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Journal of Bacteriology, November 2001, p. 6365-6371, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6365-6371.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
An Archaeal Photosignal-Transducing Module Mediates
Phototaxis in Escherichia coli
Kwang-Hwan
Jung,
Elena N.
Spudich,
Vishwa D.
Trivedi, and
John L.
Spudich*
Department of Microbiology and Molecular
Genetics, University of Texas
Houston Medical School, Houston,
Texas 77030
Received 13 November 2000/Accepted 7 August 2001
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ABSTRACT |
Halophilic archaea, such as Halobacterium salinarum
and Natronobacterium pharaonis, alter their swimming
behavior by phototaxis responses to changes in light intensity and
color using visual pigment-like sensory rhodopsins (SRs). In N.
pharaonis, SRII (NpSRII) mediates photorepellent responses
through its transducer protein, NpHtrII. Here we report the expression
of fusions of NpSRII and NpHtrII and fusion hybrids with eubacterial
cytoplasmic domains and analyze their function in vivo in haloarchaea
and in eubacteria. A fusion in which the C terminus of NpSRII is
connected by a short flexible linker to NpHtrII is active in phototaxis
signaling for H. salinarum, showing that the fusion does
not inhibit functional receptor-transducer interactions. We replaced
the cytoplasmic portions of this fusion protein with the cytoplasmic
domains of Tar and Tsr, chemotaxis transducers from enteric eubacteria.
Purification of the fusion protein from H. salinarum and
Tar fusion chimera from Escherichia coli membranes shows
that the proteins are not cleaved and exhibit absorption spectra
characteristic of wild-type membranes. Their photochemical
reaction cycles in H. salinarum and E.
coli membranes, respectively, are similar to those of native NpSRII in N. pharaonis. These fusion chimeras mediate
retinal-dependent phototaxis responses by Escherichia
coli, establishing that the nine-helix membrane portion of the
receptor-transducer complex is a modular functional unit able to signal
in heterologous membranes. This result confirms a current model for
SR-Htr signal transduction in which the Htr transducers are proposed to
interact physically and functionally with their cognate sensory
rhodopsins via helix-helix contacts between their transmembrane segments.
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INTRODUCTION |
Halobacteria exhibit phototaxis
responses to changes in light intensity and color using the
seven-transmembrane retinylidene photoreceptors sensory rhodopsins I
and II (SRI and SRII) (8). Light-activated SRI and SRII
transmit signals to their cognate transducers, HtrI and HtrII,
respectively. The Htr proteins contain two transmembrane helices and
cytoplasmic methyl-accepting and His-kinase-activating domains
(21, 38) homologous to those of chemotaxis
transducers of eubacteria, such as the
Escherichia coli and Salmonella enterica serovar
Typhimurium Tsr and Tar, chemotaxis transducers for serine and
aspartate, respectively (20, 29). Sensory rhodopsin II
from Natronobacterium pharaonis (NpSRII) is very similar in
spectroscopic and functional properties to the repellent receptor SRII
in Halobacterium salinarum, and it has been found to be more
stable in response to variation in external conditions such as
pH and ionic strength (23). The NpSRII protein mediates a
repellent response to blue-green light (maximum 
, 497 nm) when it is
coexpressed with its transducer, NpHtrII, in H. salinarum
(16). Also, when expressed in E. coli, NpSRII
is capable of binding all-trans retinal to form a
blue-green-light-absorbing pigment (9, 24).
The SR and Htr proteins appear to be subunits of a stable molecular
complex (14, 19), and transducer chimeras show that the
specificity of interaction between SRI and HtrI and between SRII and
HtrII is encoded in the transmembrane portion of the transducers
(39). Based on this finding and studies with mutants, a
model for SR-Htr signal transduction has been proposed in which the Htr
transducers physically and functionally interact with their cognate
sensory rhodopsins via helix-helix contacts within the membrane
(27). As a test of this proposal we reasoned that the
seven helices of the receptor fused to the two transmembrane helices of
the transducer should be sufficient to form a photosignal-transducing module which could couple functionally to the cytoplasmic domain of
eubacterial chemotaxis transducers, which have been shown to contain
exchangeable cytoplasmic domains with eubacterial homologs (2,
15, 35). We therefore undertook to express both the NpSRII
receptor and its transducer, NpHtrII, in a form able to mediate
phototaxis signaling in E. coli.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
E. coli strains
were grown in Luria-Bertani medium at 30 or 37°C, and halobacterial
strains were grown in complex medium (CM) (pH 6.0) medium at
37°C (28).
