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Journal of Bacteriology, June 2004, p . 4014-4018, Vol . 186,
No . 12
Cross-Regulation in Vibrio parahaemolyticus: Compensatory Activation of
Polar Flagellar Genes by the Lateral Flagellar Regulator LafK
Yun-Kyeong Kim and Linda L . McCarter*
Department of Microbiology, The University of Iowa, Iowa City, Iowa 52242
Received 26 November 2003/ Accepted 17 March 2004
Gene organization and hierarchical regulation of the polar flagellar
genes of Vibrio parahaemolyticus, Vibrio cholerae, and
Pseudomonas aeruginosa appear highly similar, with one puzzling
difference . Two
 54-dependent
regulators are required to direct different classes of intermediate
flagellar gene expression in V . cholerae and P . aeruginosa,
whereas the V . parahaemolyticus homolog of one of these
regulators, FlaK, appears dispensable . Here we demonstrate that there
is compensatory activation of polar flagellar genes by the lateral
flagellar regulator LafK .
Acting as propellers driven by rotary motors, flagella are highly
complex and efficient molecular machines . Flagellar assembly, which
occurs via a flagellum-specific type three secretion system,
initiates with the formation of a ring structure in the membrane . It
proceeds to formation of a basal body hook and culminates with the
polymerization of the extracytoplasmic propeller-like filament,
subunits of which are secreted through the basal body hook structure
(reviewed in reference 9) . The assembly of this
complex organelle requires the products of more than 35 genes .
Flagellar gene expression is carefully regulated by a number of
mechanisms such that there are sequential classes of gene expression
coupled to morphogenesis of the organelle (reviewed in reference
1) .
Although the classes of temporally expressed flagellar genes are
generally comparable (with a few variations), the specific regulators
and sigma factors controlling early, middle, and late gene expression
differ considerably . Flagellar gene organization and the specific
transcriptional regulators of polar flagellar genes appear similar in
many Vibrio and Pseudomonas species (reviewed in
reference 14) . In these organisms,
54-dependent
transcription factors regulate expression of intermediate genes,
which encode components of the flagellar export-assembly complex
and basal body hook structure . An alternate sigma factor (28)
directs late polar gene expression, including transcription of
propeller-encoding genes (e.g., flagellin genes) . In Vibrio
cholerae and Pseudomonas aeruginosa, experiments suggest that
there are four classes in the temporal hierarchy of expression,
with two subclasses of intermediate genes requiring distinct
54-dependent
regulators (5, 16) . Loss of function of
either regulatory gene blocks the flagellar gene expression pathway
(2, 8, 16,
17) . These
54-type
transcriptional regulators are also found in Vibrio
parahaemolyticus and other members of the family Vibrionaceae .
In all of these organisms, the regulators are encoded by two linked
operons: flaK and flaLM in V . parahaemolyticus,
flrA and flrBC in V . cholerae and V . fischeri, and
fleQ and fleSR in P . aeruginosa (Fig.
1) . FlrA and FleQ are essential for flagellar
formation and activate expression of certain intermediate genes,
including those encoding export and assembly components (5,
16) . FlrA and FleQ are also required for transcription of
the downstream flrBC and fleSR operons, which encode
two-component regulatory components (2,
16) . The sensor kinase component is presumably
autophosphorylated in response to as-yet-undefined signals and acts
as the phosphodonor to activate the
54-dependent
response regulator, which is necessary for expression of other
intermediate flagellar genes such as those encoding the hook basal
body (4) . This regulatory scheme is also pertinent to
Vibrio fischeri. Mutation of the V . fischeri flrA renders
the organism entirely nonmotile (15) .
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FIG . 1 . Polar and lateral flagellar regulators . (A) Physical map of the
locus encoding the V . parahaemolyticus polar flagellar
regulators . A potential rho-independent transcriptional terminator
(closed square) occurs between the coding regions for flaK and
flaL . Homologous gene clusters are found in other bacteria such as
V . cholerae (flrA flrBC) and P . aeruginosa (fleQ
fleSR) . The percent identities obtained in a pairwise BLAST analysis
(22) with the V . parahaemolyticus homolog are
provided in parentheses . FlaK, FlaM, and their homologs appear to be
transcription factors with potential
54-interacting
domains . FlaL and its homologs are potential sensor kinases . FlaM and
its homologs, in addition to containing
54-interacting
domains, are potential response regulators . The positions of insertions
introduced into the flaK coding region are indicated: a
chloramphenicol resistance cassette was inserted into the NsiI site at
position bp 200 (to make flaK1) and the HpaI site at bp 640 (to
make flaK2) . (B) The lateral flagellar regulatorygene lafK
also encodes a potential
54-interacting
transcription factor . LafK is essential for swarming but is not required
for swimming motility . The position of the insertion used to disrupt
lafK is indicated: a gentamicin resistance lacZ cassette was
introduced into the SacI site at bp 154 of the coding region . The
percent identities obtained in a BLAST analysis of LafK with its polar
flagellar homolog from each organism are shown . (C) Clustal W alignments
of FlaK and LafK (http://www.ebi.ac.uk/clustalw) .
