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Journal of Bacteriology, March 2001, p. 1655-1662, Vol. 183, No. 5
Department of Molecular Biophysics and
Biochemistry, Yale University, New Haven, Connecticut
06520-8114,1 and Izumi Campus, Meiji
University, Suginami, Tokyo 168-0064, Japan2
Received 9 October 2000/Accepted 6 December 2000
The MS ring of the flagellar basal body of Salmonella
is an integral membrane structure consisting of about 26 subunits of a
61-kDa protein, FliF. Out of many nonflagellate fliF
mutants tested, three gave rise to intergenic suppressors in flagellar region II. The pseudorevertants swarmed, though poorly; this partial recovery of motile function was shown to be due to partial recovery of
export function and flagellar assembly. The three parental mutants were
all found to carry the same mutation, a six-base deletion corresponding
to loss of Ala-174 and Ser-175 in the predicted periplasmic domain of
the FliF protein. The 19 intergenic suppressors identified all lay in
flhA, and they consisted of 10 independent examples at the
nucleotide level or 9 at the amino acid level. Since two of the nine
corresponded to different substitutions at the same amino acid
position, only eight positions in the FlhA protein have given rise to
suppressors. Thus, FliF-FlhA intergenic suppression is a fairly rare
event. FlhA is a component of the flagellar protein export apparatus,
with an integral membrane domain encompassing the N-terminal half of
the sequence and a cytoplasmic C-terminal domain. All of the
suppressing mutations lay within the integral membrane domain. These
mutations, when placed in a wild-type fliF background, had
no mutant phenotype. In the fliF mutant background, mutant
FlhA was dominant, yielding a pseudorevertant phenotype. Wild-type FlhA
did not exert significant negative dominance in the pseudorevertant
background, indicating that it does not compete effectively with mutant
FlhA for interaction with mutant FliF. Mutant FliF was partially
dominant over wild-type FliF in both the wild-type and second-site FlhA
backgrounds. Membrane fractionation experiments indicated that the
fliF mutation, though preventing export, was mild enough to
permit assembly of the MS ring itself, and also assembly of the
cytoplasmic C ring onto the MS ring. The data from this study provide
genetic support for a model in which at least the FlhA component of the
export apparatus physically interacts with the MS ring within which it is housed.
The MS ring of the flagellar basal
body of Salmonella is usually thought of in terms of its
role in motor function: as a mounting plate for the rotor element of
the motor/switch, also known as the cytoplasmic C ring. However, with
increasing attention being paid to the process of flagellar protein
export, which occurs by a type III pathway (7) and entails
delivery of the export substrates into the lumen of the nascent
structure, a second role for the MS ring is emerging, namely, as the
structure that houses the membrane components of the export apparatus.
The MS ring has a complex appearance, with two rings (M and S) and a
collar projecting beyond the cytoplasmic membrane into the periplasmic
space, yet it is constructed from subunits of a single 61-kDa protein,
FliF. It is estimated that there are about 26 subunits arranged as an
annulus that has a central pore about 10 nm in diameter
(10). The export apparatus has six integral membrane
components (FlhA, FlhB, FliO, FliP, FliQ, and FliR) in addition to some
soluble components (FliH, the ATPase FliI, and putative chaperones
FliJ, FlgN, FliS, and FliT) (1, 2, 4, 16, 18, 19, 22). We
have argued (2, 15) that the logical location for the
membrane components of the export apparatus is in a patch of
specialized membrane within the core of the MS ring, so the apparatus
can deliver its substrates into the lumen. Thus far, two of these
components (FliP and FliR) have been shown to be associated with the
basal body (2); in the case of FliR, immunoelectron
microscopy positioned it in the vicinity of the cytoplasmic face of the
MS ring.
