VARIATION IN SHAPE AND ARRANGEMENT OF BACTERIAL FLAGELLA

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 Few studies have been reported in the literature 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 reported a monotrichous organism which produced filamentous variants showing lateral flagella. The lateral flagella were interpreted to be essentially polar. Gray and Thornton (1928) described two organisms which they placed in a new genus Mycoplana. The one organism was described as having "polar, peritrichous" and the other as having "polar or peritrichous" flagella. Unfortunately, their illustrations are only drawings, 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 necessary 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 staining 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 organisms 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. Received for publication July 23, 1952 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 peritrichous flagella. They grew readily on simple peptone 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 physiologically 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 demonstrated on the heated organisms. The "H" antigens were 0.5 per cent formalin-saline suspen- 263

264 EINAR LEIFSON AND RUDOLPH HUGH [VOI,. 65 Figures 1-7 Figuire 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.

19531 VARIATION IN BACTERIAL FLAGELLA 265 sions carefully checked for tvpical flagellation. The "H" agglutinations were read after four hours and the "O" agglutinations after 20 hours. Agglutinin adsorptions were made by adding 9 parts of bacterial suspension to 1 part of serum, incubating for 2 hours at 37 C followed by centrifugation. A second adsorption was made by suspending packed organisms in the supernatant serum from the first adsorption and proceeding as before. The serum from the second adsorption was checked for the presence of antibodies for the homologous antigen, and if any were demonstrable in dilution of 1:40 further adsorption was done. EXPERIMENTAL RESULTS Experiments with Aeromonas. Several species of Aeromonas when incubated in broth at 20 C for about 16 hours showed some individuals with a single polar flagellum, some with a polar flagellum and one or more lateral flagella, and some with several lateral flagella only. When the incubation time was extended beyond 24 hours, only polar monotrichous individuals could be found. At higher incubation temperatures, such as 35 C, lateral flagella could be demonstrated only during the first 5 hours or so of incubation. At longer incubation times only polar monotrichous individuals could be found. In figures 1, 2, 3, 4, 5, and 6 is illustrated the flagellar variation of two Aeromonas species. It may be noted that the curvature of the polar and lateral flagella is alike. Experiments with media of various compositions showed a tendency for the development of a greater proportion of peritrichously flagellated individuals in a medium containing both peptone and yeast extract as compared to one with peptone only. However, in all the media tested, the relationship of flagellation to growth phase remained the same. From repeated experiments it became apparent that the organisms produced lateral flagella during the early stages of the growth cycle and predominantly only single polar flagella in the later stages. By repeated plating and fishing, pure cultures showing only one or the other type of flagellation were not obtained. Rabbit antiserum produced against a young culture with a high proportion of peritrichously flagellated individuals agglutinated the older monotrichous cultures to titer. The flagella of many strains of Aeromonas showva tendency to form loose coils or loops which are very striking (figures 5, 6, and 7). This shape does not seem to have become stabilized in the polar flagella, and individuals with the more usual curved flagella are always found interspersed among the individuals with the coiled flagella. Pure cultures with "coiled" peritrichous flagella have been observed. In figure 7 is illustrated a culture sent to us by A. J. Kluyver labeled Aeromonas liquefaciens, strain L418. The same strain also was obtained from Ellen M. Miles. Both sources indicated the culture to be monotrichous. Flagellar stains made from these cultures at various ages and temperatures of incubation failed to show any but peritrichous individuals. Evidence seems to indicate that the strain we received is a peritrichous variant, but in our hands it has shown no tendency to produce monotrichous individuals. The peculiar coiling of some of the polar flagella of other Aeromonas strains supports this view. Experiments with strain K-51 7. The original culture of this organism showed some individuals with a single polar flagellum; some with a polar flagellum and one or more, somewhat shorter and more curved, lateral flagella; and an occasional organism with lateral flagella only. By repeated plating and fishing, stra ns were obtained which showed: (1) predominantly polar monotrichous flagella, (2) predominantly mixed Figure 4. Same organism as shown in figure 3. Peritrichous flagella characteristic in the early stages of 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 same slide. The single individual is magnified 4,000 X, the filamentous 2,000 X.