The alignment symbols denoting degree of conservation are as follows: *,
residues are identical; :, conserved substitutions; and ., semiconserved
substitutions . The top alignment compares FlaK (488 amino acids) in the
top line and LafK (444 amino acids) in the lower line . The centrally
located, conserved
54-interacting
domains (pfam00158) are underlined for both regulatory proteins . Also
underlined are N terminally located conserved HTH_8 domains (pfam02954)
common to bacterial regulatory proteins in the FIS family . The bottom
alignment shows the comparison of the HTH_8 domains.
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There has been a puzzling discrepancy between observations of the
phenotype of mutants of V . parahaemolyticus that is not in
accordance with the regulatory scheme deciphered for other polarly
flagellated Vibrio and Pseudomonas species . The flaK
gene was discovered as an open reading frame encoding a potential
54-dependent
regulator located downstream of the flagellin chaperone-encoding gene
fliS (formerly named flaJ) . The gene was disrupted, but
disappointingly it was not found to be important for swimming
motility (21) . Bacteria with flaK lesions have only
slight swimming motility defects, an observation that is not
consistent with FlaK being required for intermediate flagellar gene
expression . Therefore, another regulatory scenario must be postulated
for V . parahaemolyticus . In this work, we reexamined the
phenotype of flaK mutants and elucidated a key distinction for
polar flagellar regulation in V . parahaemolyticus .
The polar flagellar regulator FlaK is homologous to P . aeruginosa
FleQ and V . cholerae FlrA, but loss of flaK is dispensable whereas
FleQ and FlrA are essential for swimming motility. In semisolid
motility plates, both polar and lateral flagellar systems contribute
to motility (3, 13) . Moreover, mutations
disabling the polar system lead to induction of the lateral
system so that impairment of polar flagellar function cannot easily
be examined in a swarming-competent strain (12) . As a
result, defects in swimming motility can best be studied in a
strain inactivated for swarming motility . Motility for strain LM1017
(Fla+Laf–) is solely the result of swimming, with
no contribution from swarming because of a lesion in the lateral
flagellar hook gene (3) . In prior work, a
chloramphenicol resistance cassette was used to disrupt the flaK-coding
region in strain LM1017 (21) . Insertions at two
restriction sites were used to create the flaK disruptions
(Fig . 1) . Mutants with flaK lesions showed
radial expansion in motility plates (Fig . 2A) and were
motile when viewed in the light microscope . To further probe
the mutant phenotype, immunoblot analysis of liquid-grown cultures
demonstrated that the strains produced polar flagellins (Fig .
2B) . Swimming motility and flagellin production were only
slightly decreased from the parental strain . For comparison, results
for the nonmotile, nonflagellated fliS mutant are shown . The
fliS strain (also derived from LM1017) contains a lesion in
a polar flagellin chaperone-encoding gene (21) .
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FIG . 2 . Introduction of mutations in flaK does not abolish
swimming motility . (A) Swimming motility in semisolid tryptone motility
agar (1% tryptone, 2% NaCl, 0.32% agar) after overnight incubation at
room temperature . Three single colonies of each strain were inoculated .
(B) Immunoblot of polar flagellin (Fla) profiles with samples prepared
from strains grown in heart infusion broth . Immunoblotting conditions
have been described (3) except that blots were
incubated with primary antiserum for 14 h . The longer incubation period
allowed detection of a cross-reacting cellular protein that serves as a
loading control . All strains were derived from strain LM1017, which
contains a defect in the lateral flagellar hook gene; therefore there is
no contribution to motility from the lateral flagella (Fla+
Laf–) . Strains: lane 1, LM1017 (flgE313L);
lane 2, LM4348 (fliS1::Camr in LM1017); lane 3, LM4347
(flaK1::Camr in LM1017); and lane 4, LM4349 (flaK2::Camr
in LM1017) . The nonmotile control strain LM4348 contains a defect in the
polar flagellar chaperone fliS (formerly named flaJ) (21).
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The lateral flagellar regulator is also homologous to FlaK.