If the model of the MS ring enclosing the export apparatus is correct,
the question arises whether there are specific interactions between the
two structures, or whether the export apparatus is better thought of as
a floating island. This report describes a number of examples where a
specific FliF mutation can be suppressed by mutations in an integral
membrane component of the export apparatus, FlhA, suggesting that the
MS ring and the export apparatus do in fact interact.
Bacterial strains, plasmids, and media.
The fliF
parental mutant strains are derived from SJW1103 (34). The
pseudorevertants derived from these fliF mutants were mapped
by P22-mediated transduction to flhA in flagellar region II.
The characteristics of the parental strains and their pseudorevertants are listed in Table 1. Chromosomal DNA
from the mutant and pseudorevertant strains was prepared by the method
of Woo et al. (32). Plasmids containing the
fliF mutant insert or the flhA isolated second site mutant inserts were cloned into pTrc99A1de4 (2),
using NdeI and BamHI sites created by PCR with
mutant chromosomal DNA as target.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.5.1655-1662.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Intergenic Suppression between the Flagellar MS
Ring Protein FliF of Salmonella and FlhA, a Membrane
Component of Its Export Apparatus
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains and plasmids used in this study
DNA techniques. PCR and cloning were performed as described previously (18) except that the Taq polymerase was from Qiagen (Valencia, Calif.). DNA sequencing was carried out with the modified T7 DNA polymerase Sequenase version 2.0 (USB Corp., Cleveland, Ohio) and various primers synthesized using an ABI model 392 DNA-RNA synthesizer (Perkin-Elmer, Foster City, Calif.).
Preparation of soluble and membrane protein fractions. The cells were inoculated into 100 ml of Luria broth and grown overnight at 37°C with shaking. After collection by centrifugation, cells were resuspended in 5 ml of 10 mM Tris-HCl (pH 8.0)-200 mM dithiothreitol and sonicated (Branson model 250 Sonifier, Danbury, Conn.). The cell lysates were centrifuged (13,000 × g, 10 min, 4°C) to pellet undisrupted cells; then the cell lysates were centrifuged at 130,000 × g for 1 h. The supernatant and pellet fractions, which contained the soluble proteins and membrane proteins, respectively, were collected separately.
Preparation of periplasmic and culture supernatant fractions. The periplasmic and culture supernatant fractions from SJW1103 (wild type), SJW2706 (fliF), MM0608 (fliF flhA), and SJW156 (flgD) were prepared as described elsewhere (18).
Preparation of hook-basal bodies. Hook-basal bodies were prepared from SJW1364 (flhA) carrying pMM106, a pET19b-based plasmid encoding N-His-FLAG-FlhA, as described by Fan et al. (2).
Antibodies. Monoclonal anti-FliF antibody and polyclonal anti-FlgD antibody are described in references 5 and 23, respectively. Polyclonal antibodies against FliG and FliN were a gift from K. Oosawa and S.-I Aizawa, Teikyo University, Japan. Anti-FLAG M2 monoclonal antibody was obtained from Sigma (St. Louis, Mo.). Antibodies were detected using an ECL (enhanced chemiluminescence) immunoblotting detection kit from Amersham (Little Chalfont, United Kingdom).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation of pseudorevertants of fliF mutants. Pseudorevertants were isolated from a total of 33 fliF mutants by streaking single colonies out on 8% gelatin agar, incubating them for up to 4 days, and looking for small swarms emerging from the streak. Cells from these swarms were picked and purified as single colonies. The locations of the suppressing mutations were broadly determined in terms of the major flagellar regions: I, II and III, which are at about 26, 42, and 43 centisomes, respectively (26). (Flagellar region III in fact consists of two subregions, IIIa and IIIb, with a region unrelated to flagellar function in between [11].)
