Increase in intracellular pH induces phosphorylation of axonemal proteins for activation of flagellar motility in starfish sperm
1 Department of Life Sciences, Graduate School of Arts and Sciences,
University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan
2 Sesoko Station, Tropical Biosphere Research Center, University of the
Ryukyus, Sesoko, Motobu, Okinawa 905-0227, Japan
* Author for correspondence (e-mail: cokuno{at}mail.ecc.u-tokyo.ac.jp)
Accepted 28 September 2005
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Summary |
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Key words: flagellar motility, regulation, axoneme, cAMP-independent, protein phosphorylation, intracellular pH, sperm, starfish
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Introduction |
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An increase in intracellular pH ([pH]i) is reported to be
involved in the activation of sperm flagellar motility in sea urchin
(Christen et al., 1982;
Lee et al., 1983
), marine
teleosts (Oda and Morisawa,
1993
) and mammals (Wong et
al., 1981
; Babcock et al.,
1983
). It is thought that cAMP-dependent protein phosphorylation
is associated with motility activation through increased [pH]i
(Brokaw, 1987
;
Goltz et al., 1988
;
Carr and Acott, 1989
), but
little attention has been given to the cAMP-independent phosphorylation
induced by the increased [pH]i. According to Carr and Acott
(1989
), two proteins are
phosphorylated in intact bull sperm during motility activation induced by an
increase in [pH]i, and the phosphorylation is probably not due to
changes in cAMP levels. However, it is not clear whether these phosphoproteins
are associated with the signaling pathway induced by an increase in
[pH]i nor whether the phosphorylation occurs independently of cAMP.
In addition, these phosphoproteins are located in the membrane
(Carr and Acott, 1989
).
In the present study, we demonstrate in starfish sperm that an increase in
[pH]i induces phosphorylation of axonemal proteins and activation
of flagellar motility independently of cAMP. It is reported that starfish
testicular sperm are generally immotile in seawater, but motility is activated
by histidine, which has a strong zinc-binding capacity
(Fujii et al., 1955). EDDA and
TPEN, high-affinity chelators of Zn2+, are also effective in
activating motility (Mohri et al.,
1990
; Morisawa, et al.,
2004
). Therefore the liberation of Zn2+ from the sperm
is thought to be a key factor for motility activation in starfish
(Fujii et al., 1955
;
Mohri et al., 1990
;
Morisawa et al., 2004
), but
downstream events in the signaling pathway for motility activation remain
unknown. As the first step, we show that an increase in [pH]i is
also involved in the activation of intact starfish sperm motility. We then
demonstrate pH-dependent but cAMP-independent activation of demembranated
sperm motility and associated phosphorylation of axonemal proteins.
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Materials and methods |
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Carboxy SNARF-1 AM and nigericin were purchased from Molecular Probes
(Carlsbad, CA, USA). [-32P]ATP was obtained from Amersham
Biosciences (Little Chalfont, Buckinghamshire, UK). All other chemicals were
of analytical grade.
All the experiments were carried out at room temperature except where noted.
pH measurement of seminal plasma
The testes prepared as described above were immediately transferred to a
1.5 ml Eppendorf tube, covered with mineral oil, and then centrifuged at
approximately 17 000 g for 5 min at 4°C. The supernatant
excluding mineral oil was used as seminal plasma. The pH of seminal plasma was
measured using a pH meter.
Motility measurement of intact sperm
Sperm were diluted into 40 µl of an experimental solution on a glass
slide (approximately 12x107 sperm cells
ml1). Movement of sperm was recorded using a video recorder
(HR-B12; Victor, Yokohama, Japan) and a CCD camera (CV-10; Video device,
Tokyo, Japan) mounted on a phase contrast microscope (Optiphoto; Nikon, Tokyo,
Japan). Recording of sperm movement was performed without a coverslip because
A. pectinifera sperm were found to adhere to the glass surface. The
percentage of motile sperm was calculated from the video recordings. Sperm
were counted as motile when they exhibited progressive movement. In case the
sperm head was attached to the glass slide, sperm exhibiting spontaneous
flagellar beating were also counted as motile.