The wild-type proteins and the fusion constructs of NpSRII and NpHtrII
were expressed under the fdx or bop promoter by
transformation of the H. salinarum strain Pho81Wr
(31) with pKJ305 derivatives (11). The NpSRII
and NpSRII-NpHtrII-StTar or -EcTsr fusion chimeras were expressed under
the plac1 promoter of pMS107 (30) in E. coli strains RP437 (wild type for chemotaxis) and UT5600
(5). E. coli DH5
was used for cloning the
cytoplasmic portion of StTar from the plasmid pMS107 (provided by
Jeffry Stock, Princeton University) and the cytoplasmic portion of
EcTsr from the plasmid JC3 (provided by John S. Parkinson, University
of Utah).
Construction of plasmids encoding the fusion and fusion chimera
proteins.
Restriction enzymes and T4 ligase were from Promega
(Madison, Wis.), and Pfu DNA polymerase was from Stratagene
(La Jolla, Calif.). Oligonucleotides were purchased from Fisher-Genosys
(The Woodlands, Tex.). Mevinolin was a gift from A. W. Alberts
(Merck Sharp & Dohme, Whitehouse Station, N.J.), and ampicillin was
from Sigma (St. Louis, Mo.).
Recombinant PCR was used to introduce 27 nucleotides encoding a
9-amino-acid residue linker (Ala-Ser-Ala-Ser-Asn-Gly-Ala-Ser-Ala;
5'-GCGTCGGCGTCGAACGGCGCGTCGGCG-3') (
10) between
the C-terminal
residue of NpSRII and the N-terminal residue of
NpHtrII (Fig.
1). The linker replaced the
C-terminal 15 residues of NpSRII,
which we have found to be dispensable
for normal signaling function
in
H. salinarum.
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FIG. 1.
Construction of the fusion proteins. Numbers indicate
numbers of residues in the indicated segment. P, G, and M fusions
(named for the final residue in the NpHtrII portion at the junctions)
are defined by the different junctions between the haloarchaeal and
eubacterial protein domains.
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Three different junctions were used for the chimera constructs (P, G,
and M fusion) (Fig.
1) for
E. coli expression based
on
studies of dimerization (
6,
32) and the conservation in
the region linking the second transmembrane helix and the methylation
domain (in bacterial chemotaxis this region is named after the
domain
present in histidine kinases, adenylyl cyclases, methyl-accepting
proteins, and phosphatase [HAMP domain] [
1]; we will
refer
to it as the stimulus relay domain [
8] to avoid
confusion with
the nine-residue linker joining NpSRII and NpHtrII in
this study).
Nucleotides encoding the N-terminal portion of NpHtrII and
C-terminal
portion of
S. enterica serovar Typhimurium Tar
(StTar) or
E. coli Tsr (EcTsr) were fused by overlapping
primers (top forward and
bottom backward primers) in which the first 20 bases derive from
N. pharaonis htrII and bases 21 to 40 are
from
tar or
tsr. The
first round of PCR was
performed at 31 cycles of 95°C for 1 min,
55°C 1 for min, and
72°C for 2 min, with a primer encoding the
N-terminal portion of
NpSRII (M fusion,
5'-GATATA
CAT-ATGGTGGGACTTACGACCTC-3')
and a backward overlapping primer (M fusion of
StTar,
5'-TCAATCAGCGAGCGTTG
CAT-ATGGTCGAAGGCCGCATAGA-3'),
along with a forward overlapping primer
(5'-TCTATGCGGCCTTCGAC
CAT-ATGCAACGCTCGCTGATTGA-3')
and primer encoding the C- terminal portion of the eubacterial
transducer (5'-GGCGGAGGCGATTTCGCCC-3'). The conditions for
the
second round of recombinant PCR (
7,
36) consisted of
31 cycles
of 95°C for 1 min, 50°C for 1 min, and 72°C for 5 min
with two
first-round PCR products. The recombinant PCR product was
purified
and digested with
NdeI (
CATATG) and
inserted into the
E. coli expression vector under the
control of isopropyl-
-
D-thiogalactopyranoside
(IPTG)
induction.
