Isolation and characterization of flagella and
flagellin proteins from the Thermoacidophilic
archaea Thermoplasma volcanium and
Sulfolobus shibatae.
D M Faguy, D P Bayley, A S Kostyukova, N A Thomas and K F
Jarrell
J. Bacteriol. 1996, 178(3):902.
These include:
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JOURNAL OF BACTERIOLOGY, Feb. 1996, p. 902–905
0021-9193/96/$04.0010
Copyright q 1996, American Society for Microbiology
Vol. 178, No. 3
Isolation and Characterization of Flagella and Flagellin
Proteins from the Thermoacidophilic Archaea
Thermoplasma volcanium and
Sulfolobus shibatae
DAVID M. FAGUY, DOUGLAS P. BAYLEY, ALLA S. KOSTYUKOVA,
NIKHIL A. THOMAS, AND KEN F. JARRELL*
Received 18 July 1995/Accepted 14 November 1995
Isolated flagellar filaments of Sulfolobus shibatae were 15 nm in diameter, and they were composed of two
major flagellins which have Mrs of 31,000 and 33,000 and which stained positively for glycoprotein. The flagellar filaments of Thermoplasma volcanium were 12 nm in diameter and were composed of one major flagellin
which has an Mr of 41,000 and which also stained positively for glycoprotein. N-terminal amino acid sequencing
indicated that 18 of the N-terminal 20 amino acid positions of the 41-kDa flagellin of T. volcanium were
identical to those of the Methanococcus voltae 31-kDa flagellin. Both flagellins of S. shibatae had identical amino
acid sequences for at least 23 of the N-terminal positions. This sequence was least similar to any of the
available archaeal flagellin sequences, consistent with the phylogenetic distance of S. shibatae from the other
archaea studied.
archaea is now available, these data are concentrated on mesophilic archaea. Little is known about the flagellar system in
thermophilic or thermoacidophilic archaea. Furthermore, no
reports have characterized the flagella from members of the
crenarcheota kingdom (which itself is exclusively thermophilic
[25]) within the archaea. The study of flagella from thermoacidophilic organisms is intrinsically important because of the
unusual stability of flagella under such extreme conditions as
well as for the insight into phylogenetic relationships within the
archaea.
(Portions of this work have been previously presented [5].)
Sulfolobus shibatae B12, obtained from D. W. Grogan (NASA
Jet Propulsion Laboratory, Pasadena, Calif.), was grown in
American Type Culture Collection culture medium 1256
(Deutsche Sammlung von Mikroorganismen und Zellkulturen
medium 88) at 728C and pH 2.0 according to the method of
Grogan (11). Thermoplasma volcanium (DSM 4299), obtained
from the Deutsche Sammlung von Mikroorganismen und
Zellkulturen, Braunschweig, Germany, was grown at 598C and
pH 2.0 as described by Segerer et al. (29).
Flagellar filaments were isolated from sheared cells and
from the culture supernatant with KBr gradients as previously
described (13, 30). Further purification of the filaments of T.
volcanium was required. Two flagellum-containing bands obtained after KBr centrifugation were collected, loaded into
centrifuge tubes containing 2 ml of 20% (wt/vol) sucrose overlayered with 5 ml of H2O, and centrifuged for 2 h at 90,000 3
g at 48C. The pellet was resuspended in T. volcanium mineral
medium and centrifuged for 6 min at 16,000 3 g at 48C in an
Eppendorf microcentrifuge. The supernatant was then centrifuged at ambient temperature in a Beckman Airfuge for 20
min at 162,500 3 g. The resulting pellet contained mainly
flagellar filaments, as determined by electron microscopy (2,
7). Flagellar filaments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) performed by the method of Laemmli (21), with a minigel system
being used. The gels were stained with a Coomassie brilliant
There have been a number of reports describing the flagella
from various members of the archaea (archaebacteria) (12).
Flagellar filaments have been characterized in species of the
genera Halobacterium (1), Methanococcus (15, 19), Methanospirillum (8, 30), Methanoculleus (17), Methanothermus (17),
and Natronobacterium (9). In all cases, the flagellar filaments
have been described as thin (10 to 14 nm) compared with
bacterial (eubacterial) flagellar filaments (20 to 24 nm) (24)
and are composed of multiple flagellins rather than the single
flagellin species common to most bacterial flagella (33). In
several cases, the component flagellins have been shown to be
glycosylated (2, 31), a feature not reported for bacterial flagellins. Furthermore, among the methanogens, the flagellar filaments composed of glycosylated flagellins were dissociated by
low concentrations of Triton X-100 while filaments from other
methanogens composed of nonglycosylated flagellins were not
dissociated by this treatment (7). Dissociation by detergent is
not a characteristic of bacterial flagella.