The effect of histidine on sperm motility was examined using artificial seawater (ASW: 430 mmol l1 NaCl, 9 mmol l1 CaCl2, 9 mmol l1 KCl, 23 mmol l1 MgCl2, 25 mmol l1 MgSO4 and 10 mmol l1 Hepes-NaOH, pH 8.2) and Na-free ASW (430 mmol l1 choline chloride, 9 mmol l1 CaCl2, 9 mmol l1 KCl, 23 mmol l1 MgCl2, 25 mmol l1 MgSO4 and 10 mmol l1 Hepes-KOH, pH 8.2), both of which contained varying concentrations (040 mmol l1) of histidine. Choline chloride solutions (CC solution, 0.5 mol l1 choline chloride and 10 mmol l1
Hepes-KOH, pH 8.2) containing 020 mmol l1 NH4Cl were used to examine the effect of NH4Cl.
Measurement of intracellular pH by fluorescence spectrophotometer
[pH]i was measured using a fluorescent pH indicator, carboxy
SNARF-1 AM. The emission spectrum of carboxy SNARF-1 undergoes a pH-dependent
wavelength shift, causing changes in the ratio of the fluorescence intensities
at two emission wavelengths, resulting in more accurate determination. In the
present study, carboxy SNARF-1 was excited at 514 nm and monitored at 580 nm
and 640 nm. The ratio of fluorescence intensities measured at the two
wavelengths (580 nm/640 nm) was then calculated.
Stock solution (1 mmol l1) of carboxy SNARF-1 AM was made in dimethyl sulfoxide (DMSO). Sperm (2040 µl) were diluted with 5 volumes of loading buffer (0.5 mol l1 choline chloride, 10 mmol l1 Hepes-KOH, pH 7.0 and 50 µmol l1 carboxy SNARF-1 AM) and incubated at 4°C in the dark. After overnight incubation, the sperm were washed with CC solution adjusted to pH 7.0 (0.5 mol l1 choline chloride and 10 mmol l1 Hepes-KOH, pH 7.0). The sperm were resuspended in the original volume (100200 µl) of CC solution, pH 7.0 and kept on ice. 12.5 µl of this suspension were added to a cuvette containing 2 ml of experimental solution: (i) Na-free ASW, (ii) ASW or (iii) CC solution. The emission spectrum of carboxy SNARF-1 was monitored using a fluorescence spectrophotometer (F-4500; Hitachi, Tokyo, Japan). After monitoring, either 10 mmol l1 histidine (i and ii) or 20 mmol l1 NH4Cl (iii) was added and emission spectra monitored again. Carboxy SNARF-1 loaded sperm retained normal motility during the measurement of [pH]i.
It is known that the fluorescence response is significantly different when
the dye is loaded in cells. In situ calibration can be performed by
using the K+/H+ ionophore, nigericin, in the presence of
120130 mmol l1 K+ (approximately
equivalent to the intracellular K+ concentration) to equilibrate
[pH]i with the controlled extracellular pH ([pH]o;
Thomas et al., 1979;
Babcock, 1983
;
Negulescu and Machen, 1990
).
12.5 µl of the suspension containing carboxy SNARF-1 loaded sperm was added
to a cuvette containing 2 ml ofcalibration solution (120 mmol
l1 KCl, 380 mmol l1 choline chloride and
20 mmol l1 Hepes-KOH, pH 6.8, 7.0, 7.25, 7.5, 7.75, 8.0 or
8.2). Nigericin (10 µmol l1) was then added and emission
spectra monitored. A stock solution (10 mmol l1) of
nigericin was made in ethanol. [pH]i was calculated from a
calibration curve of the ratio of fluorescence intensities measured at two
wavelengths (580 nm/640 nm) vs [pH]o
(
[pH]i).
Measurement of [pH]i by fluorescence microscope
Sperm were loaded with carboxy SNARF-1 as described above. The loaded sperm
suspension was added to 19 volumes of CC solution, pH 7.0, and kept on ice. 1
µl of this suspension was diluted into 40 µl of CC solution containing 0
or 20 mmol l1 NH4Cl on a glass slide. The diluted
sperm were covered with a coverslip and observed using fluorescence microscopy
(S. Kamimura, manuscript in preparation). We used micrographs representing the
fluorescent intensity at 640 nm with 470550 nm excitation.