All of the cloned PCR products were sequenced to confirm that no
mutations were introduced during the PCR
amplification.
Motion analysis.
The swimming behavior of cells was
monitored by a computerized cell-tracking system (Motion Analysis
Systems, Santa Rosa, Calif.). Early stationary phase cultures of
halobacterial cells were diluted 1:13 in fresh CM (pH 6.0), incubated
for 0.5 to 2 h at 37°C with agitation, and analyzed as described
previously (26).
Transformed
E. coli cells were grown to an optical density
at 600 nm of 0.4 in Luria-Bertani medium supplemented with 50 µg
of
ampicillin/ml at 30°C on a rotating incubator followed by the
addition of 1 mM IPTG and 1 to 2 µM all
-trans retinal. At
2 h
postinduction in the dark, cells carrying the fusion proteins
in a wild-type background (RP437) were diluted 1:10 in chemotaxis
motility buffer (20 mM potassium phosphate, 0.1 mM K-EDTA [pH
7.0])
and analyzed for their swimming responses to phototaxis
stimuli using
computerized motion analysis as described previously
(
13).
Phototaxis stimuli were delivered through a Nikon 100W He/Xe short arc
lamp. Each stimulus was a pulse of 100 ms or 2 to 4
s of 500-nm
light (500 ± 5 nm; 1.8 × 10
6
ergs/cm
2) delivered to the cell in an infrared
monitoring light (>750
nm). Data were collected and processed by a SUN
SPARC-IPC workstation
(SUN Microsystems, Mountain View, Calif.).
Membrane vesicle preparation.
The halobacterial cells
containing wild-type and fusion proteins of NpHtrII and NpSRII were
grown in 200 ml of CM in a 250-ml flask with mevinolin (1 µg/ml) at
37°C on a gyratory shaker at 240 rpm for 5 to 7 days in the presence
of the light. Membrane vesicles were prepared by sonication as
described previously (4).
Transformed
E. coli RP437 and UT5600 cells were grown at
30°C. Cells were induced by adding 1 mM IPTG and 5 µM
all
-trans retinal.
After an induction period of 3.5 h
the cells were harvested, resuspended
in 50 mM Tris containing 10%
glycerol, and stored at
20°C. Cells
were lysed by sonication at
4°C in the presence of 2 mM phenylmethylsulfonylfluoride,
1 µM
leupeptin, 1 µM pepstatin, 2 to 5 mM 1,10-phenanthroline,
300 µM
p-hydroxymecuribenzoate, and 2 to 5 mM EDTA additives at
their final respective concentrations in 50 mM Tris buffer, pH
8.0.
E. coli membranes were sedimented at 100,000 ×
g for 1 h at 4°C, and the pellet was resuspended in
buffer containing 2 M NaCl
and again pelleted by centrifugation at
100,000 ×
g for 30 min
at 4°C. Yields of 10 to 12 mg
of total membrane protein per g
of frozen cell paste were typical. The
salt wash enriched ~2-fold
the integral membrane proteins by removal
of peripheral membrane
proteins. The fusion protein was extracted from
the washed membranes
with gentle shaking in extraction buffer (1%
octyl glucoside,
300 mM NaCl, 10% glycerol, 50 mM Tris buffer [pH
8.0] containing
1 mM phenylmethylsulfonyl fluoride and 1 µM [each]
pepstatin and
leupeptin) for 4 to 6 h at 4°C, and the
solubilized fraction was
collected as supernatant after centrifugation
at 20,000 ×
g. The
yield of solubilized membrane
proteins was ~70%. Protein assays
for membrane preparations were
performed with the membrane protein
estimation kit from Bio-Rad
(Hercules, Calif.). The amount of
fusion protein was assessed by using
flash photolysis signals
at 500 nm calibrated with NpSRII in 1% octyl
glucoside, 300 mM
NaCl, and 50 mM morpholinoethanesulfonic acid (MES)
buffer, pH
6.8.
Purification of His-tagged fusion protein.
