella during the early stages of the growthcycle and predominantly only single polarflagella in the later stages. By repeated platingand fishing, pure cultures showing only one orthe other type of flagellation were not obtained.Rabbit antiserum produced against a youngculture with a high proportion of peritrichouslyflagellated individuals agglutinated the oldermonotrichous cultures to titer.The flagella of many strains of Aeromonas
showva tendency to form loose coils or loopswhich are very striking (figures 5, 6, and 7).This shape does not seem to have become sta-bilized in the polar flagella, and individuals withthe more usual curved flagella are always foundinterspersed among the individuals with thecoiled flagella. Pure cultures with "coiled"peritrichous flagella have been observed. Infigure 7 is illustrated a culture sent to us by A.J. Kluyver labeled Aeromonas liquefaciens,strain L418. The same strain also was obtainedfrom Ellen M. Miles. Both sources indicatedthe culture to be monotrichous. Flagellarstains made from these cultures at various agesand temperatures of incubation failed to showany but peritrichous individuals. Evidenceseems to indicate that the strain we received is aperitrichous variant, but in our hands it hasshown no tendency to produce monotrichousindividuals. The peculiar coiling of some of thepolar flagella of other Aeromonas strains sup-ports this view.
Experiments with strain K-51 7. The originalculture of this organism showed some individualswith a single polar flagellum; some with a polarflagellum and one or more, somewhat shorterand more curved, lateral flagella; and an occa-sional organism with lateral flagella only. Byrepeated plating and fishing, stra ns were ob-tained which showed: (1) predominantly polarmonotrichous flagella, (2) predominantly mixed
Figure 4. Same organism as shown in figure 3. Peritrichous flagella characteristic in the early stagesof the growth cycle. Photomicrograph X 3,000.Figure 5. Same organism as shown in figure 3. Multiple polar flagella with rather unusual "coiled"
shape. May be found in any stage of the growth cycle. Photomicrograph X 3,000.Figure 6. Same organism as in figure 3. The organism shown appears to be double with peritrichous
flagella on one and several coiled flagella on the other. Found in the early stages of the growth cycle.Photomicrograph X 3,000.Figure 7. Aeromnonas liquefaciens (?) (Kluyver L418) showing peritrichous coiled flagella characteristic
of this organism in all stages of the growth cycle. The two photomicrographs are taken from the sameslide. The single individual is magnified 4,000 X, the filamentous 2,000 X.
Figures 8-13Figure 8. Organism K-517 showing a typical polar flagellum. Photomicrograph X 4,000.Figure 9. Organism K-517 showing a typical polar flagellum and a single lateral flagellum. Note the
difference in curvature of the two flagella. The wavelength of the polar flagellum is about 2,u, and thatof the lateral flagellum about 1 tu. Photomicrograph X 4,000.Figure 10. Organism K-517 showing the typical polar flagellum and two lateral flagella with greater
single polar and lateral flagella, and (3) a singlestrain which showed predominantly peritrichousflagella. The different flagellar variants are illus-trated in figures 8, 9, 10, 11, 12, and 13. Onrepeated transfer in broth all the variants rapidlytended towards a more or less common de-nominator, namely, a culture showing mixedflagellation like the original. Neither the com-position of the medium nor the incubation timeand temperature had much effect on the type offlagellation. Since these variants were so un-stable, no attempt was made to study their in-dividual physiology. Rabbit antiserum producedagainst an antigen showing mixed flagellationagglutinated all variants to a comparable titer,and no indication was obtained of any antigenicdifferences in the lateral and polar flagella.However, our data cannot be consdered asconclusive in this respect.Experiments with strain H-247. The original
culture of this organism showed individuals withone of three types of flagellation: (1) predom-inantly 2 to 4 polar flagella w:th two or fewercurves (lophotrichous), (2) several lateral flagellawith typically more than two curves, and (3)both types of flagella. The difference in shapeof the polar and the lateral flagella is very strik-ing. By plating and fishing colonies, pure peri-trichous and pure lophotrichous variants wereobtained. A variant showing pure mixed flagella-tion was not obtained. The peritrichous varianthas been transferred in broth numerous timesover a period of many months without the de-velopment of any lophotrichous or mixed in-dividuals, and appears to be stable. The lopho-trichous variant is somewhat less stable andafter several passages in broth one or moretypically shaped lateral flagella in addition to thepolar flagella may be found on some individuals,and also occasionally a purely peritrichous in-dividual. Both strains show variation to filamen-tous "rough" types, but the flagellation remainsthe same. Some of the lophotrichous strainshave shown considerable tendency to produce
nonflagellated variants. Neither the mediumnor the time and temperature of incubationhave shown any definite influence on the natureof the flagellation. In figures 14, 15, 16, 17, 18,and 19 is illustrated the flagellation of the vari-ants of strain H-247.
