JOURNAL OF BACTERIOLOGY, Jan., 1966Copyright © 1966 American Society for Microbiology

Vol. 91, No. 1Printed in U.S.A.

Some Singular Properties of Bacterial Flagella, withSpecial Reference to Monotrichous Forms

F. F. ROBERTS, JR., AND R. N. DOETSCH

Department of Microbiology, Untiversity of Maryland, College Park, Maryland

Received for publication 17 August 1965

ABSTRACT

ROBERTS, F. F., JR.( University of Maryland, College Park), AND R. N. DOETSCH.Some singular properties of bacterial flagella, with special reference to monotrichousforms. J. Bacteriol. 91:414-421. 1966.-Heat (60 C for 30 min), 10 M acetamide, and8 M urea all brought about rapid and complete dissolution of flagella from mono-

trichous bacteria; hence, these flagella respond similarly to those of peritrichousforms. Chloramphenicol (103 Ag/ml) inhibited regeneration of flagella in all peri-trichously flagellated cultures; however, monotrichous forms were able to regeneratetheir flagella in a concentration 102 times that required to inhibit multiplication.Peritrichous bacteria did not synthesize flagella when infected by lytic bacterio-phages. In these experiments, the time from infection to lysis was sufficient foruninfected controls to resynthesize their flagella. Monotrichous bacteria, however,in all but one instance, were able to resynthesize their flagella before lysis occurred.A study of flagella resynthesis in a non-nutritive milieu indicated that only a smallamount of flagellum precursor is present in any given cell. The effect of temperatureon synthesis of flagella indicated that, although some bacteria multiply and are motileat a given temperature, they are unable to resynthesize their flagella at that same

temperature. This strongly suggests that initial flagellum synthesis and flagellumregeneration are not necessarily identical processes.

It has been tacitly assumed by microbiologiststhat no fundamental differences exist in themolecular plan or biosynthetic mechanisms lead-ing to the synthesis of flagella in various pro-caryotic organisms. This must not be understoodto mean that some differences, in fact, have notbeen observed. One may note, in passing, thework of Koffler (10) on the interesting propertiesof flagella from thermophilic as contrasted withmesophilic forms, or the discovery by Ambler andRees (2) of E-N-methyl lysine in Salmonellatyphimurium flagella, or the finding by Robertsand Doetsch (unpublished data) of an enzymecapable of selectively attacking flagella fromperitrichous, but not monotrichous, bacteria.The effects of chemical and physical agents on

bacterial flagella have been studied by manyinvestigators (5, 7, 14, 16, 18, 19, 20). Thesereports are, in the main, confined to two peri-trichous eubacterial species, namely, S. typhi-murium and Proteus vulgaris. A number ofgeneralizations concerning the structure andbiological properties of procaryotic flagella havebeen drawn from these reports, but, in view ofthe paucity of information on flagella of mono-

trichous organisms, we believe at least a cursoryglance is indicated.

MATERIALS AND METHODSBacteria studied. Table 1 shows the bacteria used

in this investigation and their sources.Staining methods. Flagella stains for light micros-

copy were made by Gray's (6) method. Preparationsfor electron microscopy were fixed in 10% (v/v)formalin, shadowed with chromium, and observedwith an RCA EMU 3f electron microscope.

Effect of heat. A 1-ml amount of buffered, washed-cell suspension was placed in a screw-cap tube (15 X120 mm), and heated to 60 C in a constant-tempera-ture water bath for 30 min. Samples were then re-moved, and the flagella were stained. To prepare abuffered, washed-cell suspension, bacteria were grownon Brain Heart Infusion agar (BBL) in Roux bottlesor plugged tubes (15 X 120 mm) for 4 to 24 hr at28 C. The cultures were washed from the agar surfaceby gentle addition of distilled water. The organismswere centrifuged at 3,500 X g at 5 C and washedthree times with distilled water. The final suspensionwas made in distilled water or 0.001 M phosphatebuffer (pH 6.8) to an optical density (OD) of 0.25 at400 muA (equivalent to approximately 105 organismsper milliliter).