Motility measurement of demembranated sperm
The effect of pH on reactivation of demembranated sperm was examined. Glass
slides and coverslips were coated with 1% (w/v) bovine serum albumin (BSA) to
prevent sperm from adhering to the glass surface. Sperm were suspended in 80
volumes of experimental solution: (i) CC solution, (ii) CC solution containing
20 mmol l1 NH4Cl or (iii) ASW containing 10 mmol
l1 histidine. 5 µl of the sperm suspension was gently
diluted into 40 µl of demembranation solution [150 mmol
l1 KCl, 2 mmol l1 MgCl2 1 mmol
l1 dithiothreithol (DTT), 1 mmol l1 EDTA,
0.04% (w/v) NP-40 and 20 mmol l1 Hepes-KOH, pH 8.0] and kept
on ice for 3 min. After demembranation, 2 µl of this suspension was gently
diluted into 40 µl reactivation solution (200 µmol l1
ATP, 150 mmol l1 KCl, 2.2 mmol l1
MgCl2, 1 mmol l1 DTT, 1 mmol l1
EDTA, 1 mmol l1 EGTA and 20 mmol l1
Hepes-KOH, pH 7.0, 7.2, 7.4, 7.6, 7.8 or 8.0) on a glass slide. The effect of
cAMP and cGMP was examined as follows. Sperm suspended in CC solution were
demembranated and then diluted into the reactivation solution (pH 7.0 or 8.0)
containing 10 µmol l1 cAMP or cGMP. The concentration of
Mg-ATP2 in the reactivation solution was calculated as 158
µmol l1, sufficient to reactivate the demembranated
sperm. Movement of the demembranated sperm was recorded as described above,
and reactivation rate (percentage of demembranated motile sperm) was
calculated.
Labeling of axonemal proteins with 32P
Sperm were diluted with 20 volumes of experimental solution: (i) CC
solution for preparation of immotile sperm or (ii) CC solution containing 20
mmol l1 NH4Cl for motile sperm preparation, and
centrifuged at 3000 g for 5 min at 4°C. The sperm pellet
was resuspended in 5 volumes of the fresh ice-cold experimental solution
mentioned above, and homogenized using a Teflon homogenizer for 20 strokes to
separate the heads and the flagella. The separation of heads and flagella was
checked by phase contrast microscopy. The homogenized sperm suspension was
centrifuged at 800 g for 5 min at 4°C to remove sperm
heads, and the upper part of the supernatant was carefully collected in order
to prevent the contamination of the pellet (sperm heads). Collected
supernatant was checked for head contamination using a phase contrast
microscope and centrifuged at 15 000 g for 5 min at 4°C to
recover the isolated flagella. The pellet of flagella was demembranated with
demembranation solution on ice for 10 min and centrifuged at 15 000
g for 5 min at 4°C. The pellet of axonemes was diluted
with the ice-cold, ATP-free reactivation solution (pH 7.08.0; 23
mg ml1) and [-32P]ATP (2 MBq
ml1) was added to the suspension, mixed and kept at room
temperature for 10 min. After this period, the suspension was centrifuged at
15 000 g for 5 min at 4°C. The pellet was diluted with
SDS-sample buffer, heated in boiling water for 2 min and kept at 4°C until
used.
Detection of phosphoproteins
Axonemal proteins were separated by SDS-PAGE according to Laemmli
(1970) and the phosphoproteins
detected by autoradiography. SDS-PAGE was performed on 5%15%
polyacrylamide gradient gels. The gels were stained with Coomassie Brilliant
Blue R-250, dried and exposed to X-ray film for 3 days.