Physiologically the two variants weere quitesimilar. With the usual tests, such as variouscarbohydrates, nitrate, indole, gelatin, urea,H2S, citrate, methyl red, the two variantsbehaved alike. The only difference noted was ingrowth rate in peptone or peptone-yeast extractmedia. In these media the lophotrichous variantproduced a considerably greater density after24 hours' incubation at room temperature.Physiologically both variants could be consideredas typical Alcaligenes types.
Antisera were produced in riabbits againstpure strains of each of the two variants. Table 1shows the results of agglutination and adsorptiontests. The peritrichous variant is designatedstrain H-260 and the lophotrichous, strain H-261.It is apparent from table 1 that the antigens ofstrains H-260 and H-261 are very similar. Theorganisms cross agglutinate to the same titerwith sera produced against both the boiledbacterial suspensions and the 0.5 per cent for-malin suspensions. The agglutinin-adsorptiondata confirm the similarity of the somatic anti-gens but show the presence of antigens in theflagella of strain H-261 which are not apparentin the flagella of strain H-260. Therefore we mayconclude that the variation from lophotrichousto peritrichous is accompanied by a loss of aflagellar antigenic component with little or nochange in the somatic antigens.
DISCUSSION
It is apparent from the literature and fromthe data presented in this report that bacterialflagella may undergo spontaneous variationboth in shape and arrangement. While some ofthese variations are of a temporary nature,others are more permanent mutations.
Figure 11. Organism K-517 showing the typical polar flagellum and several lateral flagella. Photo-micrograph X 4,000.Figure 12. Organism K-517 showing the typical polar flagellum and many lateral flagella with charac-
teristic curvature. Photomicrograph X 4,000.Figure 13. Organism K-517 showing typical lateral flagella only. Photomicrograph X 4,000.The organisms pictured in this plate are variants of culture H-247. All of the types shown may be
found on a single slide. Note the striking difference in curvature of the polar and the lateral flagella.The wavelength of the polar flagella is close to 3 ,u, while that of the lateral flagella is about 1 Iu.
Figures 14-19Figure 14. Typical lophotrichous individuals from a pure variant, H-261, derived from H-247. This
variant tends to dissociate into the other types shown on this plate. Photomicrograph X 4,000.Figure 16. The organism pictured has typical lophotrichous polar flagella with two, somewhat tangled,
lateral flagella. Photomicrograph X 4,000.Figure 16. This picture shows an organism, partly divided, with lophotrichous flagella at each pole
The shape of the flagella on an organism asdemonstrated by staining procedures is usuallyremarkably constant. The most important shape-characteristic appears to be the curvature,whereas length and thickness appear of minorimportance. With most types of bacteria theflagellar curvature is regular and constant.With a few types of bacteria, or in individualstrains, the flagella may show considerable
TABLE 1Agglutination titers with formalinized and boiled
antigens of strains H-260 and H-261 usingunadsorbed and adsorbed antisera
Legend: - means titer of less than 1 to 40.* Finely granular agglutination of "O" type.