The
NpSRII-NpHtrII fusion protein containing six histidine residues at the
C terminus was purified by using Ni-nitrilotriacetic acid
(NTA)-agarose beads. The halobacterial membrane was solubilized in 1%
octyl glucoside with 300 mM NaCl, 5 mM imidazole, and 50 mM MES, pH
6.0, and incubated with beads at 4°C for 16 h. The protein-bound
beads were washed with 5 mM imidazole and eluted by 300 mM imidazole,
1% octyl glucoside, and 50 mM Tris buffer, pH 7.5. E. coli
fusion chimeras containing nine histidine residues at the C terminus
were purified by Ni2+-NTA resins. The fusion
chimeras were solubilized from the E. coli membrane with
extraction buffer at 4°C for 8 to 10 h. Protein-bound beads were
incubated with 5 mM imidazole in extraction buffer to wash out proteins
bound nonspecifically. Finally, the protein was eluted by using 100 mM
imidazole in extraction buffer. Purified proteins were subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and Western blotting using anti-His-tag antibody.
Photocycle measurements.
Flash-induced absorption changes
were measured with a laboratory-constructed cross-beam flash
spectrophotometer (26), with a frequency-doubled Nd-YAG
laser (532 nm, 6 ns, 40 mJ) providing the actinic flash. Thirty-two or
sixty-four transients were recorded and averaged at a constant
temperature (18°C) with a Nicolet (Madison, Wis.) Integra
20. The amplitudes and half-life values were calculated by fitting of
single or double exponential curve fitting programs from Igor Pro
version 3.1 (WaveMetrics, Lake Oswego, Ore.).
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RESULTS AND DISCUSSION |
Expression of fusion proteins and fusion chimera proteins.
The
NpSRII-NpHtrII fusion and NpSRII-NpHtrII-StTar fusion chimera proteins
were expressed in H. salinarum and E. coli,
respectively. As an initial assessment of photochemical activity and
expression level, we analyzed H. salinarum and E. coli envelope membranes for their transient absorption changes
following a laser flash at 532 nm (Fig.
2). No flash-induced absorbance changes
in the millisecond-to-second time window were detected in membranes
from either organism when NpSRII was not present (data not shown). In
both membranes, reactions characteristic of NpSRII in its native membrane (3, 34) were observed, namely, an absorbance
transient at 400 nm with a decay half-life of <100 ms, indicative of
formation and decay of a deprotonated Schiff base form (M) of the
pigment, and a slower-decaying transient at 550 nm characteristic of
the final intermediate (O), which decays back into the dark state of
the pigment as seen at 500 nm. The O decay of NpSRII-NpHtrII in the
H. salinarum membrane is the slowest step in the photocycle (362-ms half-life) (Fig. 2). Due to the greater light scattering of the
E. coli membranes, assessment of NpSRII-NpHtrII-StTar
required addition of 1% octyl glucoside, after which clear photocycle
signals were obtained showing an O decay of 365 ms (Fig. 2). When
assayed under identical conditions (4 M NaCl and 1% octyl glucoside), the O decays of NpSRII-NpHtrII and NpSRII-NpHtrII-StTar were nearly identical at 694 and 640 ms, respectively. The NpSRII protein alone
when expressed in Pho81Wr showed photochemistry similar to that of
the fusion protein (data not shown). We observed no large effect of
NpHtrII on NpSRII photochemistry. The lack of a significant effect of
the transducer on NpSRII photocycling differs from the case of HsHtrI
and HsSRI, in which the transducer protein HtrI increases light-induced
production of the photocycle intermediate S373
(an SRI signaling conformation) and modulates the rate of return of
S373 to the prestimulus state, rendering this
return pH independent (26). Similarly, only a small effect of HsHtrII on HsSRII was observed previously for the corresponding H. salinarum proteins (22). The photocycling
rate of NpSRII is therefore not greatly affected by its being fused to
the NpHtrII protein or by its being expressed in the E. coli
membrane, despite its very different lipid composition from that of
H. salinarum.
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FIG. 2.
Flash-induced absorption difference transients.