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Results |
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Next, the effect of histidine and NH4Cl on sperm [pH]i were examined. Carboxy SNARF-1 loaded sperm were suspended in (i) Na-free ASW, (ii) ASW or (iii) CC solution, and then histidine (i and ii) or NH4Cl (iii) was added. Fig. 3A shows the [pH]i measured before and after the addition of histidine or NH4Cl. The pH of Na-free ASW, ASW and CC solution was 8.2. When sperm were suspended in the solutions, the [pH]i increased to approximately 7.37.5, but flagellar motility was not activated. Motility activation was caused when histidine or NH4Cl raised the [pH]i approximately to 7.88.1. These results suggest that the activation of sperm flagella motility was caused when the [pH]i increased up to 7.8, and also that histidine as well as NH4Cl raised the [pH]i of sperm during the motility activation. However, histidine raised the [pH]i to only approximately 7.5 in Na-free ASW, and sperm flagellar motility was generally not activated, as shown in Fig. 1B. It is possible that histidine requires extracellular Na+ to increase the [pH]i of A. pectinifera sperm.
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Effect of pH on reactivation of demembranated sperm
The experiments described here using intact testicular sperm with
NH4Cl and histidine suggest that the increase in [pH]i
of sperm was associated with the activation of sperm flagellar motility. In
order to confirm the involvement of [pH]i increase in the motility
activation, the effect of pH on the reactivation of demembranated sperm was
examined. Sperm were suspended in CC solution alone (CC treatment), CC
solution containing 20 mmol l1 NH4Cl
(NH4Cl treatment) or ASW containing 10 mmol l1
histidine (histidine treatment), and then demembranated. The demembranated
sperm were diluted into reactivation solutions of several different pH values
(7.08.0). Regardless of the treatment given to sperm before
demembranation, the ratio of the reactivated sperm increased in a pH-dependent
manner. When sperm showed no motility activation before demembranation (CC
treatment), the optimal pH range for reactivation was 7.8 or above, which was
almost the same as that reported previously in sea urchin
(Gibbons and Gibbons, 1972)
and bull (Goltz et al., 1988
).
However, sperm demembranated after motility activation
(NH4Cl-treated sperm and histidine-treated sperm) exhibited higher
reactivation rate in the lower pH range (7.07.6), resulting in a wider
optimal range of pH (Fig. 4).
These results indicate that the increase in [pH]i induced
activation of flagellar motility. It also suggests that the activation of
flagellar motility before demembranation modified the flagellar axoneme, which
enabled the demembranated sperm to be motile at the lower pH. The effect of
cAMP on the reactivation was examined at pH 7.0 and 8.0, but the percentage of
motile demembranated sperm was not improved at either pH value. Cyclic GMP
also did not improve the motility (data not shown).
|
Detection of axonemal phosphoproteins associated with motility activation induced by an increase in [pH]i
The experiments with the demembranated sperm suggested that flagellar
axonemal proteins could be modified during the motility activation. It is
known that the activation of flagellar motility is regulated by the
phosphorylation of proteins comprising the flagellar axoneme
(Inaba, 2003). Therefore,
phosphorylation of axonemal proteins during motility activation induced by the
[pH]i increase was detected using [
-32P]ATP.
Flagella were isolated from the sperm before activation (CC-treated sperm) and
after activation (NH4Cl-treated sperm), and then demembranated. The
demembranated sperm flagella were diluted with reactivation solutions (pH
7.08.0) containing [
-32P]ATP and the
32P-labeled axonemal proteins were detected.
If phosphorylation of certain axonemal proteins is associated with motility activation induced by a [pH]i increase, the following can be assumed. When the immotile sperm in CC solution were demembranated and reactivated in reactivating solution at high pH, 32P would have been incorporated into the axonemal proteins during the reactivation (CC treatment). By contrast, the incorporation should have been prevented when sperm were demembranated after motility activation (NH4Cl treatment), since the axonemal proteins would have already been phosphorylated before demembranation. However, the prevention of incorporation may not be observed when the phosphorylated proteins are dephosphorylated before reactivation.
Fig. 5A indicates that incorporation of 32P into several axonemal proteins (25, 32 and 45 kDa) occurred in a pH-dependent manner. As previously mentioned, the proportion of reactivated sperm also increased in a pH-dependent manner (Fig. 4). It is plausible that the pH-dependent phosphorylation of axonemal proteins reflects the pH-dependent reactivation of the demembranated sperm. Fig. 5B indicates that less 32P was incorporated into axonemal proteins in sperm flagella demembranated after motility activation (NH4Cl treatment). It is likely that the phosphorylation of the axonemal proteins occurred with the motility activation before demembranation and avoided the incorporation of 32P as suggested above.
