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INTRODUCTION |
Rhodobacter sphaeroides can grow either
aerobically (aerobic respiration) or anaerobically
(photosynthesis/anaerobic respiration), as do many other purple
bacteria. To adapt to these diverse environmental conditions, this
bacterium has to continuously monitor light quality and intensity as
well as oxygen tension. To adapt to the photosynthetic life style from
aerobic growth, R. sphaeroides uses, in part, a
two-component signaling system composed of a sensor histidine kinase
and a response regulator that are able to control photosynthesis gene
expression in response to decreasing oxygen tensions (1, 2). In this
system, the sensor PrrB is believed to detect changes in oxygen levels
by responding to change in either the flow of reductant or a redox
carrier through the cbb3/RdxB "redox"
centers, ultimately leading to the activation of the response regulator PrrA. Activated PrrA then serves to modulate photosynthesis gene expression and to allow cells to rapidly adapt to the new growth conditions (3). Additional regulatory factors involved in the control
of photosynthesis gene expression in R. sphaeroides have been discovered, and their interaction with the Prr sensory
transduction proteins is still under investigation (for review, see
Refs. 4 and 5).
Studies in both R. sphaeroides and Rhodobacter
capsulatus (2, 3, 6), where the two-component activation system is designated RegBA, reveal the singular importance of this two-component activation system. This system has been shown to be involved in the
control of the expression of the photosynthesis genes puf, puhA, and puc encoding the reaction center and
the light-harvesting antenna subunits (2, 7) and the cycA
gene encoding cytochrome c2 (3). This system
also serves to control those genes involved in carbon dioxide
assimilation and nitrogen fixation (8).
In general, sensor proteins are composed of two domains: the input
domain (generally within the amino terminus), which is responsible for
sensing the "signal," and the transmitter domain (generally at the
carboxyl terminus), which is responsible for communicating with the
response regulator (for review, see Refs. 9 and 10). The transmitter
domain from PrrB shows homologies to other histidine kinases (2, 6).
The PrrB polypeptide is 462 amino acids; autophosphorylation of PrrB
involves the conserved histidine residue at position 221; and PrrB has
been proposed to have both kinase and phosphatase activities (3). As to
the PrrB input domain, it is still unclear how PrrB monitors changes in
either the flow of redox through the cbb3/RdxB
redox centers or the change in concentration of a critical redox
intermediate. It has been proposed that PrrB senses changes in oxygen
tension indirectly by responding to changes in the localized redox
state/intermediate, which in turn will result from changes in oxygen
levels (11, 12). It has also been shown (11) that the Prr system is the obligatory intermediate between cbb3/RdxB and
activation of photosynthesis genes. Although the cytoplasmic domain of
RegB from R. capsulatus has been expressed and purified
(13), the intact protein from either R. sphaeroides (PrrB)
or R. capsulatus (RegB) has not yet been purified.
Genetic data from our laboratory suggest that PrrB is in close
communication with other components of the cell, such as cytochrome oxidase cbb3, RdxB, and PrrC (11, 12). Because
of their membrane localization and the nature of these proteins as
redox carriers, we believe that if there is to be a direct interaction
between these proteins, then it will likely occur at or within the
membrane space. This hypothesis is presently under genetic and
biochemical investigation, but further understanding of these processes
and the functional domains of the participating proteins requires a
greater understanding and knowledge of their structural topology. For
this purpose and as a important first step, we have begun an
investigation of the membrane topology of the PrrB polypeptide. Until
we are successful in purifying the intact PrrB protein, these indirect
approaches should provide us with sufficient information to proceed
with our functional analysis.
It was earlier suggested by Eraso and Kaplan (2) that PrrB might
possess six transmembrane-spanning domains. The model developed for
PrrB based on the hydropathy profile of the amino acid sequence
indicates six transmembrane helices. In this model, the amino and
carboxyl termini of the polypeptide are predicted to be in the
cytoplasm, with three loops located in the periplasm and two loops in
the cytoplasm. Gene fusion techniques with the Escherichia
coli periplasmically localized alkaline phosphatase and the
cytoplasmic
-galactosidase have been used as a genetic approach for
the analysis of the topology of cytoplasmic membrane proteins from
R. sphaeroides and R. capsulatus (14-17). We
therefore elected to investigate the topological model for PrrB by
making phoA and lacZ fusions at numerous
locations within the prrB gene. In this paper, we report the
result of such topological analyses of PrrB, and we propose a model for
the structure of the polypeptide in the cell membrane.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Growth Conditions--
E. coli
strains were grown at 37 °C in LB medium. DH5
-phe (1)
was used as a host for construction and expression of the prrB-phoA and prrB-lacZ fusions. R. sphaeroides strains were grown semi-aerobically at 28 °C in SIS
medium (18). Antibiotics were used at the following concentrations for
E. coli and R. sphaeroides: spectinomycin, 50 µg/ml; streptomycin, 50 µg/ml; kanamycin, 25 µg/ml; and
tetracycline, 10 and 1 µg/ml, respectively. Bacterial strains and
plasmids used in this work are listed in Table I. Alkaline phosphatase
and
-galactosidase activities were detected as blue colonies on LB
and SIS agar plates containing 5-bromo-4-chloro-3-indolyl phosphate and
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside, respectively, at a concentration of 40 µg/ml.