At the molecular level, archaeal flagellins are also quite
distinct from their bacterial counterparts. Short leader peptides have been identified on archaeal flagellins (15), while
bacterial flagellins are produced and exported from the cell
without the use of a leader peptide (23). Bacterial flagellins
have homologous regions at both the N terminus and C terminus, and these regions are known to be important in the
assembly of the flagellin into flagellar filaments (23, 33). In
marked contrast, none of the archaeal sequences described to
date have any similarity to the sequences of bacterial flagellins.
However, archaeal flagellin sequences do have extensive Nterminal homology among themselves (6, 16). Evidence that
archaeal flagellins may be related to the type IV pilin-transport
protein superfamily in bacteria has been presented (6).
Although some information on the flagellar system of the
* Corresponding author. Phone: (613) 545-2456. Fax: (613) 5456796. Electronic mail address: [email protected].
902
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Department of Microbiology and Immunology, Queen’s University,
Kingston, Ontario, Canada K7L 3N6
VOL. 178, 1996
NOTES
903
TABLE 1. Properties of flagella isolated from thermoacidophilic archaea
Organism
Optimum temp for
flagellum production (8C)
Flagellar filament
diam (nm)
Flagellin
Mr (103)
Glycosylated
flagellins
Triton X-100-sensitive
filaments
S. shibatae
T. volcanium
68
59
15
12
31 and 33
41
1
1
1
2
ples were found to be heavily contaminated after analysis by
SDS-PAGE. Therefore, these samples were further purified as
described above.
All previous reports of archaeal flagellar filaments have described diameters of 10 to 14 nm, and the flagellar filaments
from S. shibatae were only slightly wider (15 nm). However, the
ultrastructural appearance of the filaments of S. shibatae was
unusual (Fig. 1A). Generally, as observed with an electron
microscope, the surface morphology of archaeal flagella, and
certain bacterial flagella (33), is described as featureless (14).
This contrasts with, for example, Escherichia coli, for which
there are six filament surface morphotypes or patterns (33).
Unlike most archaeal flagella, the filaments of S. shibatae show
a well-defined subunit pattern when stained with uranyl acetate. Flagellum preparations of T. volcanium contained 12-nmdiameter filaments like those seen on whole cells but also, in
some cases, very thin (3- to 5-nm-diameter) filaments (Fig.
1B). The thinner filaments observed in the flagellum preparations of T. volcanium are likely to be a degradation stage, since
they are not observed on whole cells and they appear more
frequently in older preparations stored in mineral medium at
low pH. This is similar to the case with the haloalkaliphilic
archaeon Natronobacterium magadii (9). Thinner than usual
filaments have also been found on Methanothermus fervidus
(unpublished data). These small diameters (3 to 7 nm) are
more often associated with pili than flagella, although, to our
knowledge, pili have not been reported for T. volcanium or M.
fervidus.
Isolated flagellar filaments of S. shibatae were composed of
at least two flagellins (with Mrs of 31,000 and 33,000), as
determined by SDS-PAGE (Fig. 2). These flagellins appeared
to be glycosylated on the basis of both the thymol-sulfuric acid
stain (Fig. 2) and the PAS reagent stain (data not shown). The
minor higher-molecular-weight bands observed in Fig. 2, some
FIG. 1. Negative stains (1.5% uranyl acetate) of flagellar filament preparations of S. shibatae (A) and T. volcanium (B). The filament preparation of T. volcanium
has a mixture of thick (12-nm) and thin (3- to 5-nm) filaments. Bar, 100 nm.
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blue G250-perchloric acid solution (0.04% Coomassie brilliant
blue G250 in 3.5% perchloric acid [28]). The gel was microwaved in the stain for about 20 s (so that the stain became hot
but did not boil). The gel was left in the hot stain with shaking
for 2 min, and then it was destained by microwaving it in water
for 2 min. The protein bands were visible at this point, but the
gels were shaken in the hot water for an additional 15 min
before photography. The protein molecular weight markers
(Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada)
used were lysozyme (Mr 5 14,400), soybean trypsin inhibitor
(Mr 5 21,500), carbonic anhydrase (Mr 5 31,000), ovalbumin
(Mr 5 42,700), bovine serum albumin (Mr 5 66,200), and
phosphorylase b (Mr 5 97,400). Glycosylation of the flagellin
proteins was tested by either the thymol-sulfuric acid stain (27)
or the periodic acid-Schiff (PAS) stain (4) on samples separated on SDS-PAGE gels.
