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Microbiology and Molecular Biology Reviews, December 2000, p. 694-708, Vol. 64, No. 4
Department of Microbiology, University of
Washington, Seattle, Washington 98195
1092-2172/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Coupling of Flagellar Gene Expression to Flagellar
Assembly in Salmonella enterica Serovar Typhimurium and
Escherichia coli
SUMMARY
INTRODUCTION: THE FLAGELLAR REGULATORY NETWORK
FLAGELLAR PROMOTERS
Class 1 Master Operon
Class 2 and Class 3 Promoters
CENTRAL ROLE OF FLGM-
28
INTERACTION
Positive Regulation by Alternative Sigma Factor
28
Feedback Loops for Early and Middle Gene Expression
Negative Regulation
FlgM Acts as an Anti-28 Factor
FlgM Acts as an Anti-28 Holoenzyme Factor
Genetic and Molecular Analysis of FlgM-28
Interaction
Native FlgM Is an Unfolded Polypeptide
CHECKPOINT IN FLAGELLAR GENE EXPRESSION
FlgM Secreted in Response to Hook-Basal Body Completion
FlgM Secreted by a Flagellar Type III Pathway
FlgN Protein
Hook Completion and Specificity of Type III Secretion
flgM mRNA Translation Coupled to Ring Assembly
REGULATION OF NUMBER OF FLAGELLA
LINK BETWEEN REGULATION OF EXPRESSION AND EFFICIENCY
OF ASSEMBLY
CELL CYCLE CONTROL
CONCLUSION
ACKNOWLEDGMENTS
REFERENCES
SUMMARY
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How do organisms assess the degree of completion of a large structure, especially an extracellular structure such as a flagellum? Bacteria can do this. Mutants that lack key components needed early in assembly fail to express proteins that would normally be added at later assembly stages. In some cases, the regulatory circuitry is able to sense completion of structures beyond the cell surface, such as completion of the external hook structure. In Salmonella and Escherichia coli, regulation occurs at both transcriptional and posttranscriptional levels. One transcriptional regulatory mechanism involves a regulatory protein, FlgM, that escapes from the cell (and thus can no longer act) through a complete flagellum and is held inside when the structure has not reached a later stage of completion. FlgM prevents late flagellar gene transcription by binding the flagellum-specific transcription factor 
28. FlgM is itself regulated in response to the assembly of an incomplete flagellum known as the hook-basal body intermediate structure. Upon completion of the hook-basal body structure, FlgM is exported through this structure out of the cell. Inhibition of 28-dependent transcription is relieved, and genes required for the later assembly stages are expressed, allowing completion of the flagellar organelle. Distinct posttranscriptional regulatory mechanisms occur in response to assembly of the flagellar type III secretion apparatus and of ring structures in the peptidoglycan and lipopolysaccharide layers. The entire flagellar regulatory pathway is regulated in response to environmental cues. Cell cycle control and flagellar development are codependent. We discuss how all these levels of regulation ensure efficient assembly of the flagellum in response to environmental stimuli.
INTRODUCTION: THE FLAGELLAR REGULATORY NETWORK
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The synthesis and function of the
flagellar and chemotaxis system require the expression of more than 50 genes which are divided among at least 17 operons (Fig.
1) that constitute the large, coordinately regulated flagellar regulon (85). The
expression of these genes is regulated at a multiplicity of levels in
response to environmental cues, in addition to signals that are coupled to the morphological development of the flagellar organelle itself. The
elaborate regulation is accomplished via mechanisms that involve a
number of transcriptional regulators, some of which are sensitive to
one another, and at least one that directly detects the developmental state of the population of flagella expressed by a given cell. Within
the regulon, the operons are divided into three temporally regulated,
hierarchical transcriptional classes: early, middle, and late. This
hierarchical class structure is based on the effect of null mutation in
a gene of a given class on the transcription of the remaining genes in
the regulon (71). These genes were previously referred to as
class 1, class 2, and class 3, but recent findings that many genes are
transcribed from multiple promoters added confusion to this
nomenclature. The original classification was based upon a requirement
for expression of the previous transcriptional class before expression
of the next class can occur (71). In this review we will
refer to the genes as early, middle, and late and the promoters as
class 1, class 2, and class 3.
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In general, late genes are downregulated in strains defective in early
or middle genes, and neither middle nor late genes are expressed in
strains defective in early class genes. The three promoter classes that
correspond to the transcriptional classification have differences in
DNA sequence. The class 1 promoter is a single promoter that
transcribes the two early genes in the flhDC operon. As
discussed below, the flhD and flhC genes encode
transcriptional activators, and transcription of the flhDC
operon responds to many environmental cues. The FlhD and FlhC proteins
are transcriptional activators for the class 2 promoters upstream of
the middle gene operons. The protein products of middle class operons
include proteins necessary for the structure and assembly of the
hook-basal body, an intermediate structure in flagellar assembly, and
the transcriptional regulators FlgM and 28. The class 3 promoters are specific for 28 RNA polymerase. With the
exception of the hook-associated proteins (HAPs), gene products
required at the late assembly stage are transcribed only from class 3 promoters.
The transcriptional classes appeared to correspond to major steps in
morphological development of the flagellar structure (49).
However, this relatively simple transcriptional hierarchy was
complicated by the fact that many of the genes are expressed from more
than one promoter class. For example, the flgK, flgL, flgM, flgN,
fliD, fliS, and fliT genes are transcribed from both FlhDC-dependent class 2 promoters and 28-dependent class
3 promoters (32, 66, 72). The coupling of gene expression to
flagellar assembly is accomplished by the regulation of
28 activity by the anti-28 factor FlgM
(Fig. 2). Regulation of FlgM levels in
response to flagellar assembly results in the temporal regulation of
28-dependent transcription to ensure the efficiency of
assembly. In addition to complex transcriptional control, there are
layers of posttranscriptional regulation, which are also believed to ensure an efficient temporal assembly process (12, 50, 53).
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FLAGELLAR PROMOTERS
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Class 1 Master Operon
The early genes are transcribed from a class 1 flagellar promoter in a single operon, flhDC, which is sensitive to environmental and cell state sensors such as cyclic AMP (cAMP)-cAMP receptor protein (cAMP-CRP) (119, 133). The class 1 promoter is also known to require a number of other genes outside of the flagellar regulon for its expression (11, 104, 114-116). Six transcriptional start sites have been mapped in the class 1 promoter region of Salmonella enterica serovar Typhimurium (132). It has also been demonstrated that cell cycle and flagellation are interdependent and that the flhDC genes are involved in coupling these processes (96, 103). Likewise, genetic evidence suggests that a growing number of other genes are involved either directly or indirectly in transcriptional regulation of flhDC expression (5, 74, 104, 114-116).
The promoter for the flhDC master operon defines the class 1 flagellar promoter. It is a crucial regulatory point at which the decision to initiate or prevent flagellar biosynthesis can occur. It is not surprising that the class 1 promoter is where a number of global regulatory signals have input on the decision to synthesize flagella. These include cAMP-CRP (119, 133), temperature control (1, 105), heat shock proteins DnaK, DnaJ, and GrpE (116), high concentrations of either inorganic salts, carbohydrates, or alcohols (74, 115), DNA supercoiling (11, 63, 74, 115), growth phase (96), surface-liquid transition (33), phosphatidylethanolamine and phosphatidylglycerol synthesis (91, 114), and cell cycle control (90, 92, 103).