irregularity in shape. Several of the Aeromonascultures studied showed great variation in thisrespect. In these cultures individuals frequentlywere found with straight flagella, flagella hookedat the end, flagella coiled into a circle, as wellas the usual curved flagella. The dissociationinto "curly" and normal flagellar variants pre-viously reported for S. wichita has been observedalso in S. typhimurium. Variants with "curly"
flagella have not been observed in cultures ofother types of bacteria.With only a few types of bacteria is the flagel-
lar arrangement difficult to determine. Thesebacteria are usually of the peritrichous type andso poorly flagellated that it is rare to find morethan one flagellum per organism. With mosttypes of bacteria the flagellar arrangement isreadily demonstrable and generally regardedto be of considerable taxonomic importance.The data in this report show that in three dis-tinct types of bacteria the flagellar arrangementmay undergo variation of several kinds. Thesevariations may cause considerable taxonomicconfusion regardless of how common or uncom-mon they may be among bacteria in general.The formation of chains or filaments by polarflagellates may give the appearance of peritri-chous flagellation. These filaments may usuallybe recognized for what they are because of thewide spacing of the flagella, presence of non-filamentous individuals showing polar flagella,and in some types we have studied a typical,usually stouter, flagellum at the end of the fila-ment. The occurrence of lateral flagella in theearly stages of the growth cycle, as demon-strated in Aeromonas, should offer little taxono-mic difficulty provided the phenomenon is recog-nized. Organisms such as strain K-517 whichhave a mixed polar and peritrichous type offlagellation are more difficult to classify. Atpresent we shall offer no suggestions in this re-gard. Organism H-247, dissociating into stableperitrichous and somewhat less stable lopho-trichous variants, poses a serious taxonomicproblem which may challenge the foundationsof our present bacterial taxonomy. With the dis-covery of the dissociating tendency of strainH-247 we reexamined the other 14 lophotrichousorganisms in our collection. These cultures wereobtained from various parts of the world. Noneshowed the presence of lateral flagella.The commonly encountered biological muta-
tions involve phenotypic changes on a low tax-
and two more curly flagella of the peritrichous type, one of which appears to emerge from the pole.Photomicrograph X 4,000.Figure 17. The two types of flagella are well illustrated in this picture. Photomicrograph X 4,000.Figure 18. This organism shows 3 or 4 lophotrichous polar flagella and several flagella of the peritri-
chous type on each side. Photomicrograph X 4,000.Figure 19. In this picture is shown 2 peritrichous individuals from a pure culture, H-260, derived from
H-247. This organism has not shown any evidence of instability with formation of either lophotrichousor mixed flagellar types. Photomicrograph X 4,000.
onomic level. The daughter and parent typesgenerally may be recognized as varieties of acommon species. Only through an extendedseries of mutations are new species, genera,families, etc. derived. In our present bacterialtaxonomy flagellar arrangement into polar andperitrichous types is the basis for separationof many genera and some families. The questionmay well be raised how justified would we bein classifying the two variants of strain H-247into separate families or even separate genera?Our knowledge of the origin and development
of flagella is rather sketchy. The flagella appearto originate inside the cell wall and grow veryrapidly. The present concept of the cell wall asa fairly rigid, elastic structure makes it unlikelythat flagella puncture the cell wall. The alterna-tive is for the flagela to emerge at a place wherethere is not a rigid wall. Such a place maybe thegrowing end of the cell (Bisset, 1951). While theconcept of a growing end without a rigid cellwall has not been proven conclusively, it is anattractive hypothesis. Cytological studies (Robi-now, 1946; Bisset, 1950; Knaysi, 1951) have indi-cated that many bacteria are typically multi-cellular. This is particularly evident with thegram positive rods and also some gram negativerods in the early growth phases. Gram positivemotile bacilli are, to our knowledge, alwaysperitrichously flagellated. Several Aeromonascultures show peritrichous flagellation in theearly growth phases, but polar flagellation inlater growth phases. Filamentous variants ofpolar flagellates show peritrichous flagellation.These observations suggest a correlation betweenperitrichous flagellation and multicellularity.Whether or not polar flagellates are invariablyunicellular remains to be seen. However, weshould like to suggest, as a working hypothesis,that polar-peritrichous flagellar variations areessentially unicellular-multicellular variations.Two observations relative to bacterial evolu-
tion may be made from our data. (1) The varia-tion observed with strain H-247 was always in thedirection from lophotrichous to peritrichous, thelatter being the stable variant. (2) The genusAeromonas appears related to the Enterobac-teriaceae (all types peritrichous), and its ten-dency to peritrichous flagellation may indicatean evolutionary trend. These two observationsconfirm the commonly expressed belief that the
peritrichously flagellated bacteria have evolvedfrom the polar flagellated (Kluyver and vanNiel, 1936). In our present way of thinking thismay be translated into an evolution from uni-cellular to multicellular.