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PROPERTIES OF BACTERIAL FLAGELLA

TABLE 1. Bacteria used and their sources

Species Source and designation

Aeromonas hydrophila....Bacillus megaterium......

B. stearothermophilus....B. subtilis.........Chromobacteriumviolaceum.

Escherichia coli Int.......

E. coli................Proteus morganai.........P. vulgaris...............Pseudomonas aeruginosa..

P. fluorescens............

P. fragi.Serratia marcescens......Spirillum serpens........Xanthomonas

campestris..X. pruni.................X. vesicatoria............

American Type Culture Collection; 13137C. Weibull, Institute of Physical Chemistry and Biochemistry, Uppsala

University, Stockholm, Sweden; 13632American Type Culture Collection; 11330University of Maryland Culture Collection; 46-A-4

University of Maryland Culture Collection; 24-A-1J. J. Jezeski, Department of Dairy Science, University of Minnesota,Minneapolis; 86

American Type Culture Collection; 11840American Type Culture Collection; 8109University of Maryland Culture Collection; 44-1-AUniversity of Maryland Culture Collection; 5-1-AT. H. Lord, Department of Bacteriology, Kansas State University, Man-

hattan; KS-33, KS-10, KS-17University of Maryland Culture Collection; 5-2-AR. Y. Stanier, Department of Molecular Biology, University of Cali-

fornia, Berkeley; 12633J. A. Alford, U.S. Agricultural Experiment Station, Beltsville; Md. 43University of Maryland Culture Collection; 43-1-AUniversity of Maryland Culture Collection; 12638

W. L. Klarman, Department of Botany, University of Maryland; 26XW. L. Klarman, Department of Botany, University of Maryland; 27XW. L. Klarman, Department of Botany, University of Maryland; 18X

Effect of Cu, Hg, Mn, Mg, and Zn ions. Amountsof 1 ml of 0.1 or 0.01 M solutions of CuSO4.5H20,HgCl2, MnCl2*4H20, Mn2(SO4)3, MgCl2 6H2O, andZnCl2 (all chemically pure grade) were added sepa-rately to 2 ml of a washed-cell suspension in screw-captubes (15 X 120 mm). After 10 min at 25 C, sampleswere removed, and flagella stains and electron micro-graphs were made.

Effect of acetamide and urea. A 0.1-ml amount ofa buffered, washed-cell suspension was added to 1 mlof 10 M acetamide or 8 M urea in screw-cap tubes(15 X 120 mm). After 30 min at 25 C, flagella stainswere made of this suspension.

Flagella regeneration studies. Bacterial flagellawere removed in an Omni-Mixer (Ivan Sorvall, Nor-walk, Conn.) at 16,000 rev/min in an ice bath at 4 to6 C for 1 to 3 min. An Omni-Mixer container wasfilled with 30 ml of a washed-cell suspension (OD

2.0 at 400 mpu) and completeness of deflagellationwas determined as described above under Stainingmethods.

Effect of chloramphenicol. Washed-cell suspensionswere suspended in 103 jug/ml of crystalline chloram-phenicol (courtesy of H. E. Machmer, Parke, Davis& Co., Detroit, Mich.) to a final concentration of 105organisms per milliliter. An amount (30 ml) of thissuspension was deflagellated. Thereafter, the deflagel-lated bacteria were washed once and suspended inBrain Heart Infusion broth (BBL) containing 103pg/ml of chloramphenicol. Observations were madefor motility at 5-min intervals, and samples weretaken for flagella stains and electron microscope

observations. Controls of untreated bacteria weresuspended in Brain Heart Infusion broth, also con-taining 103 ,Ag/ml of chloramphenicol, and observedfor 48 hr for any change in OD (at 400 m,u). In addi-tion, deflagellated bacteria were placed in BrainHeart Infusion broth without chloramphenicol.

