Department of Biological Sciences, University
of South Carolina, Columbia, South Carolina
29208,1 and Department of Microbiology,
Molecular Biology, and Biochemistry, University of Idaho, Moscow, Idaho
838432
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TEXT |
Caulobacter crescentus is
a dimorphic bacterium that differentiates as part of its normal cell
cycle. Each cell division results in a stalked cell and a motile
swarmer cell. The swarmer cell has a single flagellum at one pole that
is responsible for motility. The flagellum is similar in structure to
enteric flagella except that the filament is composed of multiple
flagellin proteins (6, 10, 21, 35, 38). The hook-proximal
portion of the flagellar filament consists of a 60-nm segment
containing a 29-kDa flagellin (6). The next segment is 1 to
2 µm in length and consists of a 27-kDa flagellin along with
increasing amounts of a 25-kDa flagellin at its distal end. The
remaining 2 to 10 µm of the filament contains the 25-kDa flagellin.
Thus, the flagellar filament consists of at least three different
flagellar proteins in a precise arrangement.
In addition to the three flagellins found in the filament,
stationary-phase wild-type cells and some flagellar mutants synthesize a 22-kDa flagellin (15, 34). The 22-kDa flagellin is not
found in functional flagellar filaments and may result from improper processing of the 25-kDa flagellin (22, 34).
Multiple flagellin genes have been identified in two unlinked clusters
in the C. crescentus genome (11, 34). The alpha gene cluster includes the fljJ, fljK, and
fljL genes that encode the 29-, 25-, and 27-kDa flagellins,
respectively. The gene order in this region is
flaY-flaE-fljJ-fljK-fljL-flaF-flbT-flBA-flaG (30,
32). Thus, the alpha cluster of flagellin genes is embedded in a
cluster of other flagellar genes. The beta flagellin gene cluster is
approximately 1,000 kb from the alpha cluster (8). No other
flagellar genes are present at this locus.
The flagellin gene family.
We determined the nucleotide
sequences of five of the six flagellin genes in the alpha
(fljK and fljL) and beta (fljM,
fljN, and fljO) regions using the plasmids
described in Table 1. The nucleotide
sequence of the sixth gene, fljJ, had been determined previously (11), and errors in this sequence were corrected using nucleotide sequence information produced by The Institute for
Genome Research. Each region encodes a set of three independently transcribed flagellin genes. A comparison of these sequences to the
C. crescentus genome sequence being produced by The
Institute for Genome Research indicated that no additional flagellin
genes were present elsewhere in the genome. Thus, C. crescentus has six flagellin genes in two clusters of three genes.
To facilitate comparison of members of the flagellin gene family, the
nucleotide sequences of the flagellin genes were aligned. There were
455 variable sites in the 822-bp sequence. Most of the variable sites
were found in the fljJ gene, which encodes the 29-kDa
flagellin. The most similar pair of genes, fljM and fljN, contained 46 differences (5.6%), indicating that
there has been considerable nucleotide divergence among all of the
genes. Furthermore, transversions outnumbered transitions 25 to 21, suggesting either that the mutational process was approaching
saturation or that selection was occurring. A neighbor-joining tree
indicated that the three beta flagellin genes clustered with
fljK, suggesting that all four genes encode 25-kDa
flagellins (Fig. 1).
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FIG. 1.
Neighbor-joining tree indicating phylogenetic
relationships among flagellin genes. The same configuration was
obtained by maximum-parsimony analysis. Flagellin gene phylogenies were
determined using Test Version 4.0b3a of PAUP written by D. L. Swofford.
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A comparison of the derived amino acid sequences of the flagellin genes
demonstrated considerable variation among the flagellin proteins, with
FljJ differing at approximately half of the amino acid positions (Fig.
2). The two most similar proteins, FljM
and FljN, differed at 14 (5%) of 273 amino acid positions. A
neighbor-joining tree identical to that obtained with the nucleotide
sequences was obtained when the amino acid sequences were compared.
This result provides further evidence that the three beta flagellin genes encode 25-kDa flagellins. This result is consistent with previous
observations that the alpha region deletion mutants SC507 and PC7810
produce 25-kDa flagellins but not the 27- or 29-kDa flagellin (25,
34).
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FIG. 2.
Comparison of derived amino acid sequences of the six
flagellin genes. Dashes indicate deletions in the amino acid sequence
relative to one or more of the other sequences. Dots indicate identity
with the reference amino acid sequence.
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N-terminal amino acid sequences of purified flagellins.
The
25- and 27-kDa flagellins were dissociated from intact flagellar
filaments as described by Johnson et al. (16) and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the
N-terminal amino acid sequences of these purified flagellins were
determined, we found that the sequence of the 27-kDa flagellin was
identical to that predicted from the sequence of the fljL gene (Fig. 3). However, the N-terminal
amino acid sequence of the 25-kDa flagellin appeared to be a mixture of
two proteins, one encoded by the fljK gene and one encoded
by the fljM or fljN gene or both. Thus, the
flagellar filament contains multiple 25-kDa flagellins in addition to
the 27- and 29-kDa flagellins. No evidence was obtained for the
presence of the FljO flagellin. However, the fljO promoter
is expressed at approximately one-fourth of the combined rate of the
fljM and fljN promoters (see Table 2 footnote).
