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Bacterial Flagella: Structure, Function, and Composition, Study Guides, Projects, Research of Bacteriology

An in-depth analysis of bacterial flagella, focusing on their structure, function, and composition. Topics covered include the basal structure, hook portion, chemical composition of flagellar hooks, and the role of hydrophobic bonds in flagellin-flagellin interactions. The document also discusses the possible mechanism of flagellar movement and the proposed hypothesis that the energy for motility derives from an ATPase activity located at or near the basal region.

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bg1
Doll.
ZOO^.,
39:
111-118,
1072
Afti
del
XL
Convegno
del1'U.Z.I.
STRUCTURE
AND
FUNCTION OF BACTERIAL FLAGELLA
FRANCESCO
SALA
Laboratorio
di
Genctica
Biochiniica
ed
Evoluzionisticn
del
C.N.R.,
Via
S.
Epi-
fanio,
14,
27100
Pnvin (Italy)
The investigation
of
bacterial flagella is under may in several labo-
ratories. Programs aim
at
thc solution
of
great biological problcms such
as
thc moleciilar aspects of the structure and biosynthcsis
of
a
cellular
or-
ganelle, the conversion
of
chemical cncrgy into motion, the existence and
nature
of
specific mechanisms controlling cellular movement and the
evo-
lution
of
contractile proteins.
In sizc and structure bacterial flagella differ considerably from the
flagclla
of
eucariotic organisms. Bacterial flagella consist
of
a
single mi-
crotubulc
120-200
A
in diameter (BURGE and
DRAPER,
1971
;
SMITH
and
KOFFLER,
1071)
while on nlgal
or
sperm cell flagellum is
2000
A
in dia-
meter
(HorKIss,
1070)
and shows the typical
a0+2n
pattern of longi-
tudinal fibrils (essentially
n
pair
of
single tubules surrounded by nine px-
rallel doublet tubulcs) inclosed by
a
cylindrical matrix with
n
less clcar
structure.
The bacterial flagellum consists
of
three morpliologicnlly distint
parts
:
n
basal structure, closely associated with the cytoplasmic mem-
brane,
R
proximal hook portion and
a
long filament, the most prominent
fcatitrc
of
the flagellum (fig.
1).
THE
DASAL
STRUCTURE
Electron microscopy showed thnt flagella pcnetrnte the cell
wall
and nrc tightly associated with the cytoplasmic membrane
(ADRAM
et
al.,
10G5).
A
morphologically distinct basal structure has been observed by
electron microscope analysis of diffcrcnt bacteria
(ABRA~X
et
al.,
1905,
1DGG).
However major dificultcs arc still to be overcome in' order to purify
and charactcrizc sucha particle.
Obscrvation of basal structures is almost impossible in electron
micrographs of intact cells nnd
is
better achcvcd in the envelope
of
lysed
pf3
pf4
pf5
pf8
pf9
pfa

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Doll. ZOO^., 39: 111-118, 1072

Afti del XL Convegno del1'U.Z.I.

STRUCTURE AND FUNCTION OF BACTERIAL FLAGELLA

FRANCESCO SALA

Laboratorio di Genctica Biochiniica ed Evoluzionisticn del C.N.R.,Via S. Epi-

fanio, 14, 27100 Pnvin (Italy)

The investigation of bacterial flagella is under may in several labo-

ratories. Programs aim at thc solution of great biological problcms such

as thc moleciilar aspects of the structure and biosynthcsis of a cellular or-

ganelle, the conversion of chemical cncrgy into motion, the existence and

nature of specific mechanisms controlling cellular movement and the evo-

lution of contractile proteins.

I n sizc and structure bacterial flagella differ considerably from the

flagclla of eucariotic organisms. Bacterial flagella consist of a single mi-

crotubulc 120-200 A in diameter (BURGEand DRAPER, 1971 ; SMITH and KOFFLER, 1071) while on nlgal or sperm cell flagellum is 2000 A in dia-

meter (HorKIss, 1070) and shows the typical a0+2n pattern of longi-

tudinal fibrils (essentially n pair of single tubules surrounded by nine px-

rallel doublet tubulcs) inclosed by a cylindrical matrix with n less clcar

structure.