266 EINAR LEIFSON AND RUDOLPH HUGH [VOL. 65 Figures 8-13 Figure 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 that of 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 curvature. Photomicrograph X 4,000.

1953] VARIATION IN BACTERIAL FLAGELLA 267 single polar and lateral flagella, and (3) a single strain which showed predominantly peritrichous flagella. The different flagellar variants are illustrated in figures 8, 9, 10, 11, 12, and 13. On repeated transfer in broth all the variants rapidly tended towards a more or less common denominator, namely, a culture showing mixed flagellation like the original. Neither the composition of the medium nor the incubation time and temperature had much effect on the type of flagellation. Since these variants were so unstable, no attempt was made to study their individual physiology. Rabbit antiserum produced against an antigen showing mixed flagellation agglutinated all variants to a comparable titer, and no indication was obtained of any antigenic differences in the lateral and polar flagella. However, our data cannot be consdered as conclusive in this respect. Experiments with strain H-247. The original culture of this organism showed individuals with one of three types of flagellation: (1) predominantly 2 to 4 polar flagella w:th two or fewer curves (lophotrichous), (2) several lateral flagella with typically more than two curves, and (3) both types of flagella. The difference in shape of the polar and the lateral flagella is very striking. By plating and fishing colonies, pure peritrichous and pure lophotrichous variants were obtained. A variant showing pure mixed flagellation was not obtained. The peritrichous variant has been transferred in broth numerous times over a period of many months without the development of any lophotrichous or mixed individuals, and appears to be stable. The lophotrichous variant is somewhat less stable and after several passages in broth one or more typically shaped lateral flagella in addition to the polar flagella may be found on some individuals, and also occasionally a purely peritrichous individual. Both strains show variation to filamentous "rough" types, but the flagellation remains the same. Some of the lophotrichous strains have shown considerable tendency to produce nonflagellated variants. Neither the medium nor the time and temperature of incubation have shown any definite influence on the nature of the flagellation. In figures 14, 15, 16, 17, 18, and 19 is illustrated the flagellation of the variants of strain H-247. Physiologically the two variants weere quite similar. With the usual tests, such as various carbohydrates, nitrate, indole, gelatin, urea, H2S, citrate, methyl red, the two variants behaved alike. The only difference noted was in growth rate in peptone or peptone-yeast extract media. In these media the lophotrichous variant produced a considerably greater density after 24 hours' incubation at room temperature. Physiologically both variants could be considered as typical Alcaligenes types. Antisera were produced in riabbits against pure strains of each of the two variants. Table 1 shows the results of agglutination and adsorption tests. The peritrichous variant is designated strain H-260 and the lophotrichous, strain H-261. It is apparent from table 1 that the antigens of strains H-260 and H-261 are very similar. The organisms cross agglutinate to the same titer with sera produced against both the boiled bacterial suspensions and the 0.5 per cent formalin suspensions. The agglutinin-adsorption data confirm the similarity of the somatic antigens but show the presence of antigens in the flagella of strain H-261 which are not apparent in the flagella of strain H-260. Therefore we may conclude that the variation from lophotrichous to peritrichous is accompanied by a loss of a flagellar antigenic component with little or no change in the somatic antigens. DISCUSSION It is apparent from the literature and from the data presented in this report that bacterial flagella may undergo spontaneous variation both in shape and arrangement. While some of these 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. Photomicrograph X 4,000. Figure 12. Organism K-517 showing the typical polar flagellum and many lateral flagella with characteristic 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.