Flagella regeneration during infection by bacterio-phage. A washed, deflagellated suspension was addedto Brain Heart Infusion broth to a concentration of105 cells per milliliter. Amounts of 2 ml of this sus-pension were placed into screw-cap tubes (15 X 120mm); specific, lytic bacteriophages were introducedat a multiplicity ratio of 10:1; and the mixture wasincubated in the growth range of the host organism.A flagellated control with the same multiplicity ratiowas included in each experiment. At 5-min intervals,samples were removed and examined, with both lightand electron microscopes, for newly synthesizedflagella, motility, and lysis. One-step growth curveswere carried out by the method of Adams (1). Thephage and host strains are listed in Table 2.

Flagella regeneration in a non-nutritive milieu. A5-ml amount of washed, deflagellated suspensions of4-hr cultures in 0.001 M phosphate buffer (pH 6.8)was incubated in the growth range of the test bac-teria. At 10-min intervals, samples were removedand examined for flagella resynthesis and motility.

Effect of temperature on flagella regeneration.Deflagellated washed suspensions were placed inBrain Heart Infusion broth in a concentration of 105cells per milliliter in 5-mI amounts in screw-top tubes(15 X 120 mm). Replicate cultures were incubated

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ROBERTS AND DOETSCH

Clarifying tank,Sewage DisposalPlant,Laurel, Md.

Clarifying tank,Sewage DisposalPlant,Laurel, Md.

Anaerobic tank,Sewage DisposalPlant,Manhattan, Kan.

Clarifying tank,Sewage Disposal

Plant,Laurel, Md.

Clarifying tank,Sewage Disposal

Plant,Laurel, Md.

Anaerobic tank,Sewage Disposal

Plant,Manhattan, Kan.

George Hageage,Department of Micro-

biology,University of NewHampshire,

DurhamFrank M. Hetrick,Department of Micro-

biology,University of Mary-

land,College Park

Clarifying tank,Sewage Disposal

Plant,Laurel, Md.

Clarifying tank,Sewage Disposal

Plant,Laurel, Md.

Clarifying tank,Sewage Disposal

Plant,Laurel, Md.

at temperatures outside their optimal multiplicationrange for a maximum of 2 hr. At 20-min intervals,samples were removed and examined for flagellaresynthesis and motility.

RESULTS

Effects of chemical and physical agents on theflagella of monotrichous bacteria. When themonotrichously flagellated bacteria listed inTable 1 were heated to 60 C for 30 min, a com-plete dissolution of their flagella resulted. Similarresults were obtained upon exposure to 10 Macetamide or to 8 M urea for 30 min.None of the metal compounds tested, with the

exception of CuS04*5H20, showed any effects onmonotrichous bacteria that could be distinguishedfrom the controls. The presence of copper ions,prior to flagella staining, seemed to cause atwofold effect in one culture, Pseudomonasaeruginosa KS-33, even though this was not seenin any of the other cultures (Table 1). StrainKS-33 tended to form clumps of cells whenstained; however, the addition of copper ionsprior to staining resulted in the appearance ofsingle cells only. Copper ions also caused thenormal flagella to straighten out. Figure la showsthe normal flagella, and Fig. lb shows theflagella after addition of 0.01 M CUSO4- 5H20. Theabsence of this effect in the presence of Mn2(SO4)3ruled out the sulfate ion as a possible cause ofthis phenomenon, although it is possible thatmanganese interferes with the production of theeffect by sulfate, whereas copper does not inter-fere.