Mutants defective in either adenylate cyclase (cya gene) or CRP (crp gene) failed to produce flagellin, hook, or flagellin mRNA (58, 119, 133). Motile revertants of cya or crp mutants were isolated, and their mutations were found to map to the promoter region of the early flhDC operon (58, 119). These motile revertants were pleiotropic in that they also lost the high-temperature inhibition of flagellar gene expression (119). This suggests that the high-temperature inhibition of flagellum synthesis is an indirect effect on the class 1 promoter through the cAMP-CRP activator complex. A CRP binding site has been identified within the flhDC promoter regions for both E. coli and S. enterica serovar Typhimurium (121, 132).
The presence of six transcriptional start sites in the S. enterica serovar Typhimurium class 1 promoter region indicates
sites of transcriptional regulation (132). CRP binding sites
have been identified within the flhDC promoter regions for
both E. coli and S. enterica serovar Typhimurium
(9, 117, 121). One CRP binding site corresponded to a region
of near identity 58 to 84 bases upstream of the major S. enterica serovar Typhimurium transcript (P1), which was
substantially reduced in a crp mutant background (132). A second was located between the 
10 region of the
P1 transcript and the GTG start codon. Another transcript identified in
S. enterica serovar Typhimurium (P6) was dependent on a
functional hns gene for expression (132). The
H-NS protein is a bacterial nucleoid-associated protein and a
pleiotropic regulator of gene expression (27). In DNase
footprinting experiments, purified H-NS protected numerous sites on
both sides of the start of the P6 transcript (121). The
major site of transcription initiation of the E. coli flhDC
transcript was located 17 bp downstream of the S. enterica
serovar Typhimurium P1 transcription initiation site, suggesting that
some spacing differences exist between the two species (117,
132). Another metabolic intermediate, acetylphosphate, affected
expression of the flhDC operon in E. coli, and
this effect was dependent on OmpR, another pleiotropic regulatory
protein (117). The E. coli transcript was
dependent on OmpR protein for expression, and OmpR was shown to
footprint large regions upstream and downstream of the transcription
initiation site (117). Regulation of flhDC operon
expression is further complicated by the fact that it is autogenously
regulated and the nature of autogenous regulation is dependent on the
intracellular activity of 28. Under normal growth
conditions, FlhDC is an autogenous repressor of flhDC
expression, but in the presence of increased 28
activity, FlhDC is an autogenous activator of flhDC
expression (63).
The results presented above suggest a high degree of complexity for the flhDC promoter region that is only beginning to be understood. The interdependence among so many structural and physiological processes and the regulation of flhDC operon expression suggest that motility plays a crucial role in the life cycle of the cell and, as discussed later, serve to couple flagellar biosynthesis to the cell cycle. The production of flagella and resulting motility would represent a significant drain on the cell's resources. However, the ability to reach a food source ahead of competitors or to swim away from substances that adversely affect central metabolic processes would provide a significant survival advantage.
Class 2 and Class 3 Promoters
In addition to autogenous regulation, the FlhDC complex is a
positive transcriptional activator of 70-dependent
transcription from the class 2 promoters (71, 78). The FlhDC
complex activates class 2 promoter transcription by binding within the
class 2 promoter region and interacting with the C-terminal region of
the 
subunit of RNA polymerase (76). The FlhDC-dependent
transcriptional start sites have been mapped for 10 class 2 promoters,
which are shown in Fig. 3 (44, 45, 66, 78, 95). Purified FlhDC has been shown to footprint a region
between 30 and 80 relative to the transcriptional start site for
three of these class 2 promoters (Fig. 3) (78). Sequences termed TTATTCC and GCAATAA elements have been
identified in all class 2 promoters in the region where FlhDC binds
and, when deleted in the flgB promoter, are defective for
transcription (Fig. 3) (44). Operons transcribed from class
2 promoters include genes whose products are required for the
morphogenesis of the hook and basal body (49). Included in
the genes transcribed from class 2 promoters is the fliA
gene, which encodes an alternative sigma subunit of RNA polymerase,
28 (97).
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The flagellar alternative sigma factor, 28, confers
specificity for the class 3 flagellar promoters of the late genes
(43, 62, 97, 109). DNA sequence analysis of flagellar genes
revealed a novel promoter region and suggested that expression of
flagellar and chemotaxis genes might occur by an alternative sigma
factor (35). A consensus sequence of 35 (TAAAGTTTT)
N11 10 (GCCGATAA) was determined for the
28-specific class 3 promoter (Fig. 3) (35,
43). Early on, DNA sequence analysis of middle gene promoters
revealed that they contained the 10 consensus sequence of the class 3 promoter (9) and suggested a role for 28 in
middle gene expression (34). More recently it was
demonstrated that 28 will transcribe from the promoter
regions of the early and middle genes, but the transcriptional start
sites differ for FlhDC-dependent class 2 and
28-dependent class 3 promoters within the middle gene
promoter regions (43, 63, 67, 77). A negative regulator of
28, the FlgM protein (31), is transcribed
from a class 2 and a class 3 promoter (32). Among the late
genes are those transcribed only from class 3 promoters. These include
genes encoding motor torque generator subunits MotA and MotB,
chemotaxis proteins, and the flagellin proteins FliC and FljB, which
are polymerized outside the cell to form the helical filament
(85).
CENTRAL ROLE OF FLGM-
28
INTERACTION
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Positive Regulation by Alternative Sigma Factor
28
The essential positive regulatory element in the flagellar
regulatory cascade is the alternative sigma factor 28,
encoded in the fliA gene. While originally thought to be a
transcription factor only of flagellar late assembly genes, there is
evidence that 28 has recognition sequences within the
promoter regions of all three flagellar gene classes. A null mutation
in the fliA gene resulted in loss of expression of
lac operon fusions to the late genes (57, 71).
Comparison of the promoter regions for the late gene promoters revealed
a promoter consensus sequence distinct from the promoter consensus of
the primary sigma factor, 70, and similar to that of an
alternative sigma factor required for flagellar gene expression in
Bacillus subtilis, 28 (35, 43). A
thorough analysis of 28-dependent promoters reveals a
revised promoter sequence of TAAAGTTT-N11-GCCGATAA for the 35 and 10 promoter recognition sites (Fig. 3)
(43).
The fliA genes from both S. enterica serovar
Typhimurium and E. coli were cloned and sequenced, and their
28 protein products were purified and characterized
(8, 75, 98). One striking feature of the 28
protein is that it is less than half the size of the primary sigma
factor 70 (Fig. 4). The
28 protein belongs to a conserved 70
family of transcription factors (79). There are four main
regions of conservation within the 70 family, regions 1, 2, 3, and 4. DNA sequence comparison revealed that the large N-terminal
conserved portion of 70 (region 1) is not present in
28 (Fig. 4). Region 1 was found to prevent
70 from binding promoter sequences when
70 is not bound to core RNA polymerase (24,
25). Region 1 interacts with region 4 in free 70.