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Discussion |
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Activation of flagellar motility by histidine
The effect of histidine was first reported by Fujii et al.
(1955) and has been assumed to
be via chelation of Zn2+, since histidine has a strong
zinc-binding capacity and EDTA and EDDA, metal chelators, have a similar
effect (Fujii et al., 1955
;
Mohri et al., 1990
). However,
the details of the mechanism are not clear. The present experiments reveal
that flagellar motility activation by histidine depends on extracellular
[Na+] (Fig. 1) and
involves a [pH]i increase (Fig.
3A). These results suggest the possibility that histidine induces
the [pH]i increase through the Na+/H+
exchanger and causes the flagellar motility activation of starfish sperm.
Therefore, the liberation of Zn2+ from the sperm can be assumed to
induce the [pH]i increase through the Na+/H+
exchanger.
The motility activation of sea urchin sperm involves an increase in
[pH]i through the Na+/H+ exchanger
(Christen et al., 1982;
Lee et al., 1983
;
Lee, 1985
). Zn2+ is
also involved in the regulation (Clapper et
al., 1985
), but the mechanism of action is different. Sea urchin
sperm are known to initiate their motility when suspended in seawater.
According to Clapper et al.
(1985
), EDTA delays the
initiation of motility and Zn2+ prevents the inhibition. In
addition, EDTA depresses sperm [pH]i and Zn2+ reverses
the [pH]i. It is likely that in sea urchin sperm the liberation of
Zn2+ does not increase the [pH]i but, in contrast,
depresses it.
The effect of histidine on the motility activation of flagella decreased at
the end of the breeding season (data not shown), suggesting that there is an
association of Zn2+ with the maturity of sperm. The effect of EDTA
also decreases with maturity in starfish Asterias amurensis
(Mohri et al., 1990). It is
possible that the content of Zn2+ in starfish sperm or the
association with Zn2+ decreases as they reach maturity. The
Zn2+ content of starfish sperm should be measured throughout the
breeding season to examine this possibility.
Effect of [pH]i on flagellar motility activation
The flagellar motility of intact sperm was activated when histidine or
NH4Cl raised the [pH]i up approximately to 7.88.1
(Fig. 3A). These results were
confirmed by the experiments with demembranated sperm. Sperm demembranated
before motility activation (CC-treated sperm) exhibited a relatively high
reactivation rate (more than 50%) at pH 7.8 or more
(Fig. 4). It could be inferred
from these results that the range of [pH]i inducing the flagellar
motility activation of A. pectinifera sperm is approximately
8.0±0.2.
It has been reported that intracellular alkalization raises the enzymatic
activity of dynein (Christen et al.,
1983; Inaba,
2003
). The results shown in
Fig. 4 suggest that the
activation of flagellar motility before demembranation (NH4Cl and
histidine treatment) modifies the flagellar axoneme and enables the
demembranated sperm to be motile at the lower pH. The modification(s) on
axonemes caused during the motility activation might be associated with the
regulation of dynein.
The motility of demembranated bull sperm also increases with pH, and is
improved by addition of cAMP at the lower pH range. Cyclic AMP (10 µmol
l1) improves motility at pH 6.8, from approximately 10% to
80%. This indicates that cAMP is provided by adenylyl cyclase in a
pH-dependent manner and plays an important role in motility activation
(Goltz et al., 1988). In this
case, the pH-dependent motility activation reflects the pH-dependent increase
in cAMP level. Goltz et al.