Flagella regeneration. All bacteria listed inTable 1 were deflagellated and examined forability to regenerate flagella in the presence of103 pg/ml of chloramphenicol. None of theperitrichously flagellated cultures was able toresynthesize new flagella or to regain its motilityover a 48-hr observation period. However, all ofthe monotrichously flagellated bacteria were ableto resynthesize flagella and regain their motilityin the same time as normal controls withoutchloramphenicol, i.e., in less than 30 min. Thepolarly flagellated P. fluorescens 5-2-A and 12633(tuft of three to four flagella at one end) re-sponded as monotrichously flagellated species,whereas Spirillum serpens 12638 (tuft of flagellaon both ends) responded as a peritrichouslyflagellated species. Figure 2 depicts the sequenceof events in the resynthesis of flagella by P.aeruginosa 5-1-A in the presence of 103 ug/ml ofchloramphenicol.When peritrichously flagellated organisms

(Table 2) were infected with phage, none regen-erated its flagella. Contrariwise, all but onemonotrichous organism (P. fragi 43) regeneratedtheir flagella before lysis occurred. These resultsare summarized in Table 3.None of the 21 cultures examined regenerated

TABLE 2. Bacteriophages used with their hoststrains and sources

Bacterio- Host strain Source

CV-40

Pr-18

PS-5

PS-13

PS-22

PS-612

T-1

T-2

T-7L

Xa-9

xO-1

Chromobac-teriumviolaceum

24-A-1Proteus vul-

garis44-1-A

Pseudomonasaerugi-nosa

5-1-A, KS-33

P. aeruginosaKS-19, KS-

17, 5-1-A

P. fragi43

P. fluorescens5-2-A,

12633

Escherichiacoli

11840

E. coli11840

E. coli11840, 86

Xanthomonas27X

X. campestris26X

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PROPERTIES OF BACTERIAL FLAGELLA

FIG. 1. (a) Pseudomonas aeruginosa KS-33 showing normal wavelength of a single flagellum. X 21,000. (b)P. aeruginosa KS-33 after treatment with 0.01 M CuS04 .51H20 showing "straightening out" effect on theflagellum.X 15,000.

their flagella in a non-nutritive environment, bacteria to resynthesize their flagella whensuggesting the absence of any substantial flagella incubated at nonoptimal temperatures are givenprecursor "pool." in Table 4. Bacillus stearothermophilus 11330The results of experiments on the ability of synthesized flagella at 35 C, even though it was

psychrophilic, mesophilic, and thermophilic not capable of multiplying at this temperature.

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ROBERTS AND DOETSCH

Eseherichia coli Int 86 could not regenerateflagella at 28 C, even though flagella are formedduring "normal" growth at that temperature.However, when grown at 28 C, and tested forregeneration at 4 C, resynthesis of flagella did, infact, occur. P. fragi 43 responded in the samemanner as E. coli Int 86. None of the otherbacterial species resynthesized its flagella underthe conditions of the experiment. Plate counts, byuse of Brain Heart Infusion agar, after exposureof the bacteria to the various temperatures usedin testing for resynthesis, revealed no lethaleffects.

DIscussIoNIn their reactions to heat, urea, and acetamide,

flagella from monotrichous bacteria respond inthe same manner as those from peritrichousforms. It may be noted that the flagella of thermo-philic bacteria are reported to be significantlymore resistant to heat than mesophilic forms (10).Unfortunately, the counterpart of the peri-trichous thermophile, i.e., a monotrichousthermophile, has yet to be discovered.The ability of most bacteria to regenerate

flagella in a short time, when placed in a suitableenvironment, is an amazing achievement ofmetabolic synthesis. Leifson (11) reported thatflagella were synthesized at the rate of 1 ,u every2 to 3 min, but Stocker and Campbell (17)observed a rate of 0.1 ,u/min at 37 C and 0.085A/min at 25 C. Since greater than 80% of theindividual organisms in a culture may regaintheir motility after mechanical or enzymaticdeflagellation, there is no doubt that the effect ofsuch operations upon the flagella-synthesizingsites is minimal. In our study, bacteria weredeflagellated and allowed to resynthesize theirflagella three times in the absence of a significantdegree of multiplication. Age of the culture hadlittle influence on regeneration time, and 2- to24-hr cultures resynthesized flagella in the sametime period. The possibility of a critical flagellalength triggering a repressor of flagella synthesisis an attractive one, which should be investigatedfurther.