Region 4 contains the 35 promoter-binding domain. Region 1 is not
present in 28 (Fig. 4). Region 4 of 28 is
prevented from binding DNA when free from core RNA polymerase by the
interaction with the anti28 factor FlgM (17,
46; M. S. Chadsey and K. T. Hughes, submitted for
publication.)
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The 28 proteins from both E. coli and
S. enterica serovar Typhimurium are 239 amino acids in
length and were specifically required for in vitro transcription from
the class 3 tar or fliC promoter (75,
98). The fliA gene is located in the fliAZY
operon (45, 95). Class 2 and class 3 promoters transcribe
the fliAZY and fliAZ transcripts, respectively
(45). A separate promoter that is not regulated by the
flagellar regulatory system also transcribes the fliY gene
(45). Disruption of fliZ caused weak motility (45, 95), while disruption of fliY conferred no
motility defect (45). Introduction of a multicopy plasmid
carrying the fliZ expressed from vector-derived promoters
resulted in a 10-fold increase in expression of a chromosomal
fliA-lac operon fusion (68). A null mutation in
fliZ resulted in a 10-fold reduction in expression of
lac operon fusions to middle genes flgA, flgB, flhB,
fliE, fliF, fliL, and fliA but had no effect on
expression of an flhD-lac operon fusion (68).
This result suggests a positive role for FliZ in class 2 promoter
transcription. fliZ null mutants exhibited only a slight
defect in motility that could be complemented in trans
(45). This is probably why mutations in fliZ had
not been identified earlier. Kutsukake et al. (68) pointed
out that FliZ protein is predicted to contain a coiled-coil domain,
which is believed to be involved in protein-protein interactions
(83). They suggest that the role of FliZ is to modulate the
binding of the FlhD2C2 complex to class 2 promoters through a direct interaction with the
FlhD2C2 complex (68). Another
possibility is that FliZ plays a positive role in
FlhD2C2 complex formation. Either possibility provides a mechanism by which to amplify the activity of the
FlhD2C2 complex on class 2 promoter expression.
It also provides the possibility for another checkpoint in the
flagellar regulatory cascade. The FlhD2C2
complex activates transcription of the fliAZY operon, and
the 28 (FliA) and FliZ protein products act to increase
flagellar gene expression: 28 will transcribe early,
middle, and late genes, and FliZ enhances the function of the
FlhD2C2 complex on class 2 promoter transcription.
Feedback Loops for Early and Middle Gene Expression
The 28 protein is absolutely required for
transcription from class 3 promoters. However, overexpression of the
fliA gene resulted in increased expression from all middle
gene promoters (67). In vitro, E28 was shown
to transcribe the PfliA,
PfliL, and middle gene promoter regions at start
sites distinct from those used by E70-FlhDC
(77). The effect of increased transcription from middle gene
promoters by fliA overexpression was dependent on the
presence of the early (flhDC) genes (67). This
suggested that fliA overexpression led to increased early
gene transcription. Indeed, flhDC expression is elevated by
increased fliA expression (63). However, this elevation was dependent on the presence of an active flhDC
operon. In the absence of fliA, the flhDC operon
is negatively autoregulated (63, 132). These results
suggested that FlhDC is an autogenous repressor, while
28 is both an autogenous activator and an activator of
the early flhDC operon and other middle operons. Expression
of flhDC would lead to induction of class 2 promoter
transcription, including the fliA class 2 promoter. Further
increase in the expression of flhDC would then depend on the
activity of 28.
Negative Regulation
The coupling of flagellar gene expression to flagellar development by negative regulation was first suggested by Suzuki and Iino from assays of flagellin RNA levels in a variety of flagellar mutant strains (124). Mutations in any one of 11 flagellar genes examined led to a greater than 100-fold reduction in 14C-labeled amino acid incorporated into flagellin compared to incorporation into whole-cell protein. The real breakthroughs in the characterization of the flagellar regulon came with the advent of the Mud-lac fusion technology initially developed by Malcolm Casadaban (14, 15). In the early characterization of a transcriptional fusion of lac to the flagellin structural gene fliC, it was observed that loss of any one of a large number of flagellar genes (23 of the then-known 28) prevented expression of the fliC-lac fusion. Fusions of Mud-lac to all the flagellar genes in both S. enterica serovar Typhimurium and E. coli were obtained, and the effect of other flagellar genes on expression of the different fusions was determined (55, 56, 71). At the same time, many of the functions of the different flagellar genes were determined, so that a correlation between gene regulation and flagellar assembly began to unfold.
The middle genes encoded the structural genes for the hook-basal body
intermediate structure, and the late genes encoded proteins needed late
in flagellar assembly. Late genes encode hook-associated proteins FlgK,
FlgL, and FliD, the filament subunits FliC and FljB, the proton motive
force generator subunits MotA and MotB, and the chemosensory system.
FlgK and FlgL form the hook-filament junction, and the cap is composed
of FliD protein subunits. Expression of individual lac
operon fusions to each gene of the flagellar regulatory hierarchy was
determined in strains that were individually disrupted in every other
gene to determine that the flagellar regulatory hierarchy was divided
into the three main classes (Fig. 2) (71). If either
flhD or flhC, was disrupted, none of the other
genes was expressed. If any of the genes required for hook-basal body
formation was disrupted, other hook-basal body genes were still
transcribed, as was the flhDC operon, but none of the late genes was transcribed. The FlhD and FlhC proteins were envisioned as
transcriptional activators of middle gene promoters (35), and the fliA gene was shown to encode an alternative sigma
transcription factor, 28, which directed transcription
of late gene promoters (75, 97). Thus, it was easy to
hypothesize that loss of flhDC would prevent expression of
both middle and late gene promoters and that loss of
fliA would prevent expression of late gene promoters. It was more difficult to hypothesize how loss of any one of the other 34 middle genes could prevent expression of the late genes. Since the
majority of middle genes led to hook-basal body formation and the late
structural genes were needed after hook-basal body formation, it
appeared that gene regulation was coupled to flagellar morphogenesis by
negative regulation.
FlgM Acts as an Anti-28 Factor
The hypothesis that late flagellar gene expression was coupled to
flagellum assembly was demonstrated with the discovery of the negative
regulator FlgM (31, 57) and the characterization of its
activity as an anti-sigma factor (98). The anti-sigma activity of FlgM was postulated based on the following criteria: (i)
purified FlgM inhibited the in vitro transcription of the class 3 fliC promoter using 28 holoenzyme but had no
effect on the in vitro transcription of the tac promoter
using 70 holoenzyme and (ii) a cross-linked product of
FlgM and 28 was obtained both with a mixture of purified
proteins and when FlgM and 28 were cosynthesized in
vitro in either minicells or maxicells (98). Later, it was
shown that when a fusion of glutathione-S-transferase (GST)
to 28 from either E. coli or S. enterica serovar Typhimurium was isolated from crude extracts on a
glutathione column, FlgM was associated with the GST-28
fusion without the addition of a cross-linking reagent, suggesting a
strong interaction (17, 48). Electrophoresis of a mixture of
FlgM and 28 on a native gel resulted in a novel band
that separated into the individual FlgM and 28 proteins
on a denaturing gel (17). Furthermore, nuclear magnetic resonance (NMR) assays and surface plasmon resonance (SPR) assays demonstrated that the interaction between FlgM and 28
was very tight, with a dissociation constant
(Kd) on the order of 2 × 10
10 M (17, 21). This interaction is of the
same magnitude as the interaction between 28 and
S. enterica serovar Typhimurium core RNA polymerase
(Kd = 8 × 1010 M)
(17).