Absorption transients of the halobacterial (A) and E.
coli (B) membranes were measured at 400, 500, and 550 nm at
5-ms time resolution. Membranes containing the halobacterial
NpSRII-NpHtrII fusion protein were resuspended at a concentration of
0.12 mg of protein/ml in a solution containing 4 M NaCl and 25 mM Tris,
pH 6.8, and membranes containing the E. coli
NpSRII-NpHtrII-StTar fusion were resuspended at a concentration of 0.11 mg of protein/ml in a solution containing 1% octyl-glucoside, 100 mM
NaCl, and 100 mM MES, pH 6.8. The half-life values for O-rise and decay
are, respectively, 29 and 362 ms for the NpSRII-NpHtrII fusion and 50 and 365 ms for the NpSRII-NpHtrII-StTar fusion.
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From the flash photolysis data we can calculate the level of expression
of photoactive fusion proteins. Assuming that the
extinction
coefficient and flash yield from the NpSRII part of
the fusion are the
same as those in
H. salinarum membranes, we
calculate
1.6 × 10
4 molecules/cell for the fusion in
H. salinarum and 1.4 × 10
4
molecules/cell for the fusion chimera in
E. coli. These
values
are consistent with the purification yields of the
NpSRII-NpHtrII
fusion in
H. salinarum, 1.0 mg/liter of
culture, and of the NpSRII-NpHtrII-StTar
fusion in
E. coli,
of 0.8 to 1.0 mg/liter of
culture.
Polyhistidine tags were engineered on the C termini of the
NpSRII-NpHtrII fusion and NpSRII-NpHtrII-StTar fusion chimera proteins,
and Ni
2+-affinity chromatography was applied to
purify these octyl-glucoside-extracted
membrane proteins from the host
cells. The 110-kDa NpSRII-NpHtrII
fusion protein from
H. salinarum and the 72-kDa NpSRII-NpHtrII-StTar
fusion protein from
E. coli were present in imidazole-eluted material
and
migrated as single Coomassie-stained bands on SDS-PAGE (Fig.
3) and as single bands on immunoblots
(Fig.
4). There was no indication
of
degradation to smaller-size His-tagged products in either case
and in
particular no indication of cleavage of the linker that
would release
NpHtrII or NpHtrII-EcTsr molecules. The similar
flash yields (Fig.
4)
indicate uniform expression of protein in
the various fusion chimeras.
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FIG. 3.
Expression of the His-tagged fusion proteins in
H. salinarum (A) and E. coli (B) analyzed
by SDS-8% PAGE. SM, solubilized membrane; FL, flowthrough
after sample was bound to the resin; EL, eluate with imidazole. A
comparable amount of protein (10 µg) eluted by imidazole was loaded
in each case and was taken from the same samples that were used for the
absorption spectra measurement of Fig. 5. An equal number and 2.5× the
number of cell equivalents in the samples were loaded in the eluate
lanes of panels A and B, respectively.
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FIG. 4.
Immunoblot analysis of the His-tagged fusion proteins in
E. coli. An identical amount of membrane protein in each
lane (4 µg) was separated by SDS-12% PAGE. The immunoblot used
anti-His-tag antibody. Relative percent flash yields were calculated
from the maximum laser flash-induced depletion values at 500 nm. P, G,
and M fusions are defined by the fusion junction position as shown in
Fig. 1.
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The absorption spectra of the purified fusion proteins from
H. salinarum and
E. coli are very similar to each other
and to
that of purified NpSRII (
34). Each shows a
vibrational fine
structure (
31) with a main band at 501 nm
(
H. salinarum fusion)
and at 503 nm (
E. coli
fusion chimera) (Fig.
5). The presence
of
a vibrational fine structure is a characteristic feature of
SRIIs and
has not been observed in other archaeal rhodopsins.
The absorption
spectra of the proteins extracted in nondenaturing
detergent and the
flash photolysis data from the membrane preparations
argue for proper
insertion and folding of the NpSRII portion of
the fusion proteins in
both
H. salinarum and
E. coli.
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FIG. 5.
Absorption spectra of purified His-tagged fusion
proteins. (A) NpSRII protein in E. coli membrane. The
absorption maximum is at 503 nm. (B) NpSRII-NpHtrII fusion protein. The
absorption maximum is at 501 nm. (C) NpSRII-NpHtrII-StTar fusion
chimera. The absorption maximum at is 503 nm. Matching molar
concentrations of the different proteins were used in the spectral
measurements.
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Phototaxis responses in H. salinarum.