Bands selected for sequencing were transferred to polyvinylidene difluoride membrane (Bio-Rad) by the method of
Towbin et al. (32) and sequenced with a pulsed-liquid phase
sequencer (Applied Biosystems). Sequencing was performed
by the Core Facility for DNA/Protein Chemistry, Department
of Biochemistry, Queen’s University, Kingston, Ontario, Canada.
This paper describes the first characterization of flagella
from thermoacidophilic archaea (Table 1). From a phylogenetic standpoint, the flagella isolated from S. shibatae represent the first such report from a member of the crenarcheota
kingdom within the domain archaea.
Flagella were isolated from sheared S. shibatae and T. volcanium cells and culture supernatants by differential centrifugation and banding in KBr gradients. In the case of T. volcanium, two distinct bands were observed after KBr gradient
centrifugation. Both contained a large number of flagellar filaments when examined by electron microscopy, but both sam-
904
NOTES
of which stained positively for glycosylation, could represent
additional flagellin subunits, flagellin with different degrees of
glycosylation, or simply contaminating proteins. Flagellar filament preparations of T. volcanium contained one major band
with an Mr of 41,000 and several minor higher-molecularweight bands (Fig. 2). This is the first report of an archaeal
flagellum preparation which is composed of a single major
flagellin band. Of course, it is still possible that there are
multiple flagellins, each with Mrs of 41,000, that have identical
N-terminal sequences. This major band and a couple of the
high-molecular-weight minor bands (which could represent aggregated denatured flagellins) stained positively with the PAS
stain, indicating that they are likely glycoproteins (Fig. 2). The
41,000-Mr band could also be detected in whole-cell protein
preparations with the PAS stain (data not shown).
Faguy et al. (7) showed a correlation for methanogens between glycosylated flagellins (as determined by thymol-sulfuric
acid staining) and the sensitivity of the flagellar filaments to the
nonionic detergent Triton X-100. For these studies, a 50-ml
volume of isolated flagellar filaments (450 mg of protein per ml
of H2O) was mixed with 50 ml of 1% (vol/vol) Triton X-100 and
incubated for 1 h at 378C (7) and then assayed for intact
filaments by electron microscopy. The behavior of the flagellar
filaments of S. shibatae was consistent with our previous observations of methanogen flagellar filaments, with the glycosylated flagellins forming filaments which were dissociated by
Triton X-100. However, the T. volcanium flagellar filaments
were an exception in that they were not dissociated by Triton
X-100 in spite of apparently being composed of glycosylated
flagellins. Recent studies have shown that other nonmethanogenic archaea, such as Pyrococcus furiosus (18) and Halobacterium halobium (20), also have glycosylated flagellins, but
their flagellar filaments were not dissociated by Triton X-100.
The N-terminal region in archaeal flagellins is likely to be
important in the assembly of the flagellar filament. This region
is not only highly conserved among different archaeal species
but is also the most conserved region among individual gene
products within the flagellin multigene families of Methanococcus voltae and H. halobium (10, 15). The N-terminal amino
acid sequences of the 31,000- and 33,000-Mr flagellins from S.
shibatae and the 41,000-Mr flagellin from T. volcanium were
determined (Fig. 3A). The two S. shibatae flagellin sequences
were identical for the N-terminal 23 amino acids. Furthermore,
the sequences of the S. shibatae and Methanospirillum hungatei
flagellins were identical at 12 of the first 23 N-terminal amino
acid positions, and overall, 20 of 23 residues were identical or
similar by the PALIGN program of PC/GENE. The N-terminal sequences of the 31,000-Mr flagellin of M. voltae and the
flagellins of S. shibatae showed 9 identical residues out of 23,
with 15 identical or similar residues out of 23 according to the
PALIGN program. The N-terminal sequences of the 41,000-Mr
flagellin from T. volcanium and the 31,000-Mr M. voltae flagellin were identical at 18 consecutive positions (from amino acids
3 to 20), and the sequences of the T. volcanium flagellin and M.
hungatei flagellin were identical at 10 of 20 positions (Fig. 3A).
Since the sequences of only the N-terminal 20 amino acids
were determined, the identity of the sequences for the T.
volcanium and M. voltae flagellins may extend even farther.
The N-terminal sequences of the flagellins from T. volcanium
and S. shibatae do not have any similarity to those of bacterial
flagellins.