FlgM Acts as an Anti-28 Holoenzyme Factor
FlgM interacts with 28 holoenzyme in addition to
28 alone (17). FlgM prevented
28 holoenzyme from binding fliC promoter DNA
even after formation of E28 holoenzyme. Also, in native
gel assays, FlgM was found associated with E28 in
stoichiometric amounts regardless of order of addition
(28 plus core then FlgM, or 28 plus FlgM
then core), but FlgM did not associate with core RNA polymerase in the
absence of 28. There are at least three ways in which
FlgM could prevent the activity of 28 in transcription:
(i) FlgM could act as an anti-28 factor and prevent
E28 formation; (ii) FlgM could act to dissociate
E28; and (iii) FlgM could bind E28 and
prevent promoter binding. Using the SPR assay system, it was found that
FlgM would actively dissociate core RNA polymerase from
E28 while having no effect on the dissociation of
E70 (17). FlgM concentration was estimated at
400 nM, about 1,000-fold higher than the Kd of
the FlgM-28 complex (200 pM) or the
Kd of the E28 complex (800 pM).
Given that the interactions between 28 and FlgM and
between 28 and core were within a similar range, there
should be no free 28 in the cell as long as FlgM and
core RNA polymerase are in excess to 28 in the cell.
Also, small changes in FlgM concentrations approximately 1,000-fold
higher than the Kd of the FlgM-28
complex should not affect that interaction, but they may affect the
ability of FlgM to destabilize the E28 complex.
Genetic and Molecular Analysis of FlgM-28
Interaction
A deletion analysis was carried out on both FlgM and
28 to assess which portions of these proteins were
involved in direct interactions (46, 69). Mutations in
28 were obtained that express a lac operon
fusion to class 3 promoters under FlgM-inhibitory conditions
(hook-basal body mutant backgrounds) (69; Chadsey and
Hughes, submitted). These mutations resulted in 28
proteins that bypass negative regulation by FlgM. DNA sequence analysis
of the FlgM-insensitive mutations in fliA showed that they
were located in three distinct regions (2.1, 3.1, and 4) of
the 28 protein that are conserved among sigma factors
(Fig. 5) (69; Chadsey
and Hughes, submitted). Regions 2.1 and 3.1 are the regions of sigma
factors that interact with core RNA polymerase, while region 4 has been
shown to include the 35 promoter DNA-binding region. Expression of
C-terminal region 4 alone was sufficient to compete with intact
28 for binding FlgM in vivo, while expression of
N-terminal regions 2 through 3 of 28 did not compete.
SPR assays demonstrated that individuals with mutations in each of the
three regions were defective in binding FlgM. This suggested that most
of the contacts in 28 for FlgM were in region 4;
however, the V33 position in region 2.1 and the region 3.1 positions
were defective in FlgM interaction. These results were consistent with
the findings that regions 2, 3, and 4 of the B. subtilis
homologue F were found to interact with its cognate
anti-F factor, SpoIIAB (22). Mutants carrying
mutations at position H14 of 28 still bound both FlgM
and RNA polymerase with the same affinity as wild-type
28. It is possible that they represented a direct
interaction of regions 2 and 3 with FlgM or an allosteric effect of
regions 2 and 3 on region 4. It is not known how the H14 substitution
mutations overcome FlgM inhibition. They may affect the ability of FlgM to destabilize the E28 complex or the stability of
28 itself. It is noteworthy that mutations in
28 region 4.2 that overcome FlgM inhibition do not
reside within the DNA recognition helix (see Fig. 5); there are no
mutations between positions 213 and 226. This was because
28 mutants insensitive to FlgM inhibition in vivo were
also selected to retain the ability to transcribe class 3 promoters.
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The C-terminal half of the 97-amino-acid FlgM protein was found to
interact directly with 28 (30, 46). Single
and double amino acid changes in FlgM that inhibited
anti-28 activity in vivo were identified at amino acid
positions 57, 58, 63, 66, 67, 76, 77, and 82 (21). Mutant
analysis suggested that the anti-28 region of FlgM lay
between amino acids 42 and 86 (21, 46). NMR analysis showed
that FlgM resonances extending from V41 through K97 were all affected
by binding to 28 (21). SPR analysis
demonstrated that the single-amino-acid changes L66S and I82T resulted
in a fourfold increase in the Kd of FlgM for
28 (17). These results suggested that amino
acids 41 through 97 of FlgM directly contact 28 in
region 4 and possibly in regions 2.1 and 3.1. It remains possible that
the defect in the ability of 28 region 2.1 and 3.1 mutants to bind FlgM may result from an allosteric effect on region 4.
Native FlgM Is an Unfolded Polypeptide
Using multidimensional NMR, Daughdrill et al. made the unexpected
discovery that free FlgM protein resided in a mostly unfolded state
under physiological conditions (21). It was only after binding 28 that residues 41 through 97 of FlgM attained
a folded conformation, while the N-terminal 40 amino acids remained
unstructured in the presence or absence of 28. These
results suggest that the stability of FlgM within the cytoplasm is
dependent on the interaction with other proteins. Less FlgM was present
in strains lacking 28, and stability measurements of
FlgM in this background indicated that some of this instability is due
to an increase in FlgM turnover (50).
The N terminus of FlgM encodes the secretion signal (described below). Deletions of the N-terminal portion resulted in a significant reduction in its cellular levels (20, 46) that may have resulted from increased turnover when the unstructured N terminus was not efficiently bound by another protein. This has yet to be determined.
CHECKPOINT IN FLAGELLAR GENE EXPRESSION
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FlgM Secreted in Response to Hook-Basal Body Completion
The remarkable finding that FlgM was regulated by secretion was a
clear example of transcriptional regulation in response to cell
development as opposed to regulation in response to cellular physiological status. As a result, genes expressed from
28-dependent class 3 promoters, whose products are
required at later assembly stages, are not activated until a
morphogenic requirement is fulfilled: the completion of the hook-basal
body intermediate structure. It was difficult to understand how the
presence of the 30 hook-basal body genes could be sensed by a single
protein, given that the combined product of those 30 genes was a
structure and not the chemical product of a metabolic pathway. The FlgM protein senses the developmental state of the flagellum by itself being
a substrate for secretion through the flagellum-specific type III
secretion pathway (42, 64). When the hook-basal body structure is complete and therefore competent for secretion of the
hook-associated proteins and flagellin, it also becomes competent for
secretion of FlgM (Fig. 2). The onset of FlgM secretion results in a
decrease in its cellular concentration and a concomitant derepression
of 28-mediated expression of the class 3 transcripts
(50, 53), whose structural products include the HAPs, the
flagellins, and motor force generators (49). This finding
also challenged the established paradigm that development proceeds in
response to gene expression, since in the case of bacterial flagella it
is development that cues gene expression (81).