None of the fliF mutants yielded suppressor mutations lying in region I. For 30 of them, all of the suppressor mutations lay in region III. Since fliF itself lies in region IIIb, many of these suppressors may have been intragenic, and none were analyzed further in this study. The remaining three fliF mutants (SJW2706, SJW2713, and SJW2715) between them gave rise to a total of 24 examples of intergenic suppressors in region II, in addition to a total of 229 examples of suppressors in region III. Finer deletion mapping of two of the region II suppressors indicated that they lay in or close to flhA, a gene which encodes an integral membrane component of the flagellar export apparatus (18); DNA sequence analysis later confirmed that all of the suppressors we identified lay in flhA (see below).Motility of SJW2706 (fliF) and its fliF
flhA pseudorevertants.
DNA sequence analysis of the three
parental fliF strains revealed that they were identical (see
below). Strain SJW2706 (fliF) was chosen for further study.
SJW2706 swarmed very poorly at 30°C, whereas the pseudorevertants,
illustrated by MM0608, swarmed considerably better (Fig.
1), although still only at about 20% of
the wild-type rate (estimated after 15 h at 30°C [data not
shown]). When examined in liquid culture by dark-field light
microscopy, SJW2706 was essentially nonmotile, with only an occasional
cell swimming feebly. Few if any flagella could be seen. With MM0608
and the other pseudorevertants that were examined, ca. 20 to
50% of the cells were swimming. Swimming speed was less than that of
wild-type cells, and there were only ca. two to three flagella per
swimming cell. Wild-type SJW1103, in contrast, was vigorously motile,
with well-formed bundles containing ca. five to seven flagella. Thus,
the swarming behavior can be simply explained by essentially total
failure to assemble flagella in the case of the parental
fliF mutant and partial success in assembling flagella in
the case of the pseudorevertants.
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The parental FliF mutation and the suppressing FlhA mutations. FliF is a protein containing 560 amino acids, or 559 after cleavage of the N-terminal methionine (9). (In this report, numbering starts with Met-1; the sequence in databases such as SwissProt starts with Ser-2.) The fliF mutation in all three parental strains was found to be the same, an in-frame deletion of six nucleotides (bp 521 to 526) about a third of the way into the gene, resulting in loss of two amino acids, A174 and S175.
Flagellar region II contains a mixture of flagellar, motor, chemotaxis, and receptor genes. Classical genetic deletion mapping had indicated that for at least two of the region II pseudorevertants, the suppressing mutations lay in or near flhA. DNA sequence analysis was carried out on the suppressor alleles of all 24 pseudorevertants and resulted in identification of 19 of the mutations (Table 2). They were all missense mutations, lying within flhA, as the deletion mapping had suggested. Two of the mutations were encountered several times, so that only 10 were distinct. Three of those were different nucleotide changes within the same codon, two of them giving the mutation F138L and the other giving F138C. Thus, amino acid changes were seen at only eight FlhA positions.
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Comparison of swarming abilities of the pseudorevertants. With the knowledge of the second-site flhA mutations responsible for suppression, we examined whether these could be ranked or compared in any useful way in terms of their abilities to suppress. After 15 h at 30°C, among those giving the poorest swarming was MM0663, whose FlhA mutation was S272N. It swarmed at about 75% of the rate of MM0608 (V268L) (whose swarming was shown in comparison with the wild-type strain and the parental fliF mutant in Fig. 1). The best swarming was observed with MM0624 (I148S), which swarmed at about 140% the rate of MM0608. Thus, although there were differences, they were small compared with the roughly fivefold difference between the wild-type strain and the pseudorevertants as a group.
Construction and properties of second-site flhA mutants. The second-site mutations from a subset of the pseudorevertants (MM0608, MM0609, MM0610, MM0621, MM0635, MM0652, and MM0654) were transferred by phage P22-mediated transduction into KK1012 (lacking region II [14]) to construct strains containing only the second-site flhA alleles: MM1608, MM1609, MM1610, MM1621, MM1635, MM1652, and MM1654, respectively. (The flhA mutations of MM1635, MM1652, and MM1654 subsequently proved to be identical.) These strains, illustrated by MM1608 (Fig. 1), swarmed at essentially wild-type levels, indicating that these second-site flhA mutations by themselves have no phenotype.