The strong sequence similarity of the flagellins of T. volcanium and M. voltae is consistent with the 16S rRNA sequence
data which place Thermoplasma spp. as close relatives of methanogens and halophiles in spite of their very different physiologies (25). The N-terminal sequences of the S. shibatae flagellins are the least similar compared with the previously reported
sequences, which are all from methanogens and halophiles
(members of the euryarchaeota kingdom of the domain archaea). This finding is again consistent with 16S rRNA sequence data which place Sulfolobus spp. in the crenarchaeota
kingdom, the second of the two major lineages of the archaea
(34). Thus, the flagellin N-terminal region is conserved in both
subdivisions of the archaea and likely is common to all archaeal flagellins.
With the data presented in this paper, 22 N-terminal sequences of archaeal flagellins from 10 different organisms are
FIG. 3. (A) N-terminal amino acid sequences of S. shibatae and T. volcanium
flagellins aligned with the M. voltae 31,000-Mr flagellin sequence, the 24,000-,
25,000-, and 35,000-Mr flagellin amino acid sequence of M. hungatei, and the
deduced amino acid sequence of the H. halobium flaA gene. The halophile
sequence is aligned on the basis of the presumed cleavage site of a leader peptide
(16). The M. hungatei sequence was reported by Kalmokoff et al. (16). The M.
voltae sequence was reported by Kalmokoff and Jarrell (15), and the H. halobium
sequence was reported by Gerl and Sumper (10). Identical amino acids in the
same position for all the flagellins are shaded dark, while similar amino acids
(according to PALIGN) are lightly shaded. (B) Archaeal flagellin consensus
pattern. The standard International Union of Pure and Applied Chemistry oneletter codes for the amino acids are used. Ambiguities are indicated by listing the
acceptable amino acids for a given position vertically. Each amino acid position
in the pattern is separated from its neighbors by a hyphen. Where there is more
than one amino acid for a position, the choices are listed in decreasing frequency
of occurrence. The pattern is based on 22 flagellin sequences from 10 different
organisms (M. voltae, Methanococcus jannaschii, Methanococcus deltae, Methanococcus vannielii, Methanoculleus marisnigri, M. hungatei GP1 and JF1, H. halobium, T. volcanium, and S. shibatae).
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FIG. 2. SDS-PAGE of flagellar filament preparations of S. shibatae and T.
volcanium. Lane 1, Coomassie brilliant blue stain of S. shibatae flagellar filaments; lane 2, thymol-sulfuric acid stain of S. shibatae flagellar filaments; lane 3,
Coomassie brilliant blue stain of T. volcanium flagellar filaments; lane 4, PAS
stain of T. volcanium flagellar filaments. Molecular weight markers are shown in
lane M.
J. BACTERIOL.
VOL. 178, 1996
NOTES
This research was supported by an operating grant from the Natural
Sciences and Engineering Research Council of Canada (to K.F.J.).
D.M.F. and D.P.B. were recipients of Ontario Graduate Scholarships.
We thank the Human Frontier Science Program for a short-term
fellowship which enabled A.S.K. to work in the laboratory of K.F.J.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
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known. In several other cases, such as those involving H. halobium (10), Natronobacterium pharaonis and N. magadii (26),
and M. fervidus (unpublished observations), the N-terminal
sequences of the flagellins could not be obtained, indicating
that the proteins were likely blocked. The available sequences
include ones from members of both the crenarchaeota and
euryarchaeota. Because of the high sequence conservation, it is
possible to formulate a consensus sequence for archaeal flagellins for amino acid positions 3 to 23 (Fig. 3B). Over this stretch
of 21 amino acids, 6 positions are invariant in all flagellin
sequences. Another 10 positions have 1 of only 2 different
amino acids, while in the remaining 5 positions, 1 of only 3
amino acid choices is found.
On the basis of 16S rRNA sequence data and other properties, Woese et al. (34) proposed that all organisms be
grouped in three domains: bacteria, eucarya, and archaea, with
the last including methanogens, extreme halophiles, and the
genera Thermoplasma and Sulfolobus. Another proposal, based
on sequence data for EF-1a and EF-2 and other considerations, groups the eubacteria, methanogens, halophiles, and
genus Thermoplasma into a superkingdom called parkaryotes
and the eucaryotes and the eocytes (including the genus Sulfolobus) into the superkingdom called karyotes (22). The high
degree of conservation in the flagellin sequence of S. shibatae
and the methanogen and halophile flagellin sequences supports the distinct nature of the archaea as proposed by Woese
et al. (34) and is inconsistent with the alternative hypothesis.
Recently, the flagellin sequence (GenBank accession number
U17575) was obtained for Aquifex pyrophilus, a hyperthermophilic bacterium which represents the deepest branch of the
bacteria (3), and it has a definite bacterial flagellin sequence.
Thus, the flagellin sequence can be said to be a positive defining characteristic of the archaea, similar to the isopranyl lipids
and uniquely modified nucleotides in tRNA (35).
905