Further proof that FlgM was secreted through the hook-basal body structure came with the ability to artificially induce the flagellar regulon (52). The flhDC operon was placed downstream of the tetracycline-inducible tetA promoter. In this construct, the flagellar transcriptional hierarchy was induced following the addition of tetracycline to the medium. These cells were motile in the presence of tetracycline and nonmotile without inducer present. Following addition of tetracycline, the class 1 (flhDC operon) promoter was immediately transcribed; this was followed by flgM gene transcription at 15 min (transcribed from both class 2 and class 3 promoters); also at 15 min after induction, transcription of the middle class fliA gene was induced; and 30 mins after induction, transcription of the late fliC gene occurred (52). Induction of fliC gene expression 30 min after flhDC induction coincided with the appearance of FlgM anti-sigma factor in the spent growth medium and with the completion of the hook-basal body structure. At 45 min after induction, nascent flagellar filaments were visible under the electron microscope (52). These results support a model in which the flagellar regulatory hierarchy of S. enterica serovar Typhimurium is temporally regulated following induction and both FlgM secretion and class 3 promoter transcription occur upon completion of the hook-basal body intermediate structure.
FlgM Secreted by a Flagellar Type III Pathway
Bacteria translocate proteins by a number of distinct pathways. Translocation systems that secrete proteins to the external environment have been categorized into four main groups, designated types I, II, III, and IV (13, 80), to distinguish them from the signal sequence-dependent, or sec, pathway by which many periplasmic and integral membrane proteins traverse or are inserted into the cytoplasmic membrane, respectively (94).
The type III secretion pathway appears to be most commonly found in pathogenic bacteria (19, 41, 122, 123). The protein components of this type of secretion system have been identified in several species, and in most cases they are both structurally and functionally homologous to components required for flagellar basal body formation (106, 126). These proteins are presumed to form the conduit by which flagellar proteins are secreted (84). Homologous proteins have been shown to form the type III secretion structures for the secretion of virulence determinants (54, 61, 125). Despite the growing number of proteins that have been identified as type III secretory proteins, no clear consensus has emerged for the sequence of the secretion signal. In several examples, the amino-terminal domains of type III secretory proteins have been shown to be sufficient for translocation (46, 73, 87, 110, 120) or to contain information that is essential for secretion (59). However, in some instances, mutations in other regions of type III secretory proteins have also been shown to have an impact on the efficiency of their secretion (38, 73). Paradoxically, short amino-terminal regions of some type III isecretory proteins have been shown to be sufficient to direct secretion (110, 131) but have also been shown to be dispensable for secretion (18). A remarkable finding by Anderson and Schneewind was that a signal for type III-directed secretion could be found in both the 5'-untranslated region (5'-UTR) of the mRNA of the type III secretion substrate and the amino acid sequence of secretion substrates (6, 7). Two distinct amino acid regions have been identified within type III secretion substrates from Yersinia that are required for secretion in addition to mRNA signals (18). This suggested that multiple secretion signals exist for some type III secretory proteins and that some type III secretion substrates may be secreted by more than one type III mechanism (18, 118).
With few exceptions, it is thought that all flagellar proteins,
including the transcriptional regulator FlgM, are secreted by a type
III pathway that involves passage through the center of the flagellar
substructures (84). Following completion of the hook-basal
body, secretion of the FlgM anti-28 factor occurs by
this pathway (52, 108). The work presented below is a
characterization of the determinants contained within FlgM that are
required for it to be specifically recognized and secreted by this
pathway (20).
Studies on the determinants for FlgM secretion by the flagellar type
III secretion pathway suggest a mechanism that is similar to the
results reported for the Yersinia type III secretion system. Evidence suggests that FlgM secretion can occur through both amino acid- and mRNA-dependent pathways. FlgM secretion does not need to be
coupled to translation. Replacement of the 5'-UTR of flgM with the 5'-UTR from either the lac operon or the arabinose
operon allowed FlgM secretion (20). Evidence also suggests
that multiple amino acid regions can serve to direct FlgM secretion.
Deletion of amino acids 3 through 11, 17 through 52, or 55 through 92 still allowed secretion even when the 5'-UTR was from the
lac operon (20). It is still possible that the
mRNA signal lies between amino acids 11 and 17. The efficiency of FlgM
secretion was influenced by the 5'-UTR. FlgM translated from the class
2 flgAMN transcript was found primarily in the cytoplasm,
whereas FlgM translated from the class 3 flgMN transcript
was found primarily in the external environment (50). Thus,
during hook-basal body assembly, when the role of FlgM is to inhibit
28 activity, FlgM is made from a transcript where
translation is not efficiently coupled to secretion. After hook-basal
body completion, 28-dependent transcription occurs, but
FlgM is also produced from a 28-dependent class 3 transcript. However, FlgM expressed from this transcript is coupled to
its secretion from the cell. Why express FlgM from a class 3 transcript
if it is destined to be secreted? Loss of FlgM results in an increase
in the number of flagella (67) and an increase in their
length (C. Jones and S. Aizawa, unpublished observations), resulting in
reduced motility. The coupling of class 3 flgM translation
to secretion may provide a mechanism by which to regulate the final
length of the flagellum for optimum motility.
FlgN Protein
As mentioned above, evidence exists for independent mRNA and amino acid type III-dependent secretion signals. The N terminus of all late assembly secreted proteins, FlgM, FlgK, FlgL, FliD, FliC, and FljB, is required for secretion, yet there is no homology between the N termini of these proteins. One characteristic common to the late assembly secreted proteins is that their N termini are all disordered in structure (4, 21, 127-129). Perhaps the lack of structure in the N termini of the late assembly secreted proteins provides a polypeptide secretion signal in addition to a possible mRNA signal. Another plausible source for the secretion signal lies outside the secreted proteins themselves in another set of proteins, the type III secretion chaperones. Type III secretion chaperones are known to bind type III secretion substrates, and this binding is thought to be required for secretion of type III secretion substrates and to prevent proteolytic degradation of the secretion substrates in the cytoplasm (10). The FlgN and FliT proteins have been reported to be type III secretion chaperones for the flagellar late assembly secreted proteins (29). However, FlgN and FliT were found to bind the C-terminal region of their target proteins, not the N-terminal secretion signal domain. The flagellum is hollow, and subunits pass through a central channel of 25 to 30 Å (23, 93, 107). The size of the channel requires that the subunits pass through as partially folded monomers. The FlgN protein is known to bind the FlgK and FlgL proteins, while the FliT protein binds FliD (29). Binding of FlgN or FliT to the unstructured N terminus of late assembly secreted proteins might protect them from proteolytic degradation. Also, binding of FlgN or FliT to these proteins suggests that a secretion signal that directs FlgK, FlgL, and FliD (and other late assembly secreted proteins) to the flagellum-specific secretion apparatus could lie in its associated chaperone protein in addition to an amino-terminal secretion signal. Once brought to the secretion apparatus, the unstructured nature of the N termini of the late assembly secreted proteins maintained by chaperone binding could then serve to facilitate passage into the secretion channel.
