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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.
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