What is distinctive about V . parahaemolyticus? One clear difference
between V . parahaemolyticus and the other polarly flagellated
bacteria is that V . parahaemolyticus possesses dual flagellar
systems: the polar system is utilized for swimming in dilute
liquid environments, and the lateral (or peritrichous) flagella are
employed for swarming, i.e., movement over surfaces or through
viscous conditions (reviewed in reference 11) . The two
flagellar gene sets are distinct, and in fact they are found on
different chromosomes (10) . Approximately 50 polar
and 40 lateral flagellar genes have been identified (7,
13, 20) . Flagellar gene mutants
that have a cell defective for swimming motility are not affected in
swarming motility and vice versa . We have recently elucidated the
flagellar hierarchy for the lateral system (20) . To our
surprise, regulation appeared different from the FlhDC-mediated
regulation of peritrichous flagella observed for many swarming
bacteria including Escherichia coli, Salmonella enterica serovar
Typhimurium, and Proteus mirabilis (6) . Like the
polar system, the lateral flagellar system requires rpoN
(encoding
54)
and a
54-dependent
transcriptional activator . The lateral transcriptional regulator LafK
is homologous to the polar flagellar regulators (Fig . 1) .
In a pairwise BLAST comparison (22), LafK showed 43%
identities and 66% positives with FlaK (E value = 2e-60) . A
sequence alignment for FlaK and LafK is shown in Fig . 1C; not
only is there high similarity observed within the central
54interaction
domains but there is also considerable alignment in the C-terminal
helix-turn-helix domains . This observed similarity between polar
and lateral flagellar regulators suggested the hypothesis that
perhaps LafK was responsible for compensating for the loss of FlaK .
The lafK gene can substitute for loss of flaK function.
To test the idea that the lateral flagellar regulator was responsible
for polar flagellar gene expression, a mutant with defects in both
flaK and lafK was constructed . The lafK1::Genr
allele and methods for introduction onto the chromosome via allelic
exchange have been described (19,
20) . Introduction of lafK1::Genr into a
swarming-competent strain abolishes swarming motility (20)
and production of lateral flagella (Fig . 3A, compare the
mutant in lane 2 with the parent in lane 1) . These lanes in the
immunoblot also show no effect by lafK1 on levels of polar
flagellin in the flaK+ strain . Introduction of the
lafK1 allele into the swimming-only strain LM1017 (strain 3)
to make LM7010 had little effect on swimming motility (Fig.
3B, strain 4) or polar flagellin production (Fig.
3A, lane 4) . In contrast, introduction of the
lafK1 allele into the flaK-defective strain LM4347 (Fig.
3, strain 5) to make the double mutant strain
LM6856 (Fig . 3, strain 6) decreased polar flagellin
production and swimming motility . These results indicate that the
lateral flagellar regulator LafK can compensate for loss of FlaK and
direct transcription of polar flagellar genes .
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FIG . 3 . flaK or lafK mutants swim, but flaK lafK
mutants are impaired for swimming motility . (A) Polar (Fla) and lateral
(Laf) flagellin profiles in immunoblots with samples prepared from
strains grown overnight on HI plates . Strains: lane 1, LM5674 (wild
type, Fla+ Laf+); lane 2, LM7011 (lafK1::Genr
in LM5674); lane 3, LM1017 (flgE313L); lane 4, LM7010
(lafK1::Genr in LM1017); lane 5, LM4347 (flaK1::Camr
in LM1017); and lane 6, LM6856 (lafK1::Genr flaK1::Camr
in LM1017) . (B) Swimming motility in semisolid tryptone motility agar .
Plates were inoculated with three single colonies of each strain and
incubated at room temperature overnight . Strains are numbered as for
panel A.
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FlaK cannot substitute for LafK. To confirm and extend the
above observations, flaK and lafK mutations were
recombined singly and in combination into the wild-type chromosome .
Our initial studies were performed in a swimming-only strain
background that was disabled for swarming motility . This background
is most suitable for directly assessing effects on the polar system
as contributions from the lateral system mask motility defects in
semisolid "swim" plates; however, such a background is not suitable
for determining effects on the lateral system . Figure 4
shows that mutations in the polar flagellar genes flaK or
fliS do not affect the capacity of strains to swarm . The figure
also illustrates the complication of dual flagellar systems and the
compensatory action of lateral flagella in swim plates . The rates of
radial expansion of flaK or fliS strains in swim plates
are equivalent and indistinguishable from those of the wild-type
parental strain, even though the fliS strain is completely
blocked for polar flagellin synthesis and nonmotile in the absence of
lateral flagella while the flaK strain synthesizes almost
normal amounts of polar flagellin and can swim in the absence of
lateral flagella (Fig . 2 and similar immunoblot
data not shown for wild-type backgrounds) . Nevertheless, movement on
swim and swarm plates is effectively stopped by the introduction of
the lafK1 allele into flaK or fliS strains,
whereas the lafK1 allele abolished swarming in the wild-type
background but had no effect on swimming motility (Fig .