The flgN gene is transcribed in the middle class
flgAMN operon in addition to the 28-dependent
class 3 flgMN transcript (32, 50, 72, 111). Mutations in flgN result in a poorly motile phenotype with
only one or two flagella per cell instead of the normal complement of
six to ten (72). A closer examination of flagellar
substructures revealed that flgN mutant cells possessed
hook-basal bodies, but only a few of these had attached filaments. This
was consistent with the idea that FlgN binds FlgK and FlgL both to
escort these proteins to be secreted and to prevent their intracellular
degradation. The defect in motility of flgN mutants can be
overcome by loss of FlgM, suggesting that an increase in
28-dependent expression of late genes can bypass the
flgN defect. Overexpression of the flhDC operon
also overcomes the motility defect of an flgN mutation
(28, 50). The end result of flhDC overexpression
is also likely to be an increase in late gene expression. If FlgN
served to protect FlgK and FlgL from proteolytic degradation, then
overproduction of FlgK and FlgL could lead to increased intracellular levels of FlgK and FlgL and the ability to synthesize normal flagella.
In addition to a role as a type III secretion chaperone,
flgN has also been identified as a regulator of FlgM levels
in the cell. Evidence suggested that FlgN was a translational regulator of flgM gene expression from the class 3 promoter transcript
(50). Loss of flgN also led to increased
transcription of the class 3 fliC promoter in a hook-basal
body mutant strain (50). Strains defective in
flgN show reduced levels of intracellular FlgM levels, which
would account for increased class 3 promoter transcription in
flgN null strains (50). However, a
lacZ translational fusion to flgM was not
expressed in an flgN hook-basal body double mutant strain
even though this fusion was transcribed at wild-type levels. Finally,
FlgM protein produced in strains defective in flgN was still
secreted, suggesting that FlgN is not required for FlgM secretion
(50). The fact that FlgN acts on class 3 flgMN
translation suggests that some 28-dependent class 3 transcription does occur prior to hook-basal body completion and this
transcription is important to the efficient coupling of flagellar
development to regulation of gene expression.
The finding that FlgN is a type III chaperone protein for FlgK and FlgL and a translational regulator of flgM transcribed from its class 3 promoters suggested a model for type III secretion that is inclusive of both mRNA and amino acid secretion signals (50). A type III secretion substrate could be targeted to the secretion apparatus by chaperone binding, especially if the chaperone is located at the base of the type III secretion apparatus. In addition, a requirement for type III secretion chaperones for translation of secretion substrates suggests a possible role to keep substrates translated at the base of the apparatus from diffusing away prior to secretion. Perhaps type III secretion chaperones such as FlgN have two roles in secretion. One role is to escort secretion substrates to the secretion apparatus. Another role is to help translate secretion substrates and to keep newly translated substrates from diffusing away from the base of the type III secretion apparatus prior to secretion. After translation, FlgN could bind the C terminus of FlgK and FlgL (and FlgM?) at the flagellar type III secretion apparatus and prevent these proteins from diffusing from the base of the flagellar structure before they can be secreted.
There is likely to be a redundancy in the secretion signal. One is an
mRNA secretion signal that favors the coupling of translation to
secretion. The second secretion signal would be an amino acid signal in
the N terminus of the secreted protein that could be a structural
signal (disordered) and/or in the amino acid sequence of the chaperone
or translational regulatory proteins recognized by the type III
secretion apparatus protein. A model for the coupling of the
translation of class 3 transcripts to secretion of the substrate
protein by FlgN is presented in Fig. 6.
The mRNA transcript is translated at the base of the flagellar
secretion apparatus by the action of the chaperone protein and the Flk
protein. Flk, discussed below, is currently thought to bind ribosomes
to direct the localized translation of specific mRNA transcripts at the cytoplasmic face of the inner membrane (50, 53). In
conjunction with FlgN, Flk will localize the translation of the class 3 flgMN transcripts at the base of the flagellar secretion
apparatus. Binding of FlgN to the C-terminal regions of the secreted
proteins prevents their escape from the base of the secretion apparatus before they can be secreted.
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Hook Completion and Specificity of Type III Secretion
Hook completion is a key checkpoint in flagellar development. It signals FlgM secretion and initiation of flagellin gene expression. FlgM responds to completion of the basal body-hook structure. Still, it remains to be determined what senses completion of the hook and signals the type III secretion apparatus to "stop" secretion of hook subunits and "start" FlgM and late assembly protein secretion. The first clue came with the isolation of mutations in a gene responsible for hook length control, fliK. fliK mutants were identified as having a polyhook phenotype (100). In a wild-type strain, hooks grow to a length of about 55 nm (36), whereas in an fliK null mutant strain, hook length varies from 40 to 900 nm and the mutants are nonmotile (36). Results from several labs support a model in which the switch to late protein secretion is from signals sent to the type III secretion apparatus upon completion of the hook-basal body (36, 70, 88, 130). Because fliK mutants are defective at filament assembly, they are nonmotile. Motile revertants of an fliK null allele have been isolated and include extragenic suppressors that map exclusively to the flhB locus (36, 70). FlhB is a component of the type III secretion apparatus. It has been hypothesized that FliK measures hook length and signals the FlhB component of the type III flagellum-specific secretion apparatus that the hook is complete. The type III secretion machinery switches substrate specificity from secretion of hook-type subunits to secretion of FlgM and late assembly proteins (60, 88).
It was thought that the flhB mutants that allowed secretion of late assembly substrates in the fliK mutant strains (flhB/FliK bypass) were no longer able to distinguish between middle and late class secretion substrates. However, in a strain possessing the flhB/FliK bypass mutation and also defective in the hook (flgE) gene, FlgM was not secreted (65). Kutsukake reported that an additional null mutation in the rflH locus was also required to allow FlgM secretion in the flgE flhB/FliK bypass double mutant background. He proposed that RflH acted as a gate to prevent secretion of late class secretion substrates prior to hook-basal body completion (65). This suggests that the secretion apparatus possesses a physical barrier to secretion of late substrates prior to hook-basal body completion in addition to secretion signal specificity by FliK and FlhB. Null mutations in the rflH locus, called flk, were isolated as causing a defect in flgM translation in strains defective in P- and L-ring assembly (51, 53). Thus, the physical barrier to secretion of FlgM by RflH (Flk) may be associated with a role in cotranslation secretion of FlgM (discussed below).
The mechanism of hook length measurement is not known. However, recently it has been shown that the FliK protein is secreted as a hook-type protein (89). Prior to hook completion, FliK was found in the spent growth medium, but in strains with intact hooks, FliK was found in the cytoplasm (89). This result suggested that secretion of FliK itself was dependent on hook length. Once the hook has reached its proper length, FliK was retained in the cytoplasm, where it could interact with FlhB of the type III secretion machinery to change secretion specificity from hook-type secretion substrates to filament-type secretion substrates, including FlgM. It remains to be determined how a completed hook can prevent the secretion of FliK.
flgM mRNA Translation Coupled to Ring Assembly
The flk gene was identified as a positive regulator of
the activity of FlgM at an assembly step just prior to hook-basal body completion, at the point of assembly of the P- and L-rings
(51). FlgM inhibition of 28-dependent class 3 flagellar gene transcription was relieved in P- and L-ring assembly
mutants (flgA, flgH, or flgI) by introduction of
a null mutation in the flk gene (51). Strains
defective in hook-basal body synthesis exhibit an increase in the
intracellular levels of FlgM, presumably due to the inability of FlgM
to be secreted. In P- and L-ring mutant strains, the addition of a
recessive mutation in flk resulted in a reduction in
intracellular FlgM levels down to those seen in wild-type
(Fla+) strains (53). The reduction in
intracellular FlgM levels by mutations in the flk gene was
concomitant with a significant reduction in flgM-lacZ mRNA
translation, expressed from the class 3 flgMN transcript, in
P- and L-ring mutant strains, while the translation of
flgM-lacZ mRNA from the class 2 flgAMN transcript
was unaffected. No reduction in either flgAMN or
flgMN mRNA stability was measured in the absence of Flk in
Fla+, ring mutant, or hook-basal body deletion strains.