In 1961, Quadling and Stocker (14) reportedthat chloramphenicol inhibited flagellar resyn-thesis in S. typhimuriumn. Since then, this drug

has been used by other investigators to preventflagella resynthesis in various peritrichousbacteria. However, our work revealed that 103,g/ml of chloramphenicol did not inhibit flagellaresynthesis in monotrichous bacteria. This anti-biotic was tested on 8 species, 3 genera, and 12strains, and all were able to resynthesize theirflagella with the exception of S. serpens 12638.This bacterium, with a tuft of flagella at bothends, is intermediate between the true polarlyflagellated monotrichous bacteria and the peri-trichous forms. Strains 5-2-A and 12633 of P.fluorescens, with a tuft of flagella at only one endof the cell, are more closely related to true mono-trichous forms, and, indeed, they resynthesizedtheir flagella in the presence of chloramphenicol.This drug immediately and completely halted themultiplication of the test bacteria; however,motility was not so rapidly inhibited. Permeabilityeffects cannot account for this phenomenon,since the synthesis of cellular protein was inhib-ited. According to Rendi and Ochoa (15), theinhibitory action of chloramphenicol may be dueto interference with the attachment of messengerribonucleic acid (RNA) to ribosomes. Yee andGezon (21) suggested that suppression of proteinsynthesis may result from the channeling ofavailable nitrogen to RNA. Neither of theseexplanations accounts for the selectivity of proteinsynthesis we observed in the resynthesis of mono-trichous flagella, i.e., synthesis of flagella proteinin the face of inhibition of cytoplasmic protein.The site of the flagella-synthesizing mechanism

may be the basal granule, and perhaps it isimpervious, in some unknown manner, to chlor-amphenicol; or the synthesis of this special typeof protein (flagellin) is "different" and henceunaffected by the drug. Murray and Birch-Andersen (13) noted that the area around thebasal granule of S. serpens was relatively ribo-some-free, and perhaps this observation lendssome credence to our latter hypothesis.Cohen (3, 4) found that in phage-infected

bacteria, total protein synthesis proceeds in thehost cell at the same rate as before infection. Thisis not to say that the rate of cellular proteinsynthesis is unaltered, and Monod and Wollman(12) showed that phage-infected E. coli could nolonger be induced to form f3-galactosidase. The

FIG. 2. (a) Pseudomonas aeruginosa 5-1-A before deflagellation in 1,000 ,g/ml of chloramphenicol. X 8,000.(b) P. aeruginosa 5-I-A immediately after deflagellation in 1,000 jAg/ml of chloramphenicol. X 23,000. (c) P.aeruginosa 5-I-A with short, iiarrow diameter flagellum, synthesized in the presence of 1,000 ,ug/ml of chloram-phenicol 10 miii after deflagellation. X 37,000. (d) P. aeruginosa 5-1-A showiiig continued flagellulm synthesis inthe presence of 1,000 ,ug/ml of chloramphenicol 20 miii after deflagellation. X 16,000. (e) P. aeruginosa 5-1-Ashowing continued flagellium synthesis in the presence of 1,000 Ag/ml of chloranmphenicol 30 miii after deflagella-tion. X 28,000. (f) P. aeruginosa 5-1-A showing flagellum of normal length synthesized in the preseiice of 1,000,Ug/ml of chloI-amphenlicol 40 miii after deflagellation. X 33,000.

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ROBERTS AND DOETSCH

Chromobacter-ium viola-

ceum24-A-1

Proteus vulgaris44-1 -A

Pseudomonasaeruginosa

5-1-AKS-33

P. aerugintosaKS-19KS-175-1 -A

P. fragi43

P. fluorescens5-2-A12633

Escherichia coli11840

E. coli11840

E. coli11840

86

Xanthomonaspruni

27XX. campestris26X

min

42

45

7060

757050

62

5055

39

41

47

42

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65

Te infection on granule, and it might direct the synthesis of)I2 flagellin only. The basal granule may be im-

lagellar regeneration permeable to phage deoxyribonuclease, andtime* hence afford protection to the internal basal

granule DNA. Other "satellite" DNA systemsmin have been reported, such as the sex factor, an

autonomous element existing in a free cytoplasmicstate in bacteria.