Complementation, dominance, and multicopy properties of the
suppressor flhA mutations.
To examine the properties
of the suppressor flhA alleles expressed in
trans, the wild-type flhA gene and several of the
suppressor flhA alleles were cloned into pTrc99A. Even
without induction, pTrc99A-based plasmids result in expression levels
that are considerably higher than those from the chromosome
(21). The insert consisted of the entire flhA
gene (bases 1 to 2079, with no flanking material from either
flhB or flhE [Table 1]). These plasmids were
introduced into SJW2706 (fliF), MM0608 (fliF
flhA), and a flhA null mutant, SJW1364. The resulting
transformants were inoculated onto soft tryptone agar plates and
incubated at 30°C (Fig. 2).
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Complementation, multicopy, and dominance properties of the parental FliF mutant protein. We wished to know whether the parental FliF mutant protein would, in high copy number, improve the function of the parental mutant itself, and also whether it would exert negative dominance over wild-type cells (generating poorly motile first-site phenotype) or second-site flhA mutants (generating pseudorevertant phenotype).
The parental mutant fliF allele from SJW2706 was cloned into pTrc99A to give plasmid pMMiF2706, which was then used to transform various hosts. The swarming of the parental mutant SJW2706 or two pseudorevertants that were tested (MM0608 and MM0635) was not improved by overproduction of mutant FliF (data not shown). In the wild-type and second-site mutant backgrounds, the level of swarming was reduced markedly, to about 40% of that of the same cells transformed with vector (Fig. 3). Thus, although there was clearly dominance, it was not complete, since that would have resulted in the much slower swarming of the parental mutant (in the case of the wild-type host) or the pseudorevertant (in the case of the second-site host).
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The parental FliF mutation does not affect C-ring assembly.
fliF null mutants fail to assemble the C ring, which is a
peripheral membrane structure consisting of FliG, FliM, and FliN; thus,
the C ring associates with the membrane only via the MS ring
(13). Formation of the C ring is essential not only for motor function but also (for poorly understood reasons) for export and
further flagellar assembly. To examine whether the parental FliF
mutation affects C-ring formation, cells were lysed and separated into
a high-speed pellet fraction (containing membrane and
membrane-associated material) and a supernatant fraction (containing
soluble cytoplasmic proteins). The fractions were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
immunoblotting with anti-FliF, anti-FliG, and anti-FliN antibodies
(Fig. 4). SJW1103 (wild type) and null
mutants SJW1684 (fliF) and SJW1364 (flhA) were
used as controls.
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The parental fliF mutant is defective in export and second-site flhA mutations partially suppress the defect. We next examined whether the failure of the parental fliF mutant to swarm is a result of failure to export substrates and whether the second-site suppressor flhA mutations restore the ability to export. FlgD, the hook-capping protein, was chosen as a test substrate. Periplasmic and culture supernatant fractions of SJW2706 and MM0608 were prepared as described before (18), and immunoblotting was carried out with polyclonal anti-FlgD antibody. SJW1103 (wild type) and SJW156 (flgD) were used as positive and negative controls, respectively.
FlgD was not detected in either the periplasmic or the culture supernatant fractions of the parental fliF mutant (Fig. 5, lanes 3 and 4). We conclude that its poor flagellation and motility result directly from poor flagellar protein export. FlgD was exported into both fractions by the intergenic fliF suppressor mutant (lanes 5 and 6), indicating that the second-site flhA mutation can suppress the defect in flagellar protein export of the parental fliF mutant. However, suppression was not complete: both the overall level of FlgD export and the amount found in the supernatant (lane 6) versus the periplasm (lane 5) were much lower than with wild-type cells (lanes 2 and 1). The latter aspect can be explained by the fact that impaired export of periplasmic components (the rod proteins) results in doubly impaired export of external components like FlgD.