This led to the conclusion that the reduction in intracellular FlgM
levels by mutation in the flk gene occurs only at the level
of flgM mRNA translation (53). The mechanism by
which Flk couples flgM translation to P- and L-ring assembly
is not understood. Flk is thought to be anchored to the cytoplasmic
membrane by a C-terminal 18-amino-acid hydrophobic amino acid segment
(53). It has homology to a region of the S1 RNA-binding
protein that interacts with the 30S subunit of the ribosome. It is
tempting to speculate that Flk can substitute for S1 to bring the
ribosome to the cytoplasmic membrane to translate proteins that need to
be localized there (Fig. 6). In the case of FlgM, it may facilitate the
coupling of FlgM translation to FlgM secretion by the flagellar
secretion apparatus.
Both Flk and FlgN were found to regulate translation of flgM expressed from the class 3 flgMN transcript in hook-basal body mutant strains. However, in strains possessing intact hook-basal bodies, loss of either flgN or flk had no effect on translation of flgM from the class 3 transcript (50, 53). This suggests that the effect of Flk and FlgN on translation or cotranslation secretion is redundant to the normal flagellar cotranslation secretion mechanism. It is likely that the flagellar basal structure includes the ability to couple translation to secretion in order to ensure the efficiency of assembly. Such a possibility remains to be addressed.
REGULATION OF NUMBER OF FLAGELLA
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The exact regulatory requirements leading to the evolution of the
interwoven regulatory circuits of the FlhD-FlhC complex, 28, and FlgM are not yet completely clear. They may
promote ordered and efficient assembly of the flagellum by precisely
timing the accumulation of precursor molecules in response to the
developmental state of the structure. These regulatory systems may also
provide a means of independently regulating the number and length of
flagella. Flagellar biogenesis is intimately related to cell division
and regulation of flagellar gene expression. The growth of the filament is independent of the cell cycle, and filament length appears to be
under a localized control mechanism at the base of each flagellum,
whereas the number of filaments (or flagellar basal bodies) is
dependent on cell cycle (3). Flagellum number can be
increased by increasing either FlhDC levels or 28
activity. A mutation in flgM has been reported to increase
flagellum number by two- to threefold in S. enterica serovar
Typhimurium (67). Surface-induced differentiation of
S. enterica serovar Typhimurium and E. coli from
swimmer cells to swarmer cells also resulted in at least a twofold
increase in flagellum number (33). This transition resulted
from an increase in flhDC transcription. Since
28 is now known to activate transcription of the
flhDC operon, the number of flagella per cell might
ultimately be regulated at the level of 28 activity.
LINK BETWEEN REGULATION OF EXPRESSION AND EFFICIENCY
OF ASSEMBLY
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It is known that structural subunits are assembled into the flagella in a specific order. Therefore, if precursors accumulate as cellular pools that are not predisposed to incorporation into any particular growing flagellum, then either ordered protein expression or ordered secretion or both may occur. It is not known how precursor bias is accomplished in flagellar synthesis. The transcriptional hierarchy of the flagellum pathway is one mechanism that leads to the efficient assembly of the flagellum following induction of the pathway. FlgM is secreted following the completion of the hook-basal body intermediate structure (52, 108). This results in the full induction of class 3 promoter transcription. Thus, transcription of the filament genes fliC and fljB does not happen until a flagellar motor is in place onto which the filament can be assembled outside the cell. Given that the filament comprises a significant fraction of the total cell protein and at high concentrations will autopolymerize (82), it makes sense to link filament production to secretion competence.
Efficiency of assembly is further enhanced by the ability to change the protein substrate specificity at the hook-basal body assembly stage. The HAPs FlgK, FlgL, and FliD (the flagellar cap) are transcribed from class 2 promoters (66), yet they are in the late class of secretion substrate (37, 39, 40). During hook-basal body assembly, these proteins are produced in the cytoplasm (66). Once the hook is completed, the secretion apparatus substrate specificity changes to allow secretion and assembly of FlgK, FlgL, and FliD. If the HAPs were transcribed only from a class 3 promoter, as is the case for the filament, then the HAPs and the filament subunits would be produced at the same time and the HAPs might compete with filament subunits for secretion. Any filament subunits secreted prior to assembly of the HAPs would be discarded into the external medium and would lead to inefficient assembly.
In addition to the transcriptional hierarchy and secretion hierarchy, there are additional layers of posttranscriptional control that further increase the efficiency of flagellar assembly. As mentioned above, the Flk protein is hypothesized to regulate flgM mRNA translation in response to completion of the P- and L-rings and the elongation of the hook outside the cell (51, 53). In this way, intracellular FlgM levels may be lowered to allow some filament transcription just prior to hook completion. Thus, Flk may sense that hook-basal body completion is imminent and allow some filament subunits to be produced and ready for secretion as soon as they are needed. Otherwise, if filament transcription occurred only after hook-basal body completion and FlgM secretion, then a time lag would ensue in the assembly process. This time lag would include the time required for filament transcription and translation following FlgM secretion. By reducing the translation of flgM during hook elongation, some class 3 translation could allow a small amount of filament to be ready for secretion as the assembly of the HAPs is completed so that the efficiency of assembly of the entire flagellum is maximized.