(1988
) assumed that dynein was
phosphorylated in a cAMP-dependent manner, and in that state dynein activity
was not limited to an alkaline pH range. However, cAMP did not improve the
motility of demembranated starfish sperm in the present experiments. It is
possible that dynein is phosphorylated through a cAMP-independent mechanism in
starfish.
pH-dependent and cAMP-independent protein phosphorylation
In salmonid fish and mammals, it is reported that cAMP is necessary for the
reactivation of demembranated sperm
(Morisawa and Okuno, 1982;
Ishida et al., 1987
) and that
cAMP-dependent phosphorylation is associated with the flagellar motility of
sperm (Tash et al., 1984
;
Morisawa and Hayashi, 1985
;
Hayashi et al., 1987
;
Si and Okuno, 1995
; Inaba et
al., 1998
,
1999
;
Itoh et al., 2001
; Fujinoki et
al., 2001
,
2003
). In starfish A.
pectinifera, pH-dependent reactivation of demembranated sperm was
observed without cAMP (Fig. 4),
and axonemal proteins (25, 32 and 45 kDa) were phosphorylated during the
pH-dependent reactivation (Fig.
5). These results suggest that pH-dependent and cAMP-independent
phosphorylation is involved in the motility activation of starfish sperm
flagella.
It was thought that cAMP-dependent protein phosphorylation is associated
with the motility activation through an increase in [pH]i
(Brokaw, 1987;
Goltz et al., 1988
;
Carr and Acott, 1989
), but
little attention has been given to the cAMP-independent phosphorylation
induced by the [pH]i increase. Whether the increasing
[pH]i induces the phosphorylation of dynein component(s) or other
axonemal proteins independently of cAMP remains unknown. Two proteins in
intact bull sperm are phosphorylated during motility activation induced by an
increase in [pH]i, and the phosphorylation is thought to be
cAMP-independent because increasing [pH]i does not raise the cAMP
level (Carr and Acott, 1984
,
1989
). However, it is not clear
whether these phosphoproteins are associated with the signaling pathway
induced by an increase in [pH]i, since the [pH]i of
intact sperm loaded with 32PO4 is raised and the
phosphoproteins labeled during the motility activation are detected.
Therefore, independence from cAMP is also not clear. In addition, the
phosphoproteins are located in the membrane
(Carr and Acott, 1989
). This is
the first report describing the pH-dependent and cAMP-independent
phosphorylation of axonemal proteins in motility activation.
The detergent-soluble fraction contains cAMP-dependent protein kinase and
its potential substrate. They together give cAMP-sensitivity to the
demembranated sea urchin and starfish sperm
(Ishiguro et al., 1982).
Whether cAMP-dependent protein kinase and/or the substrates were removed from
the axoneme during the demembranation was not investigated. This study does
not contradict the association of cAMP with motility activation induced by
increasing [pH]i, but proposes another, new mechanism regulating
motility activation. It is conceivable that cAMP-dependent phosphorylation and
pH-dependent phosphorylation regulate flagellar motility together. Actually,
we detected cAMP-dependent phosphorylation in addition to pH-dependent
phosphorylation when cAMP was added to reactivation solution (pH
7.08.0; A. Nakajima et al., manuscript in preparation). Cyclic AMP did
not improve the percentage of motile demembranated sperm but might be
associated with the regulation of other motility factors. How pH-dependent
phosphorylation relates to cAMP-dependent phosphorylation in the regulation of
flagellar motility will need to be examined.
Using another species of starfish, A. amurensis, the outer dynein
arms were extracted from the axoneme using high salt solution and fractionated
by centrifugation on sucrose density gradient. A protein in the dynein
fraction, thought to be a light chain of the dynein, was phosphorylated during
the pH-dependent and cAMP-independent motility activation of demembranated
sperm flagella (A. Nakajima et al., manuscript in preparation). It has been
reported that the outer dynein arms of human sperm performed pH-dependent
activation, since reactivation of sperm lacking the outer dynein arms was not
pH-dependent, in contrast to reactivation of normal sperm
(Keskes et al., 1998). The
present study suggests a possibility that the increase in [pH]i
activates dynein through the pH-dependent and cAMP-independent phosphorylation
of dynein component(s) and/or other axonemal proteins.
Conclusion
The present study demonstrates that an increase in [pH]i induces
the phosphorylation of axonemal proteins and flagellar motility activation in
starfish sperm. It is possible that an increase in [pH]i regulates
the flagellar motility activation through phosphorylation. Moreover, the
pH-dependent phosphorylation and motility activation are independent of cAMP,
suggesting the involvement of a new mechanism for regulation of flagellar
motility.
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Acknowledgments |
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References |
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