The bacterial flagellum consists of three morpliologicnlly distint

parts : n basal structure, closely associated with the cytoplasmic mem-

brane, R proximal hook portion and a long filament, the most prominent

fcatitrc of the flagellum (fig. 1).

THE DASAL STRUCTURE

Electron microscopy showed thnt flagella pcnetrnte the cell wall

and nrc tightly associated with the cytoplasmic membrane (ADRAM et al.,

10G5). A morphologically distinct basal structure has been observed by

electron microscope analysis of diffcrcnt bacteria ( A B R A ~ X et al., 1905,

1DGG). However major dificultcs arc still to be overcome in' order to purify

and charactcrizc sucha particle.

Obscrvation of basal structures is almost impossible in electron

micrographs of intact cells nnd is better achcvcd in the envelope of lysed

112

cells (figs. 1 and 2). Consequently description of size and shapc vary con-

siderably since, most likely, the structure itself deteriorates under the

unfavourablc conditions for specimen preparation. This appears to be the

reason why no rigorous dcscription of the basal structure of various Ba-

cillus species has been given yct (SMITH and KOFFLER,1071).

ABBAM et al. (1065) observed that in Profeus vulguris some flagella

originate from spherical structures the diamctcr of which is similar to or

only slightly larger (110-140 A) than the diameter of the filament portion.

of the flagellum. However it was observed that in the same cell most flagella

originate in largcr structures (200-700 A in dinmeter). Thc speculation

was that the larger bodies may not be real structural entities but perhaps

artefacts resulting from the folding of the cell membrane on the smaller

spherical basal body. On the other hand, subscquently ABRAM(1008)

obscricd that cells of Profeus vulgaris damaged by intcraction with Bdello-

vibrio bacleriovorics have flagella which originate in spherical bodies of

uniform size (800430A in diameter). Such bodies appear to enclose a disc, or a double disc (160-180 A in diameter and 40-GO A thick). REMSEN et

al. (10G8) described the basal structure of Eclothiorhodospira tnobilis as

consisting of a pair of discs with a diameter of 200-250 A and connected

by a thin rod.

At the present our knowledge concerning the function of the basal

structure is limitcd to several hypothesis. It seems reasonable to postulate

one or more of the following functions : simple anchoring element, site of

synthesis of flagellin and of its polymerization into the flagellum, site

responsible for the rotation of the flagellum, site generating the encrgy

and/or regulating the movement of flagella (SMITE and KOFFLEB,1071).

In any case the close association of the basal structure with the cyto-

plasmic membrane might bear a functional sigxiificancc.

THE HOOK PORTION

Morphology and finc structure differentiate the portion of the fi- lament which connects the basal structure to the proximal end of the fi-

lament (fip. 2 ). In llncillus putnilus such portion is hook shaped (u the

hook D) and is 580-740 A in length and 120-150 A in diameter (ABRAM

et ul., 1070). Exposure of flagella from cells of B. pumilus to various relatively

mild chemical and physical treatments (acid, alcohol, heat) results in the

disintegration of the flagellar filaments, whcrcas the hook portion remains

intact (ABRAM et al., 1970). Under certain conditions the flagella are made

Fig. 1 - A ccll of Proleits culgcrris nrgntivcly stuiticd with potnssiuin pliospl~otungstntc ( x 65,000). (Courtcsy of Ilr. 11. I<OFFLEI~ and D. A D n. u I ).

Fig. 2 - I.‘lagclla ussocintccl with frngnirnts of tlic cytoplasriiic iiiciiibrnnc in plingc- Iyscd cclls of Ilnei//us slecrrol/~er.,,roj)/~ilits 194. .\lost of tlic flagclln originntc in splicricnl or disc s l i a l d bodies, tlic bnsnl structures. A prosiiiinl Iiook- slinpctl region, connecting tlic bnsnl structurc to tlic filnmrnt, is clcarly visible. Scgntivcly stninctl with potnssiunl I)Iiosl)liotuligstntc ( x 85,000). (Courtcsy of Dr. D. Aniuii, A. 1 :. \‘.-rr~ii and 11. liour~cn).