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Attempt to obtain biochemical evidence for FlhA association with the basal body. Two membrane components of the export apparatus (FliP and FliR) have been shown previously to be associated with the basal body (2). The genetic evidence described above provides strong reason to believe that FlhA interacts with the basal-body MS-ring protein FliF. We sought biochemical evidence for such an interaction, by preparing hook-basal bodies from a flhA mutant transformed with a plasmid producing FlhA tagged with the FLAG epitope (6) and, as a control, wild-type cells transformed with vector. The samples were then subjected to SDS-PAGE and immunoblotting. With monoclonal anti-FliF antibody, a strong band at the position expected for FliF was seen with both hook-basal body samples. With monoclonal anti-FLAG antibody, there was a weak signal at the position expected for FLAG-tagged FlhA in the case of the sample from the mutant transformed with the plasmid producing FLAG-tagged FlhA and no signal with the control (data not shown). Thus, we have obtained weak biochemical evidence for FlhA being in physical association with the basal body.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Both the first-site FliF mutation and its suppressors are rare. In our initial survey, we found that the majority of fliF mutants giving rise to suppressors produced ones that were in flagellar region III and may well have been intragenic. Only 3 out of 33 fliF mutants gave suppressors that were in a different flagellar region and therefore necessarily were intergenic, and even here the intergenic suppressors were much less common than ones in the same region. Since these three mutants proved to be identical, the true numbers we obtained are 1 out of 31. Of the 19 suppressor mutations identified, two were encountered several times (Table 2). Also, three different mutations were found within a particular codon (number 138), but these gave rise to only two distinct amino acid changes. The end result is that we found nine distinct amino acid changes, involving eight positions. Thus, both the parental and the suppressing mutations appear to be fairly rare events, indicating that the interaction between the MS ring and the export apparatus may be quite a restricted one.
Position of the parental FliF mutation.
As is evident from the
complex shape of the MS ring (30), the FliF protein
subunits from which it is built must themselves have a complex
architecture. This makes it difficult to predict where the
two-amino-acid deletion (A174 and S175) in the mutant FliF protein is
located within its three-dimensional structure. However, on functional
grounds (suppression by a mutation in FlhA), it seems reasonable to
propose that the deletion may be on the inner surface of the annular
pore of the MS ring (Fig. 6). Apparently, it does not greatly affect the intersubunit interface within the MS
ring, or the interface between the MS ring and the C ring, since both
of these structures assemble in the FliF mutant (Fig. 4).
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-lactamase and -galactosidase fusions and concluded that
Q129 (equivalent to Salmonella E137) and E332 (equivalent to
Salmonella E367) lie in the periplasm. The result with E332
would seem to preclude a second span as early in the Salmonella sequence as position 199. However, given the
complex shape of the FliF subunit which gives rise to the quite bulky M-ring feature of the MS ring, it may be that FliF has an unusual transmembrane structure and crosses the membrane more often than the
two times indicated by computer algorithms. With this reservation, we
tentatively conclude that the parental mutation (deletion of amino
acids 174 and 175) lies in the periplasmic domain of FliF.
Conservation of FliF sequence among species is generally quite weak,
based on CLUSTAL W alignments (28) of the 11 FliF
sequences available in the SwissProt database (version 39). However,
the parental FliF deletion (A174 and S175) belongs in one of the
regions that is most strongly conserved, and within this fairly
conserved region it contributes to a short conserved motif
A-S(A)-V(I)-X-V(L/I), where A is conserved in all 11 sequences, S and the first V are both conserved in 10 of the 11 sequences, and the second V is conserved in 3 of the 11 sequences.
Thus, it appears to represent an important part of the FliF sequence,
such that deletion could cause a significant conformational change and
so affect the interface with FlhA.
Positions of the suppressor FlhA mutations.