SUARY
Variation of both flagellar shape and arrange-ment is shown to occur in three distinct typesof bacteria. Lateral flagella are produced fre-quently in young cultures of Aeromonas whichproduce only polar, monotrichous flagella inolder cultures. In a culture of a yellow, nonwatersoluble, pigmented organism were found in-dividuals with polar, peritrichous and mixedflagellation. The polar and lateral flagella showeda distinct difference in curvature. Stable variantscould not be obtained. A polar (lophotrichous)to peritrichous type of variation was shown tooccur in a culture physiologically classified asAlcaligenes. Various intermediary stages withboth types of flagella on the same individualwere demonstrated. The peritrichous variantappeared to be stable but the lophotrichousvariant tended to produce individuals withmixed flagellation and also an occasional in-dividual with peritrichous flagella. The tax-onomic implications of these phenomena arediscussed. A hypothesis is presented to the effectthat all flagella may have a polar origin and thatperitrichous flagellated bacteria are multicellular.Two observations are made bearing on theevolutionary trend in bacteria from polar flagel-lated to peritrichouis flagellated types.
REFERENCESBISsET, K. A. 1950 The cytology and life-history
of bacteria. Williams and Wilkins Co., Balti-more, Md.
BISSET, K. A. 1951 The development of thesurface structure in dividing bacteria. J.Gen. Microbiol., 5, 155-158.
GRAY, P. H. H., AND THORNTON, H. G. 1928Soil bacteria that decompose certain aromaticcompounds. Zentr. Bakt. Parasitenk., Abt.II, 78, 74-96.
KLUYVER, A. J., AND VAN NIEL, C. B. 1936Prospects for a natural system of classifica-tion of bacteria. Zentr. Bakt. Parasitenk.,Abt. II, 94, 369-403.
KNAYSI, G. 1951 Elements of bacterial cytology,2nd ed. Comstock Publishing Co., Inc.,Ithaca, N. Y.
ment of bacterial flagella. J. Bact., 62, 377-389.
MILES, E. M., AND MILES, A. A. 1951 The iden-tity of Proteus hydrophilus Bergey et al. andProteus melanovogenes Miles and Halnan, andtheir relation to the genus Aeromonas Kluyverand van Niel. J. Gen. Microbiol., 6, 298-306.
MUDD, S., AND WARREN, S.- 1923 A readily culti-
vable vibrio filterable through berkefeld "V"candles, Vibrio percolans (new species). J.Bact., 8, 447-458.
ROBINOW, C. F. 1946 Addendum to The bacterialcell, by Rene J. Dubos. Harvard UniversityPres, Cambridge, pp. 355-377.
STANIER, R. Y., AND ADAMS, G. A. 1944 Thenature of the Aeromonas fermentation. Bio-chem. J., 38, 168-171.