Consequently, it is possible that the FljO flagellin was present in the
flagellar filament at levels too low to be detected.
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FIG. 3.
N-terminal amino acid sequences of purified flagellin
proteins. An amino acid pair, e.g., A/S, indicates that two amino acids
were found at the same position, suggesting that the protein sample
consists of a mixture of two proteins.
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Flagellar filaments containing multiple flagellins have been found in a
variety of other eubacterial genera, including Yersinia (18), Vibrio (20, 24),
Helicobacter (17), Campylobacter (12), Agrobacterium (4), and
Rhizobium (1, 27). However, in contrast to that
in the C. crescentus flagellar filament, the physical
arrangement of these multiple flagellins within the corresponding flagellar filaments is not known. In C. crescentus, the most
divergent flagellin, FljJ, is found in small quantities in the region
closest to the flagellar hook (6). Thus, FljJ is used to
initiate flagellin subunit assembly and then other flagellins are
assembled once a short segment is completed. These observations suggest
that the divergent FljJ flagellin is a specialized flagellin that is designed to facilitate the first steps of filament assembly and attachment to the hook. Similarly, the moderately diverged FljL flagellin is located between the FljJ flagellin and the 25-kDa flagellins that form the main part of the filament. Thus, it may serve
as an adapter between the two disparate flagellins. Mutants that do not
produce the FljJ and FljL flagellins assemble a shortened flagellum and
exhibit reduced motility (25, 33). Thus, an abnormal
flagellar filament is produced in the absence of these two flagellins.
Origin of the 22-kDa flagellin.
The origin of the 22-kDa
flagellin protein has been a mystery since it was first observed
(13). To determine whether the 22-kDa flagellin was derived
from one or more of the six flagellin genes, we purified the 22-kDa
flagellin from the insoluble red material produced by the SC3845 mutant
strain cells as described by Smit et al. (37) and determined
its N-terminal amino acid sequence using an automated amino acid
sequencer (Beckman Coulter, Fullerton, Calif.). The results were
identical to those obtained with the 25-kDa flagellins (Fig. 3),
indicating that the 22-kDa flagellin was derived from at least two of
the 25-kDa flagellin genes. To confirm this result, we isolated the
22-kDa flagellin from SC3794, a strain that contains a deletion of the
alpha region. The N-terminal sequence of this 22-kDa flagellin was
identical to that of the FljM and FljN flagellins, and the sequence
corresponding to the deleted fljK gene was not present. This
result proves that both the fljK gene and at least one of
the beta region genes are the source of the 22-kDa flagellins.
Regulation of flagellin gene expression.
Previous studies have
demonstrated that the fljK and fljL genes are
expressed from 
54 promoters and that they require both
the RpoN protein, encoding 54, and the FlbD activator
protein for expression (2, 29). However, inspection of the
regions upstream from the beta cluster flagellin genes revealed no
sequences that resemble a 54 promoter. This result is
consistent with the observation that flagellin gene expression does
occur at a low level in rpoN and flbD mutants
(26; J. Malakooti and B. Ely, unpublished data). To
determine experimentally whether 54 is required for
expression of the beta cluster flagellin genes, we cloned each of the
promoters in front of a lacZ reporter gene and measured
-galactosidase levels in various genetic backgrounds (3).
Expression of the fljMNO genes was only 10 to 20% of that measured with the fljK gene in a wild-type background (Table
2). However, when expression in an
rpoN mutant background was compared, the fljM,
fljN, and fljO promoters were expressed at 20 to
50% of the rate measured for their expression in wild-type cells. In
contrast, the fljK and fljL genes were expressed
at less than 2% of the rate measured in wild-type cells. Similar
results were obtained when flagellin promoter expression was measured
in a flbD mutant (Table 2). Thus, neither RpoN nor FlbD is
required for fljMNO expression, and expression of these beta
region flagellar genes can account for the residual flagellin synthesis
observed in rpoN and flbD mutants. As with
fljK, expression of the fljMNO promoters was not
affected by the presence of other flagellar mutations (Table 2).
Previous studies have demonstrated that flbT mutants
overproduce the 25-kDa flagellin proteins (15, 30). The FlbT
protein must be involved in flagellin mRNA turnover, since flagellin
mRNA is more stable in an flbT mutant strain than in a
wild-type strain (23). When expression of the
fljM, fljN, and fljO promoters was
measured in an flbT mutant background, expression was 45 to 90% of that measured in wild-type cells. Since expression from the
fljK and fljL promoters is reduced 10-fold in
flbT mutants, the beta cluster flagellin genes are likely to
be responsible for most of the overproduction of flagellin observed in
flbT mutants.
Nucleotide sequence accession numbers.
The flagellin gene
nucleotide sequences in Fig. 2 have been assigned GenBank accession
numbers AF089835 and AF040268.
We thank Nelida Caballero and Yoshi Ishikawa for expert technical
assistance and Nina Agabian for providing clones and unpublished information.
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