Computer Hydropathy Analyses--
The PrrB protein sequence from
R. sphaeroides was analyzed using the protein structure
programs provided through the Internet by EMBL, Heidelberg; the Protein
Sequence Analysis from the Biomolecular Engineering Research Center,
Boston; the SOSUI from Tokyo University of Agriculture and Technology;
and the algorithm developed by Kyte and Doolittle (19).
Molecular Biology Techniques--
Standard methods were
performed, if not otherwise indicated, according to Sambrook et
al. (20). Enzymes and chemicals were purchased from New England
Biolabs Inc. (Beverly, MA), Stratagene (La Jolla, CA), Promega
(Madison, WI), and Sigma. Plasmid DNAs were purified using the Bio-Rad
Quantum prep plasmid kit. DNA was treated with restriction enzymes and
other nucleic acid-modifying enzymes (Klenow fragment, alkaline
phosphatase, T4 DNA polymerase, and T4 DNA ligase) according to the
manufacturer's specifications. DNA fragments were analyzed on agarose
gels, and different restriction fragments were purified using the
Geneclean kit (BIO 101, Inc.). DNA sequencing was performed using an
ABI 373A automatic DNA sequencer (Applied Biosystems Inc., Foster City,
CA) at the DNA Core Facility of the Department of Microbiology and
Molecular Genetics, University of Texas Health Science Center (Houston,
TX). Site-directed mutations were constructed using the Stratagene
Quick Site-directed mutagenesis kit according to manufacturer's instructions.
Conjugation Techniques--
Plasmid DNA was mobilized into
R. sphaeroides strains using the tri-parental conjugation
system previously described by Davis et al. (21), with
E. coli HB101 (pRK2013) as a helper strain. Exconjugants
were selected on SIS plates supplemented with the appropriate
antibiotics and the desired chromogenic substrate.
Construction and Screening for prrB-phoA and prrB-lacZ
Fusions--
The ApaI/KpnI fragment from pUI
plasmids (see Table I) containing the three different reading frames
coding for the mature alkaline phosphatase was cloned into the multiple
cloning site of pBBR1MCS-2. The resulting plasmids are termed pSOP.
These plasmids are replicative in both E. coli and R. sphaeroides. The prrB-phoA gene fusions were
constructed as follows. The prrB gene was digested with the
restriction enzymes cited in Table II, and purified prrB DNA
fragments were treated with Klenow fragment or with T4 polymerase when
appropriate to generate blunt-ended fragments, which were ligated into
the SmaI restriction site of the pSOP plasmids to generate
in-frame prrB-phoA fusions. The orientations of the fusions were determined by restriction analyses, and the reading frame was
confirmed by DNA sequencing using the phoA primer
(5'-ACCGCCGGGTGCAGTAATAT-3').
To construct prrB-lacZ fusions, the prrB gene was
digested with different enzymes, and the purified prrB
fragments were cloned in frame with the lacZ gene in plasmid
pUI523A. The orientations and the in-frame reading of the fusions were
analyzed by restriction analysis and then by DNA sequencing using the
lacZ primer (5'-GGGATGTGCTGCAAGGCG-3'). All the
prrB-phoA and prrB-lacZ constructs were made in a
way that the expression of the these fusions is under the control of
the prrB promoter. The resulting plasmids (Table
I) encode mature alkaline phosphatase and
-galactosidase, the former lacking its signal sequence fused to
varying lengths of the N-terminal ends derived from PrrB.
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Table I
Bacterial strains and plasmids
The abbreviations used below are as follows: Apr,
ampicillin-resistant; Kmr, kanamycin-resistant; Smr,
streptomycin-resistant; Spr, spectinomycin-resistant;
TCr, tetracyclin-resistant.