Pho81Wr
cells carrying either NpSRII, NpSRII and NpHtrII, or the NpSRII-NpHtrII
fusion were analyzed for their response to blue-green (500 nm)
photostimuli. As expected, Pho81Wr cells expressing NpSRII alone did
not show a response; their frequency of swimming reversals, by which
they reorient swimming direction when exposed to a repellent stimulus,
was unaffected by a pulse of light (Fig.
6A). The cells exhibited a strong
repellent response, evident as a high reversal frequency in the
population of cells peaking at ~400 ms after the pulse, when the
wild-type N. pharaonis NpHtrII and NpSRII proteins were
heterologously coexpressed in H. salinarum (Fig. 6B),
confirming functional interaction in vivo between NpSRII, NpHtrII, and
the H. salinarum cytoplasmic signal transduction proteins,
as reported in a previous study (16). Furthermore, the
NpSRII-NpHtrII fusion mediates normal repellent phototaxis responses in
H. salinarum (Fig. 6C). Therefore, the flexible linker does
not inhibit the functional interaction between the receptor and the
transducer.
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FIG. 6.
Phototaxis responses for H. salinarum.
Two seconds after initiation of data collection, a 500-nm photostimulus
was delivered to the cells for 100 ms. Five points per second were
collected and used to calculate the reversal frequency. Response data
are the averages for 16 repetitive stimuli delivered every 1 min.
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Phototaxis responses in E. coli.
The
NpSRII-NpHtrII-StTar fusion chimeras and the NpSRII-NpHtrII-EcTsr
fusion chimeras were tested for function in E. coli cells by
monitoring their swimming behavior in response to a step-up and
step-down to blue-green light. E. coli cells reorient their swimming direction by an erratic subsecond motion called tumbling, and
attractant and repellent chemotaxis stimuli, respectively, inhibit and
augment tumbling frequency in a population of cells. Tumbling frequency
was not affected by light in cells containing the fusion chimeras
without the addition of retinal (shown for NpSRII-NpHtrII-StTar in Fig.
7A). Also, no light effect was observed without induction (Fig. 7B), nor was it observed with either of the P
fusions (Table 1 and Fig. 7C and D). These four sets of data (Fig. 7A
to D) illustrate the variation in the signal/noise ratio that we
observed with assays of the cell populations used in these experiments,
ranging from the most stable (Fig. 7D) to least stable (Fig. 7A)
signals that we obtained.
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FIG. 7.
Phototaxis responses by E. coli
containing fusion chimeras. One-half second after
initiation of data collection, cells with 1 µM retinal (except row A)
were stimulated with 2 s of 500-nm continuous light for recording
the attractant or repellent responses of Tar-M fusion (A and B), Tar-P
and Tsr-P fusion (C and D), Tar-G and Tsr-G fusion (E and F), and Tar-M
and Tsr-M fusion (G and H). Tracking was set at 15 frames per second,
and the rcd (degrees/second) was plotted. Response data are the
averages for 32 repetitive stimuli delivered every 30 s.
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A similar population to which retinal had been added exhibited
suppression of tumbling by 500-nm light and a clear enhancement
of
tumbling behavior peaking ~1 s after the light was extinguished
(Fig.
7G). Light is therefore acting as an attractant stimulus
through the
NpSRIItr-NpHtrII-StTar molecule. On the other hand,
the
NpSRII-NpHtrII-EcTsr protein mediates a repellent response
to the same
light, evident as an increase in tumbling frequency
during the
illumination period (Fig.
7F and H). Repellent responses
(Fig.
7E) are
also seen for the fusion chimera with the Tar cytoplasmic
domain when
the splice point is changed (G fusion) (Fig.
1 and
Table
1). The differences in motility responses
among constructions
with differing junctions in the signal relay domain
(Table
1)
point to this region of the transducers as being important in
controlling the conformations of their CheW/A kinase-binding domains.
The responses shown in Fig.
7 are small in comparison to those of
E. coli bacteria stimulated with photoreleased
chemoeffectors
(
13), which produced ~10-fold-larger
rate-of-change-of-direction
(rcd) amplitudes. The rcd as well as
the signal/noise ratio are
limited by the sluggish motility of
the cells, which is caused
by the high concentration of retinal that
must be added to reconstitute
the receptor in
E. coli in
vivo. A more quantitative assessment
of SR-mediated
responses in
E. coli would be facilitated by use
of a strain
engineered to produce retinal endogenously, such as
has been
accomplished recently (
37) using plasmid-encoded
-carotene
and oxidative cleavage
enzymes.