268 EINAR LEIFSON AND RUDOLPH HUGH [VOL. 65 Figures 14-19 Figure 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

1953] VARIATION IN BACTERIAL FLAGELLA 269 The shape of the flagella on an organism as demonstrated by staining procedures is usually remarkably constant. The most important shapecharacteristic appears to be the curvature, whereas length and thickness appear of minor importance. With most types of bacteria the flagellar curvature is regular and constant. With a few types of bacteria, or in individual strains, the flagella may show considerable TABLE 1 Agglutination titers with formalinized and boiled antigens of strains H-260 and H-261 using unadsorbed and adsorbed antisera ANISEIRUM ANTIGEN Arboiled0 2 orm2l- Against withbed i0ed boiled f2- fo2 inized.inized 260 boiled none 2,560 2,560 _ 260 boiled 261 boiled - - _ 261 boiled none 2,560 2,560 80* 160* 261 boiled 260 boiled - - 40* 40* 260 formal- none 5,120 5,120 5,120 5,120 inized 260 formal- 261 for- 160 40 - - inized malinized 261 formal- none 2,5602,560 5,120 5,120 inized 261 formal- 260 for- 40 80-1,280 inized malinized Legend: - means titer of less than 1 to 40. * Finely granular agglutination of "O" type. irregularity in shape. Several of the Aeromonas cultures studied showed great variation in this respect. In these cultures individuals frequently were found with straight flagella, flagella hooked at the end, flagella coiled into a circle, as well as the usual curved flagella. The dissociation into "curly" and normal flagellar variants previously reported for S. wichita has been observed also in S. typhimurium. Variants with "curly" flagella have not been observed in cultures of other types of bacteria. With only a few types of bacteria is the flagellar arrangement difficult to determine. These bacteria are usually of the peritrichous type and so poorly flagellated that it is rare to find more than one flagellum per organism. With most types of bacteria the flagellar arrangement is readily demonstrable and generally regarded to be of considerable taxonomic importance. The data in this report show that in three distinct types of bacteria the flagellar arrangement may undergo variation of several kinds. These variations may cause considerable taxonomic confusion regardless of how common or uncommon they may be among bacteria in general. The formation of chains or filaments by polar flagellates may give the appearance of peritrichous flagellation. These filaments may usually be recognized for what they are because of the wide spacing of the flagella, presence of nonfilamentous individuals showing polar flagella, and in some types we have studied a typical, usually stouter, flagellum at the end of the filament. The occurrence of lateral flagella in the early stages of the growth cycle, as demonstrated in Aeromonas, should offer little taxonomic difficulty provided the phenomenon is recognized. Organisms such as strain K-517 which have a mixed polar and peritrichous type of flagellation are more difficult to classify. At present we shall offer no suggestions in this regard. Organism H-247, dissociating into stable peritrichous and somewhat less stable lophotrichous variants, poses a serious taxonomic problem which may challenge the foundations of our present bacterial taxonomy. With the discovery of the dissociating tendency of strain H-247 we reexamined the other 14 lophotrichous organisms in our collection. These cultures were obtained from various parts of the world. None showed the presence of lateral flagella. The commonly encountered biological mutations involve phenotypic changes on a low taxand 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 peritrichous 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 lophotrichous or mixed flagellar types. Photomicrograph X 4,000.