Kerridge (9) raised the incubation temperatureNo regenera- of a S. typhimurium culture to 44 C to prevent

tion flagellar synthesis, and it is well known that most

No regenera- strains of P. vulgaris produce flagella in abun-tion dance at 28 C, but only a few at 38 C. The

excellent review by Kerridge (8) summarizesmany observations on effects of temperature on

30 flagella formation in S. typhimurium, and it30 should be consulted for data representative of a

30peritrichous organism. In our experiments,

30 deflagellated, psychrophilic, and mesophilic30 bacteria were incubated at or above their normal

multiplication temperatures, and thermophilesNo regenera- were incubated below their optimal multiplication

tion temperatures. B. stearothermophilus 11330, athermophile, was able to resynthesize flagella at a

30

30

No regenera-tion

No regenera-tion

No regenera-tion

No regenera-tion

30

30

* Time required for bacteriophage-infectedcultures to synthesize sufficient flagella to regain25% motility.

results of our experiments on flagella synthesisduring phage infection suggest that monotrichousforms may synthesize flagellin in a differentmanner from that of cellular protein in general.There was a correlation between the latent periodand flagella regeneration. With bacteria in whichflagella regeneration occurred, the latent periodaveraged 61 min, but with those showing noregeneration, the latent period averaged 41 min;nevertheless, noninfected peritrichous forms wereable to regenerate their flagella within this time.

It may be that replicating "satellite" deoxy-ribonucleic acid (DNA) is present in the basal

TABLE 4. Effect of temperature onflagellar regenerationi*

Growth Regen- FlagellarSpecies th eration synthesistemp after 2 hrt

C C

Aeromonias hydrophilia 28 45 None13137

Bacillus stearothermo- 55 35 Synthesisphi/is 11330

Escherichia coli Int 86 4 28 None4 45 None

28 4 Synthesis28 28 None28 45 None

Proteus morganiii 8109 28 45 NoneP. vulgaris 44-1-A 28 45 NonePseudomonas aeruginzosa 28 45 None5-1-A

P. fluorescetis 12633 28 45 NoneP. fragi 43 4 28 None

4 45 None28 4 Synthesis28 28 None28 45 None

Xanthomonas campestris 28 45 None26X

* Deflagellated bacterial cultures, grown at thetemperature indicated above, were incubated atspecified regeneration temperatures in BrainHeart Infusion broth.

t The demonstration of flagella on 25% of thecells after 2 hr was considered to be indicativeof new flagella synthesis.

TABLE 3. Effect of bacteriophagflagellar regeneratio

Phage Host strain Latent F. _~~~~pro

CV-40

Pr-18

PS-S

PS-13

PS-22

PS-612

T-1

T-2

T-7L

Xa-9

XO-1

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PROPERTIES OF BACTERIAL FLAGELLA

temperature 20 C lower than its optimum (55 C),even though it could not divide at that tem-perature. None of the mesophilic bacteria grownat 28 C could resynthesize their flagella at 45 C.Two cold-tolerant cultures, E. coli Int 86 and P.fragi 43, could resynthesize flagella at 4 C ifgrown at 4 or 28 C, but could not resynthesizetheir flagella at 28 C if grown at 4 or 28 C. Thegeneralization that all bacteria are able to resyn-thesize their flagella, therefore, appears to requirequalification. From this work, it seems that eventhough flagella synthesis and regeneration occursin the same bacterium, these processes may not,in fact, be identical.

ACKNOWLEDGMENTS

This investigation was supported by Public HealthService training grant 5 TI GM 615-04 from theDivision of General Medical Sciences.We thank W. L. Wallenstein for his contributions

in the electron microscopic work.