These results demonstrate that the fusion chimeras functionally couple
to the CheW/A/Y taxis machinery in
E. coli. One interesting
question in these experiments was whether the repellent nature
of the
NpSRII-NpHtrII signal seen in
H. salinarum would translate
to a repellent signal in
E. coli regardless of the origin of
the
cytoplasmic domain. The answer is that either attractant or
repellent
signals may be generated depending on the particular
cytoplasmic
domain selected and the position of the splice point. For
example,
Tar-G and Tar-M (Table
1) mediate opposite responses (Fig.
7E
and G), despite the fact that they differ only in the splice position,
with the latter containing 23 more NpHtrII residues and 23 fewer
StTar
residues than the
former.
The irregular nature of the sign of the response may indicate that
subtle forces set the bias in the kinase-activating versus
kinase-inhibiting conformations of the two cytoplasmic domains
that
couple to CheW/A. Both Tar ("taxis to aspartate and
repellents")
(
25) and Tsr ("taxis to serine and
repellents") are dual-function
attractant or repellent receptors
depending on the nature of the
chemoeffector, and therefore an
equilibrium mixture of their two
conformations must be maintained in
order to be able to shift
in either direction depending on the ligand.
A metastable equilibrium
between two conformations that can be shifted
by small energy
changes in the proteins has been indicated by studies
of mutant
chemotaxis receptors (
18,
33) and by our studies
of response
inversion in the SRI-HtrI complex in
H. salinarum (
12). SRI-HtrI
is also a dual-function
attractant and repellent receptor; the
sign of the signal in this case
depends on the spectral quality
of the light, which determines whether
one or two photons are
absorbed. Single mutations, resulting in one
case in an addition
of a single methyl group to residue I61V, I64V, or
V71I in HtrI,
switches the complex from repellent to attractant
signaling in
response to orange light activation (
12).
The haloarchaeal transducer chimeras demonstrated that the SRI and SRII
interaction specificities are encoded in the transmembrane
portions of
HtrI and HtrII, respectively (
39), suggesting that
the
surfaces of physical interaction between the SR and Htr proteins
also
reside in the transmembrane region. However, the presence
of a receptor
interaction domain on the cytoplasmic portion of
the transducers, as
was suggested based on deletion experiments
(
14), could
not be completely ruled out by the swapping of HtrI
and HtrII portions,
if such a domain were to be conserved between
HtrI and HtrII and not
exhibit SR specificity. The results obtained
here strongly support the
chimeral evidence for interaction between
the transmembrane helices,
because one would not expect any natural
interaction between NpSRII and
the Tar or Tsr cytoplasmic domains
from enteric eubacteria. Evidently
the seven helices of the NpSRII
receptor fused to the two transmembrane
helices of the transducer
constitute a membrane-embedded mobile
photosignaling module sufficient
in itself to convert light into a
conformational signal, which
is transmittable to evolutionarily very
distant transducer cytoplasmic
domains. The function of this module in
E. coli provides a potential
in vitro assay of the SR-Htr
signal relay based on the
E. coli chemotaxis system, for
which in vitro reactions are well established
(
17).
| |
ACKNOWLEDGMENTS |
We thank Shahid Khan for his current motion analysis program of
E. coli swimming behavior and Michael Manson, John S. Parkinson, and Jeffry Stock for E. coli and S.
enterica serovar Typhimurium plasmids and strains. We also
thank Jun Sasaki for early work on htrII-sopII cloning
from N. pharaonis DNA and Chii-Shen Yang for comments.
This work was supported by National Institutes of Health grant
R01GM27750 to J.L.S.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Molecular Genetics, University of TexasHouston
Medical School, JFB 1.708, 6431 Fannin, Houston, TX 77030. Phone: (713) 500-5458. Fax: (713) 500-5499. E-mail:
John.L.Spudich{at}uth.tmc.edu.
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Journal of Bacteriology, November 2001, p. 6365-6371, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6365-6371.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