Another posttranscriptional regulatory mechanism that enhances the assembly process is at the level of FlgE (hook) expression. Strains defective in basal body assembly showed reduced levels of hook protein present in the cells compared to a strain defective in HAP1 (FlgK) assembly (49). Transcription of flgE was unaffected by basal body mutations (12, 71). Recent studies of hook (FlgE) levels in different basal body-defective strains suggest that accumulation of intact hook protein is dependent upon successful assembly of previously translated flagellar components (12). In strains that were unable to assemble either the MS-ring (fliF), the switch complex (fliG, fliM, fliN), or a functional flagellar type III secretion apparatus (flhA, flhB, fliE, fliH, fliI, fliJ, fliO, fliP, fliQ, and fliR), hook protein was present at levels comparable to those in the wild type (12). However, negligible FlgE protein was detected in strains deficient for any structural or enzymatic component of basal body assembly (flgBCDFGJ) (12). Intermediate levels of FlgE were observed in strains blocked for ring or hook assembly (flgAIHD) (12). Wild-type levels were observed in strains missing the hook-filament junction proteins (flgKL) and in strains that did not have a functional flagellar type III secretion system (flhAB) (12). A threefold increase in FlgE was observed in a strain deleted for the hook length regulator FliK (12). These data suggested that FlgE expression was posttranscriptionally regulated in response to the stage of basal body assembly. FlgE translation or stability was decreased in the absence of assembly of prior hook-basal body components. This regulation of FlgE did not occur until after initiation of the basal body, because wild-type levels of the FlgE protein were present in strains unable to begin flagellar assembly (fliF mutant, MS-ring negative). The C-ring and type III secretion apparatus can be mounted onto the MS-ring once it is embedded into the inner membrane (2). Type III secretion mutant strains had wild-type levels of the FlgE protein, but mutants blocked for assembly of the basal body structural components had negligible levels of the protein (12). This suggests that either the MS-ring or the type III secretion apparatus was required for regulation of FlgE expression or stability until completion of the flagellar basal body. Once the MS-ring and the type III secretion apparatus were complete, FlgE protein levels were negatively regulated until initiation of hook assembly (12). The FlgE protein is the major component of the hook-basal body structure and the last assembled component of the hook-basal body. By preventing the accumulation of FlgE protein from the cell during rod assembly, the FlgE subunits would not compete with rod subunits that are required prior to hook assembly and cotranscribed with the flgE gene. An alternative possibility is that hook subunits and rod subunits are continuously secreted during hook-basal body assembly and that prior to rod formation, hook subunits are proteolyzed in the periplasm. This would alleviate a requirement for different secretion signals that direct rod subunits to be secreted prior to hook subunit secretion.
CELL CYCLE CONTROL
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The bacterium Caulobacter crescentus differentiates from a motile swarmer cell into a nonmotile stalked cell (reviewed in references 26, 99, and 113). Synthesis and loss of the flagellum have been clearly demonstrated to be coupled to an asymmetric cell division, and extensive and elegant studies have elucidated many of the mechanisms that couple flagellar biosynthesis to the cell cycle. This has resulted in C. crescentus's being generally regarded as a model developmental system, and the characterization of flagellar regulation has been used to elucidate the mechanism of cell cycle control in Caulobacter (47, 99). Work in E. coli has also shown that flagellar biosynthesis and cell division are coregulated (102, 103), although it has proven more difficult to separate S. enterica serovar Typhimurium and E. coli cells by stage of development than C. crescentus. However, in an elegant study using a membrane filtration technique, Prüss and Matsumura were able to synchronize an E. coli culture (102). The expression of the flhDC operon increases to a maximum during mid-log-phase growth, decreases during late log phase, and then increases to half-maximal levels during stationary phase (102). Expression of a middle flhBAE operon was also growth phase dependent but reached its peak of expression in late log phase (5). Prüss and Matsumura demonstrated that expression of the early flhDC operon, the middle flhBAE operon, and the cells' swimming speed depended on the stage of the cell cycle that the cells were in (102). Expression of flhDC peaked at the middle of a cell cycle, followed by a peak in flhBAE expression half a cycle later (102). This demonstrates the temporal nature of flagellar gene expression in E. coli. Swimming speed was highest at the time of cell division. This suggests that swimming speed is dependent on the formation of new flagella following cell division and that it takes a full cycle to form functional flagella. These results also support earlier findings that expression of flagella decreased in conditional cell division mutants under nonpermissive conditions (96).
In S. enterica serovar Typhimurium, flagellar growth was synchronized by placing the flhDC operon under control of the tetA promoter (52). Placement of the flagellar regulon under control of the inducible tetA promoter allowed the determination of whether multiple flagella can grow simultaneously versus sequentially on a single cell. As described above, the flhDC master operon is influenced by a number of global regulatory signals. Placement of the flhDC structural genes under control of the tetA promoter allowed synchronous flagellar growth following the addition of tetracycline without interference from known or unknown regulatory signals that affect the expression of the flhDC promoter (52). Electron micrograph sampling following induction by tetracycline demonstrated that multiple flagella appeared simultaneously on the cell. However, it took about two cell cycles before the cells exhibited strong swimming behavior following induction of flhDC. It could be that this represents an artifact of using the tetA promoter to induce the flhDC operon, or the increase in swimming speed shown in E. coli was due to induction of flagella from two previous cell cycles.
Prüss and Matsumura also demonstrated that FlhD in the absence of FlhC is a regulator of cell division (103), and this regulation occurs through induction of the acid response gene cadA, encodong lysine decarboxylase (101). They hypothesize that cells degrade serine and synthesize acetylphosphate, which is used to phosphorylate OmpR. Shin and Park showed that acetylphosphate inhibited flhDC expression through OmpR (117). Production of acetylphosphate might serve as an indicator of the metabolic state of the cell or its local environment. FlhD is required to sense serine depletion through acetylphosphate, resulting in a reduction in the rate of cell division. How FlhD regulates cadA expression and how CadA might affect cell division are unclear.
The multiple control mechanisms described for the flagellar regulon appear to make sense only if different motors in the same cell are built simultaneously. These results suggest that normal induction of flagellar biosynthesis would be intimately associated with the cell cycle.
CONCLUSION
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Bacterial flagella are complex structures requiring the input of over 50 genes for their assembly and function in response to the presence of attractants or repellents in the environment. Research over the last few years has revealed that regulation of the flagellar regulon is also highly complex. The efficiency at which the flagellar and chemotaxis genes are regulated throughout the assembly process and the sensitivity of the flagellar regulatory complex to numerous global regulatory systems suggest that the efficient coupling of the flagellar regulatory system to flagellum assembly is under enormous selective pressure in the environment.
The flagellar genes are transcriptionally regulated through an elaborate combination of mechanisms that create a regulon sensitive to environmental conditions, the physiology of the cell, and the developmental state of the motility and chemotaxis systems. Continued study is likely to unveil additional mechanisms, possibly including posttranscriptional mechanisms, in addition to the secretion-dependent regulation of FlgM. These may include regulation of protein stability or the efficiency of translation of individual cistrons. The numerous flagellar genes with nonstructural and unknown functions are candidates for regulators mediating nontranscriptional effects.
The exact regulatory requirements leading to the evolution of the
interwoven regulatory circuits of the FlhD-FlhC complex, 28, and FlgM are not yet completely clear. They may
promote ordered and efficient assembly of the flagellum by precisely
timing the accumulation of precursor molecules in response to the
developmental state of the structure. These regulatory systems may also
provide a means of independently regulating the number and length of
flagella. It is certain that flagellation is a significant biosynthetic undertaking for the cell and that the more rapidly these complicated organelles can be synthesized, the faster an individual cell can swim
toward a more favorable growth environment. This seems to have placed
speed and efficiency at the forefront of selective pressures that
resulted in such an intricate and versatile regulatory system. Given
the importance of a large extracellular organelle system that
constitutes a significant fraction of the total cell protein of
flagellated cells, it is not surprising that novel regulatory
mechanisms have been discovered during the study of the flagellar
regulons of bacteria.
ACKNOWLEDGMENTS
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We thank Shin-Ichi Aizawa for comments and suggestions.
This work was supported by Public Health Service grant GM56141 from the National Institutes of Health to K.T.H. G.S.C. is a recipient of a PHS National Research Award (G32 T GM07270).
FOOTNOTES
* Corresponding author. Mailing address: Department of Microbiology, Box 357242, University of Washington, Seattle, WA 98195. E-mail: hughes{at}u.washington.edu.
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