LiTERATURE CITED

1. ADAMS, M. H. 1959. Bacteriophages. IntersciencePublishers, Inc., New York.

2. AMBLER, R. P., AND M. W. REES. 1959. Epsilon-N-methyllysine in bacterial flagella protein.Nature 186:86.

3. COHEN, S. S. 1947. Synthesis of bacterial virusesin infected cells. Cold Spring Harbor Symp.Quant. Biol. 12:35-49.

4. COHEN, S. S. 1948. Synthesis of bacterial viruses;synthesis of nucleic acid and protein in Escheri-chia coli infected with T2r+ bacteriophage. J.Biol. Chem. 174:281-293.

5. ERLANDER, S. R., H. KOFFLER, AND J. F. FOSTER.1960. Physical properties of flagellin fromProteus vulgaris, a study involving the applica-tion of the Archibald sedimentation principal.Arch. Biochem. Biophys. 90:139-153.

6. GRAY, P. H. H. 1926. A method of staining bac-terial flagella. J. Bacteriol. 12:273-274.

7. KERRIDGE, D. 1959. Synthesis of flagella byamino acid-requiring mutants of Salmoniellatyphimurium. J. Gen. Microbiol. 21:168-179.

8. KERRIDGE, D. 1961. The effect of environment on

the formation of bacterial flagella. Symp. Soc.Gen. Microbiol. 11:41-68.

9. KERRIDGE, D. 1963. Flagellar synthesis in Sal-monella typhimurium: the incorporation ofisotopically labelled amino acids into flagellin.J. Gen. Microbiol. 33:63-76.

10. KOFFLER, H. 1957. Protoplasmic differencesbetween mesophiles and thermophiles. Bac-teriol. Rev. 21:227-240.

11. LEIFSON, E. 1931. Development of flagella ongerminating spores. J. Bacteriol. 21:357-359.

12. MONOD, J., AND E. L. WOLLMAN. 1947. L'inhibi-tion de la croissance et de l'adaption enzy-matique chez les bacteries infectees par lebacteriophage. Ann. Inst. Pasteur 73:937-956.

13. MURRAY, R. G. E., AND A. BIRCH-ANDERSEN.1963. Specialized structure in the region of theflagella tuft in Spirillum serpens. Can. J. Micro-biol. 9:393-401.

14. QUADLING, C., AND B. A. D. STOCKER. 1961. Anenvironmentally-induced transition from flagel-lated to non-flagellated state in Salmonellatyphimurium: the fate of parental flagella atcell division. J. Gen. Microbiol. 28:257-270.

15. RENDI, R., AND S. OCHOA. 1962. Effect of chlor-amphenicol on protein synthesis in cell-freepreparations of Escherichia coli. J. Biol. Chem.273:3711-3713.

16. RINKER, J. N., AND H. KOFFLER. 1951. Prelimi-nary evidence that bacterial flagella are not"polysaccharide twirls." J. Bacteriol. 61:421-431.

17. STOCKER, B. A. D., AND J. C. CAMPBELL. 1959.The effect of non-lethal deflagellation onbacterial motility and observations on flagellarregeneration. J. Gen. Microbiol. 20:670-685.

18. WEIBULL, C. 1948. Some chemical and physico-chemical properties of Proteus vulgaris. Bio-chim. Biophys. Acta 2:351-361.

19. WEIBULL, C. 1949. Chemical and physico-chemi-cal properties of Proteus vulgaris and Bacillussubtilis. A comparison. Biochim. Biophys.Acta 3:378-382.

20. WEIBULL, C., AND A. TISELIUS. 1945. The acidhydrolysis of bacterial flagella. Arkiv KemiMineral. Geol. 20:3.

21. YEE, R. B., AND H. M. GEZON. 1963. Ribonucleicacid of chloramphenicol-treated Shigella flex-neri. J. Gen. Microbiol. 32:299-306.

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