4, top row) . We interpret these results to indicate that (i) LafK
is not necessary for swimming motility, (ii) FlaK cannot compensate
for the loss of LafK by acting on the lateral system, and (iii) LafK
is essential for swarming . In summary, the results obtained with the
Fla+ Laf+ background are consistent with our
findings obtained with the Fla+Laf– background,
specifically that both flaK and lafK must be mutated to
cause severe loss of movement in swimming motility plates .
Furthermore, the data extend our findings to demonstrate that that
loss of LafK is sufficient to eliminate swarming motility, suggesting
the FlaK has no significant compensatory role in the lateral system .
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FIG . 4 . In the wild-type background, the flaK mutation has a
phenotype only in the context of a lafK mutation . Two single
colonies of each strain were inoculated on a swarm plate (2.5% heart
infusion, 2% NaCl, 1.5% agar [DIFCO]) and into a swim plate (semisolid
tryptone motility agar; 0.325% agar [Difco]) . Plates were incubated at
room temperature overnight . Strains: LM5674 (wild type), LM7011 (lafK1::Genr),
LM4471 (flaK1::Camr), LM7305 (flaK1::Camr
lafK1::Genr), LM4472 (fliS1::Camr),
and LM7395 (fliS1::Camr lafK1::Genr).
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Although the introduction of the lafK defect caused a profound
loss of motility and polar flagellin production in flaK strains,
there was slight residual motility observed in swim plates and
some low-level polar flagellin synthesis . In the light microscope,
the majority of the cells (>99%) were nonmotile; however an
occasional, highly motile bacterium could be observed . The phenotype
was observed in both the wild-type and LM1017 backgrounds and with
two flaK alleles (data not shown) . It is not clear why these
strains were not completely defective for polar motility; however, it
is probably not due to reversion or suppression . Residual motility
was observed in the presence of growth in gentamicin and
chloramphenicol to maintain selection for both alleles; it was
observed as slow radial expansion in a swim plate and was not due to
flares of motility; and if one retested the periphery of the swim
zone, the rate of swimming was slow and equivalent to that of the
starting strain . Similar residual motility (or rather, lack of a
completely nonmotile phenotype) has been reported for flrA
mutants of V . cholerae but was not observed in V . fischeri
(8, 15) .
Thus, the mystery and apparent discrepancy between polar flagellar
regulation of V . parahaemolyticus and regulation in other
Vibrio or Pseudomonas species can be in part accounted for
by the unique, compensatory ability of the second flagellar system of
V . parahaemolyticus . LafK was absolutely required for
expression of the lateral flagellar genes and swarming motility . In
contrast, FlaK was not absolutely required for expression of the
polar flagellar genes and swimming motility . Mutants with flaK
defects can still swim (albeit with a slightly slower expansion rate)
and produce polar flagellins (albeit with a slight decrease in the
amount of protein) . Introduction of the lafK defect into
flaK strains caused a significant decrease in swimming motility
and production of polar flagellins . The overlapping activity of the
lateral and flagellar regulators is not reciprocal, as flaK
cannot compensate for lafK . Thus, this shared regulation
occurs in only one direction . It will be interesting to explore what
this regulation means in terms of flagellar gene expression and
motile behavior . Although distinct gene sets encode each flagellar
system, the constituents of the navigation system (i.e., chemotaxis
signal transduction) are shared (18) . There is
only one set of central cytoplasmic chemotaxis phosphorelay-encoding
genes, and these are found in association with polar flagellar genes
(10) . Future work will examine the effect of these
mutations on chemotaxis gene expression . Perhaps this
cross-regulation of polar flagellar genes by the lateral flagellar
regulator is a mechanism to ensure expression or upregulation of the
chemotaxis genes when the bacterium is growing on a surface and
locomotion is driven by the numerous lateral flagella .
This work was supported by National Science Foundation award number
MCB-0315617 .
* Corresponding author . Mailing address: Department of
Microbiology, The University of Iowa, Iowa City, IA 52242 . Phone: (319)
335-9721 . Fax: (319) 335-7679 . E-mail:
linda-mccarter@uiowa.edu .
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