FlhA is a
692-amino-acid integral membrane protein with a large soluble domain
encompassing the C-terminal half of the protein (residues 328 to 692)
(Fig. 7). Within the N-terminal half, the membrane protein topology algorithm TopPred2, using a span length of 19 amino acids, predicts eight 
-helical transmembrane spans (TM1 to
TM8) at the following approximate residue positions: TM1, 21 to 39;
TM2, 46 to 64; TM3, 68 to 86; TM4, 124 to 142; TM5, 211 to 229; TM6,
251 to 269; TM7, 288 to 306; and TM8, 309 to 327. TM7 is less strongly
predicted than the others and is predicted weakly or not at all by some
other algorithms (e.g., TMHMM [27]). Two experimental
arguments, however, support its existence, which would place the
C-terminal domain in the cytoplasm rather than in the periplasm.
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perhaps just one. Probably
because of a quite tight geometry, a FlhA subunit will interact with
whichever FliF subunit it happens to be close to.
The single parental FliF mutation has given rise to suppressors in
predicted periplasmic loops, transmembrane spans, and cytoplasmic loops
of FlhA, and so it is clear that they cannot all correspond to
immediately proximal interactions. Rather, we imagine that the FliF
mutation causes a displacement that can be compensated for in various
ways by the FlhA mutations in its N-terminal transmembrane domain. The
absence of any examples in the C-terminal cytoplasmic sequence suggests
it may constitute a distinct domain that is insensitive to the detailed
state of the MS ring.
A cartoon illustrating our overall interpretation of the FliF-FlhA
suppression data is given in Fig. 6.
Interpretation of multicopy and dominance effects.
The
multicopy and dominance effects observed, and our interpretation of
them, are illustrated in cartoon form in Fig.
8. For convenience, mutant FliF and FlhA
will be referred to as FliF* and FlhA*, respectively. We observed
no examples of simple multicopy effects. In other words, overproduction
of FliF* in the absence of FliF had no effect on swarming [cf. cases
(v) and (ii) or cases (vi) and (iii)]. The same was true of
overproduction of FlhA* in the absence of FlhA [cf. cases (vii) and
(iii)].
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Is FlhA unique in suppressing mutation in FliF? There are six integral membrane proteins (FlhA, FlhB, FliO, FliP, FliQ, and FliR) that are associated with flagellar protein export. Why has only FlhA emerged from this suppression study? To avoid analyzing potential examples of intragenic suppression, we set aside the many suppressor mutations that were in the same flagellar region (region III) as the parental fliF one. However, this also meant that we eliminated the possibility of encountering suppressors in the four export genes that lie in region III: fliO, fliP, fliQ, and fliR. Thus, of the export genes, only suppression by flhA and flhB could have been detected by our strategy. It is still noteworthy, however, that of the region II suppressors, no examples were found in flhB.
In independent studies using a different parental FliF mutant, H. Komatsu and K. Oosawa (personal communication) have found examples of intergenic suppression involving the Mot and switch proteins. Taken together, the data suggest that the inner pore of the MS ring interacts with the export apparatus, while its outer circumference and its cytoplasmic face interact with the motor.| |
ACKNOWLEDGMENTS |
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We thank Hitomi Komatsu and Kenji Oosawa for sharing data prior to publication on suppression of FliF mutations by mutations in the Mot and switch proteins, and we thank Jorge Galán for providing unpublished data on the topology of Salmonella InvA.
This work has been supported by USPHS grants GM40355 and AI12202.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Biophysics and Biochemistry 0734, Yale University, P.O. Box 208114, 266 Whitney Ave., New Haven, CT 06520-8114. Phone: (203) 432-5590. Fax: (203) 432-9782. E-mail: robert.macnab{at}yale.edu.
Present address: Protonic Nanomachine Project, ERATO, JST, Seika,
Kyoto, 619-0237, Japan.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, March 2001, p. 1655-1662, Vol. 183, No. 5
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.5.1655-1662.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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