The study of the common aspects of thc primary structurc of various fla-

gcllins is expcctcd to help in the rccognition of the portions of the molc-

cule of most functional and structural relevance. Peptide maps, produced

after tryptic digestion of flagellins from different bactcrin have been found

to differ (ABRON,1966; 31. FARQUIIAR and 11. KOFFLER, personal com-

munication). The amino-acid composition of flagcllin from diffcrcnt bac:

teria is known (SMITE and KOFFLER,1071) : each flagcllin has a unique

composition exccpt that some intercsting properties arc in common : all

flagellins so far examined contain no cysteinc or tryptophan residues, mhilc

tyrosine, proline and hystidinc are present in low amount. Metliionine has

been shown to be thc NH, - terminal amino acid in flagellins from different

Bacillus spp. (SALA and KOFFLER,1067; J. STENESII and H. KOFFLER,

personal communication) while alanine is the NH, - tcrminal amino acid

in P. vulgutis (CHANGE et at., 1960). However the functional and structural

implications of the .established characteristics of the primary structure of

bacterial flagella arc not yet understood.

KLEINel al. (1960), have shown that flagellin isolated from various

strains of B. puniilus contains 2 1 3 2 % of a-helix. Furthermore they have

demonstrated that the polymerization of flagellin into flagclla is accom-

panicd by conformational changes : the structure of the molccule is essen-

tially unfolded at pII2 while it becomes helical in thc range of pH4 to 10.

The self assembly of flagellin into flagellar filaments is optimal at pIF 5,

(ABRAM and KOFFLER,1064), thus suggesting that the reaggregation pro-

cess requires the flngellin molcculc to have thc conformation characteristics

of neutral pH values. Furthermore this indicates that the information for

thc three-dimensional structure of flagcllin is encodcd in its primary

structure. Indeed this mas confirmed by the finding that flagellin is ca-

pablc of self-assembly into flagellar filanicnts also when obtained by de

novo synthesis in a ccll-free system (SAM el al., 19G8 ; GAERTNER et al., 1968).

A furthcr important aspect of the chemistry of flagellin is the nature

of the forces that hold the flagellin molecules together within the flagellum.

The absence of cysteinc from all the flagellins so far examined rule out disul-

fide bonds. The rolc of hydrophobic bonds in flagellin-flagcllin interactions

appears significnnt (KOFFLER,1057; BlAnTxNEz el nl., 1067; SMITE and

KOFFLER,1071) :agents such as urea, detergents and guanidine hydrochlo-

ride, afkcting hydrophobic bonds, largely disintegrate flagella. On the

0 t h hand salts, which arc known to stabilizc hydrophobic bonds by de-

creasing the solubility of non-polar groups in the more polar solvent also

stabilize flagellar filaments and causc aggregation of flagellin into flagella.

An insight into the bonds involved in intcrmolccular interactions in

115

flagellin may be gained through thc use of chemically or genetically mo-

dified flagellin. A promising approach is that of introducing selective chc-

mica1 modifications in the side chains of tlic amino acid residucs of the

flagellin molecule. Tctranithromethnne is one of such reagents. I n its

presence tyrosine is modified into nitrotyrosinc :tyrosine is one of tlic most

hydrophobic amino acids and in globular proteins has becn shown to

participate in intra- as wcll as intcr-molecular hydrophobic interactions.

blodification of flagellin of Bncillus stearothermopliilus with tctranithro-

methane allowcd YARDROUCXIand KOFFLER (1071) to establish that at

least one of the tyrosine residues reacting when free flagcllin is nitrated

is involved in flagellin-flagcllin interactions in the flagellum.