270 EINAR LEIFSON AND RUDOLPH HUGH [VOL. 65 onomic level. The daughter and parent types generally may be recognized as varieties of a common species. Only through an extended series of mutations are new species, genera, families, etc. derived. In our present bacterial taxonomy flagellar arrangement into polar and peritrichous types is the basis for separation of many genera and some families. The question may well be raised how justified would we be in classifying the two variants of strain H-247 into separate families or even separate genera? Our knowledge of the origin and development of flagella is rather sketchy. The flagella appear to originate inside the cell wall and grow very rapidly. The present concept of the cell wall as a fairly rigid, elastic structure makes it unlikely that flagella puncture the cell wall. The alternative is for the flagela to emerge at a place where there is not a rigid wall. Such a place maybe the growing end of the cell (Bisset, 1951). While the concept of a growing end without a rigid cell wall has not been proven conclusively, it is an attractive hypothesis. Cytological studies (Robinow, 1946; Bisset, 1950; Knaysi, 1951) have indicated that many bacteria are typically multicellular. This is particularly evident with the gram positive rods and also some gram negative rods in the early growth phases. Gram positive motile bacilli are, to our knowledge, always peritrichously flagellated. Several Aeromonas cultures show peritrichous flagellation in the early growth phases, but polar flagellation in later growth phases. Filamentous variants of polar flagellates show peritrichous flagellation. These observations suggest a correlation between peritrichous flagellation and multicellularity. Whether or not polar flagellates are invariably unicellular remains to be seen. However, we should like to suggest, as a working hypothesis, that polar-peritrichous flagellar variations are essentially unicellular-multicellular variations. Two observations relative to bacterial evolution may be made from our data. (1) The variation observed with strain H-247 was always in the direction from lophotrichous to peritrichous, the latter being the stable variant. (2) The genus Aeromonas appears related to the Enterobacteriaceae (all types peritrichous), and its tendency to peritrichous flagellation may indicate an evolutionary trend. These two observations confirm the commonly expressed belief that the peritrichously flagellated bacteria have evolved from the polar flagellated (Kluyver and van Niel, 1936). In our present way of thinking this may be translated into an evolution from unicellular to multicellular. SUARY Variation of both flagellar shape and arrangement is shown to occur in three distinct types of bacteria. Lateral flagella are produced frequently in young cultures of Aeromonas which produce only polar, monotrichous flagella in older cultures. In a culture of a yellow, nonwater soluble, pigmented organism were found individuals with polar, peritrichous and mixed flagellation. The polar and lateral flagella showed a distinct difference in curvature. Stable variants could not be obtained. A polar (lophotrichous) to peritrichous type of variation was shown to occur in a culture physiologically classified as Alcaligenes. Various intermediary stages with both types of flagella on the same individual were demonstrated. The peritrichous variant appeared to be stable but the lophotrichous variant tended to produce individuals with mixed flagellation and also an occasional individual with peritrichous flagella. The taxonomic implications of these phenomena are discussed. A hypothesis is presented to the effect that all flagella may have a polar origin and that peritrichous flagellated bacteria are multicellular. Two observations are made bearing on the evolutionary trend in bacteria from polar flagellated to peritrichouis flagellated types. REFERENCES BISsET, K. A. 1950 The cytology and life-history of bacteria. Williams and Wilkins Co., Baltimore, Md. BISSET, K. A. 1951 The development of the surface structure in dividing bacteria. J. Gen. Microbiol., 5, 155-158. GRAY, P. H. H., AND THORNTON, H. G. 1928 Soil bacteria that decompose certain aromatic compounds. Zentr. Bakt. Parasitenk., Abt. II, 78, 74-96. KLUYVER, A. J., AND VAN NIEL, C. B. 1936 Prospects for a natural system of classification 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.

19531 VARIATION IN BACTERIAL FLAGELLA 271 LEIFSON, E. 1951 Staining, shape, and arrangement of bacterial flagella. J. Bact., 62, 377-389. MILES, E. M., AND MILES, A. A. 1951 The identity of Proteus hydrophilus Bergey et al. and Proteus melanovogenes Miles and Halnan, and their relation to the genus Aeromonas Kluyver and van Niel. J. Gen. Microbiol., 6, 298-306. MUDD, S., AND WARREN, S.- 1923 A readily cultivable vibrio filterable through berkefeld "V" candles, Vibrio percolans (new species). J. Bact., 8, 447-458. ROBINOW, C. F. 1946 Addendum to The bacterial cell, by Rene J. Dubos. Harvard University Pres, Cambridge, pp. 355-377. STANIER, R. Y., AND ADAMS, G. A. 1944 The nature of the Aeromonas fermentation. Biochem. J., 38, 168-171.