The specific geometry in which subunits are arrangcd is a further

problem in the study of flagclla. The problem has becn ap roached with

the aid of electron microscopy, X-ray and optical diffraction. Electron

micrographs of flagellar filaments have shown that the ccntcr of the fla-

gellum is either empty or consists of different material than flagellin (KER-

RIDGE cf al., 1062 ; BURGEand DRAPER, 1071). Thc malls of the tube arc

composed of flagellin molccules arranged in a rcgulnr fashion. AsnAx D.

and H. KOFFLER(personal communication) have shown that under appro-

priate experimental conditions (mild acid treatment) flngcllar filaments of

B. pumilus unravel into six fibers with cach fiber composcd of single strands

of ovoid subunits. Under alkaline conditions thc same filaments release

transverse slices consisting of six exagonally arranged ovoid subunits

surrounding an hollow center. If artefacts arc ruled out, such findings

further support the conclusion that tlic flagellar filament of B. pumilus

is composed of ovoid flagellin moleculcs regularly arranged around an

hollow center.

P.

MECHANISM OF MOVENENT OF FLAGELLA

Tlic basic questions concerning flagellar movcmcnt arc still unanswe-

red. No conclusive evidence has yet been given about the contractile pro-

perty of flagellar filaments.

Basically bacterial flagella and muscles differ a t least in two respects :

first the muscle is a two component system in which contraction is strictly

dependent on the interactions bctmeen two proteins : actin and myosin,

whilc flagella are defined as a one component systcm. Furthermore muscular

fibers, but not flagella, clearly show an ATPasc octivity on which contraction

is dependent. NEWTONand KERRIDGE (1005) suggested that in order for

filaments to contract each flagellin molecule must posscss both contractile

117

whether thc two proteins occur in tlic same or in different filaments of the

same cell has recently been solvcd by OZLER et al. (1071) who dcrnonstrated

that both flagcllin A and 13 are located within the samc flagellum. The

experimental evidence was that all flagellar filaments arc coated on the

entire length when treated with specific anti-A or anti-13 scra.

I n conclusion, while research has produced a good deal of evidence

on the physical-chemical nature of the protein componcnts of bacterial

flagella, much remains to be learnt about the mechanism of movement

of flagella.

Furthcr questions concern the existence, nature and location of

specific sensory mechnnisms, controlling the movement of flagella. For

instance it is well documented that bacterial cells move toward and away

from a given chemical environment (ARMSTROXC and ADLER, 10G0). In every

case, the nature of tlic specific recognition sitcs as well as the mechanism

by which tlic signal from the receptor is transmitted to the flagella is

unknown.

BIBLIOGRAPHY

ABRAYD., 1908 - Slrucfural features of fhe sifes of origin of flagella offached lo cells and

membrane fragmenfs of Protcus vulgaris. Bact. Proc., p. 80.

ABRA~ID. nnd KOFFLERII., 1004 - In vitro formation of flagella-like filamenfx and ofher

sfrucfutes from flagellin. J. 3101. Biol., 9 : 1GS-185.

ADRAJID.; KOFFLERII. nnd V A ~ A.R E., 10G5 - Basal sfrucfure and aflachrnenf

of flagella in cells of Proteus vulgaris. J. Bact., DO: 1337-1854.

ADRAXD., BfiTcrrEx J. R., KOFFLER 11. nnd VAT~ERA. E., 1070 - Diflerentiafion ltcifhin

the Laclerial flagelluni and isolafion of fhe prorimal hook. J. Bact., 101 : 250-201.

ABRAHD., VAITER A. E. and KOFFLERXI., 1000 - rfttachmcnf and slrucluralfeafures

of flagella of certain bacilli. J. Dnct., 91 : 2045-2068.

ARROX11. E., l0GG - X S. Thcsis: Purduc University, Lafnyctte, Indiana, U.S.A.

ARslSTRONG J. B. and ADLERJ., 1000 - Localion of genes for molilily and chemofaris on

the Escherichia coli genefic map. J. Bact., 97: 150-101.

DURGER. E. and DRAPERJ. C., 1071 - Sfrucfure oJ bucferial flagella from Salmonella

typliimurium : eflecfs of hgdrafion and some sfains on fhe equaforial A’-ray diflracfion

paflerns of cad films. J. i’tfol. DioI., 56 : 21-34.

Camo J. Y.,B n o m D. M. and G u z m A. N., lQG0 - Characlerfzafion of the subunifs

01 the j?ugelIa of Protcus vulgnris. J. Biol. Clicm., 244 : 6106-5200.

DOETSCUR. N.,19GO - Some speculations accounting for fhe movement of bacferialflagella.

J. Theor. Biol., 1 1 : 411417.

GAERTNER E II., 1900 - Ph. D. Thesis : Purduc University, Lnfnyette, Indiana, U.S.A.

GAERTNERP. XI., KOFFLER 11. and SALAF., 1968 - Biosynfhesis offlagellin. In : Sum-

posiuw on Jbrow proleins. Ed. Crewther 1Y. G., Aust. Amd. Scl., Cnnberra, p. 99-

118

IIormNs J. M., 1970 - Subeidiary wmponcnfs o j theflagella of Chlamydomonas reinhardii.

J. Cell sci., 7 : 823-880.

KERRIDGED., HORNER. JV. nnd GLAUERTA. hf., 1062 - Structural components offla-

gella jrom Salmonella typhirnurium. J. Mol. Biol., 4 : 227-238.

I ~ I D.,N FOSTERJ. F. and KOFFLER H., 1969 - Changes in polarimctric paramefns

associafed with lhe polymerization ojflagellin i n f oflagellar $laments. Dioch. Biophys. Res. Comm., 36: 844-850.

KOBAYASUXT., RINKERJ. N. and KOFFLER H., 1050 - Purification and chemical pro-

perlies o j flagellin. Arch. Biochern. Biophys., 84 : 842-3G2.

KOFFLERH., 1957 - Protoplasmic diflerenccs behceen mesophilo and thermophiles. Bact.

Rev., 21: 227-240.

B ~ R T I X E ZR. J., BROWN D. M. and GLAZERA. N., 10G7 - Thejormation of bacferid$u-

gella. I I I. Characterization o j the flagella o j Bacillus subtilis and Spirillum serpcns.

hIITcnEN J. R., SMITU R. W. and KOFFLER H., 1070 - The nature of bacterial flagellar

hooks. J. Cell. Diol., 4 7 : 1423.

NEWION B. A. and I ~ R R I D G E D., l9G5 - Flagellar and ciliary niooemenl in microor-

ganisms. Syrnp. SOC.Gen. Microbiol., 1 G : 220-240.

OILER L., KAKAF., SMITER. W. and KOFFLERII., 1071 - The presence ojjlagellins A

and D in the jlageIlum of Bacillus pumllus 101. I3act. Proc., 23 : 27.

REXSEN C. C., WATSONS. IV., WATERBURYJ. B. and TRUPERII. G., 1008 - Fine stru-

cfure o j Ectothiorliodospira rnobilis Pclsh. J. Bact., 93 : 2374-2302.

SAM F., GAERTNERF. II. and KOFFLER H., 1968 - The synthesis in vitro ojflagellin

by a cell-jrce eztracf jron, Bacillus purnilus. Giorn. Bot. Ital.. 109 : 327-335.

SALAF. and KOFFLERII., lOG7 - Enzymic synthesis ojjlagellin. Fed. Proc.. 26 : 812.

SMITI~R. \V. nnd KOFFLER II., 1060 - Sel/-aesembly o j bacterialflagellar~lamen&. Abstr.

3rd Int. Biophys. Congr., Cambridge, p. 15.

SJUTU R. \V. nnd KOFFLER II., 1971 - Dacterial flogella. I n : Adv. Nierob. Plrysiol.

Ed. Rose A. H. nnd Wilkinson J. F., Amd. Press. N.Y., 8 : 219-339.

SULLXVAN A., Bux J., SUZUSIH. and SXITER. W., l0G0 - Possible phase oariafion in

Bacillus purnilus. Uact. Proc., p. 30.

YARBROUGEL. R. and KOFFLER H., 1071 - hlodification ojflagellin a n d f l a g e l l a r $ h m e ~

/rom cells o j Bacillus stearotherrnophilus 2184 by ktranilromethane. (Manuscript

In preparation).

J. m i. Biol., 2 8 : 45-51.