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Site-directed Mutagenesis--
Since some regions at the 5'-end
of the prrB gene did not contain any useful restriction
sites, oligonucleotides were designed to introduce mutations leading to
individual blunt-ended restriction sites (NaeI,
SmaI, and RsaI). Six prrB mutations
were generated using site-directed mutagenesis. The position and the
sequence of each oligonucleotide as well as the residue substitutions
are indicated in Table II. The constructed plasmids were confirmed by
restriction analysis and then by DNA sequencing as described above.
Alkaline Phosphatase and
-Galactosidase Assays--
Alkaline
phosphatase activity was determined as described by Brickman and
Beckwith (22). E. coli or R. sphaeroides cells carrying prrB-phoA plasmids were grown in LB or SIS medium
containing the appropriate antibiotics. When the culture reached
A600 nm = 0.5, 1 ml of each culture was
centrifuged and washed once with 1 ml of ice-cold 1 M
Tris-HCl (pH 8.0). Pellets were resuspended in 1 ml of the same buffer;
a drop of 0.01% SDS and chloroform was added; and the samples were
incubated at 37 °C for 5 min to permeabilize the cells. 0.5 ml of
the samples were then mixed with 0.5 ml of 0.8 µg/ml substrate for
the alkaline phosphatase assay (p-nitrophenyl phosphate in 1 M Tris-HCl (pH 8.0); Sigma) and incubated at 37 °C. When
a yellow color started to appear, the time was noted, and the reaction
was stopped by adding 200 µl of 0.5 M
K2HPO4 (pH 8.0). Cell debris was removed by
centrifugation, and the absorbance of the supernatants at 420 nm was recorded.
-Galactosidase activity was determined on permeabilized cells
according to Miller (23). 1 ml of exponential growing culture was
washed with 1 ml of ice-cold 100 mM
K2HPO4 (pH 7.6). Pellets were resuspended in 1 ml of the same buffer. Cells were permeabilized with a drop of 0.01%
SDS and chloroform at 37 °C for 5 min. 0.9 ml of the samples were
then mixed with 0.1 ml of substrate for the
-galactosidase assay
(o-nitrophenyl
-D galactoside (0.8 µg/ml); Sigma) and
incubated at 37 °C. When a yellow color started to appear, the time
was noted, and the reaction was stopped by adding 200 µl of 1 M Na2CO3. Cell debris was removed
by centrifugation, and the absorbance of the supernatants at 420 nm was
recorded. Alkaline phosphatase and
-galactosidase activities were
calculated as A420 nm × 1000/time (min) × volume (ml) × A600 nm as described (24).
Immunoblot Analyses--
Cells (E. coli and R. sphaeroides) harboring the different prrB-phoA encoding
plasmids were grown to early logarithmic phase. 10 µl of each culture
were resuspended in loading buffer (60 mM Tris-HCl (pH
6.8), 10% glycerol, 2% SDS, 0.1% bromphenol blue, and 3%
-mercaptoethanol) and boiled for 3 min, after which 5-µl samples
were loaded onto SDS-polyacrylamide gels (12%). After running of the
gels, the proteins were transferred to nitrocellulose membranes by wet
electrotransfer in transfer buffer (50 mM Tris, 380 mM glycine, 0.1% SDS, and 20% methanol). PhoA fusion
proteins were detected with polyclonal antibody (at a dilution of
1:5000) directed against alkaline phosphatase and with goat anti-rabbit immunoglobulin conjugated to alkaline phosphatase as the secondary antibody (at a dilution of 1:25000). PrrB-PhoA polypeptides were detected on the protein blots by the alkaline phosphatase color detection system (Promega).
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RESULTS |
Analyses of prrB-phoA Fusions--
To identify the hydrophobic
segments of the PrrB polypeptide that could act as
transmembrane-spanning regions, hydropathic profile computer analysis
of the amino acid sequence of PrrB was performed. This analysis
suggested that PrrB is a membrane-bound protein and that the first 190 amino acids of a total length of 462 amino acids from the N-terminal of
the protein should form the membrane-spanning domain as shown in Figs.
1 and 2.
According to these analyses, both the amino and carboxyl termini of the protein should be located in the cytoplasm, and the protein should cross the membrane six times and should therefore possess three periplasmic and two cytoplasmic loops. To investigate these
predictions, a series of 18 prrB-phoA gene fusion-derived
proteins were constructed (Fig. 1). The design of the hybrid proteins
was such that at least one fusion was placed in each loop facing either
the periplasm or the cytoplasm. Additional fusions were placed in the
putative transmembrane domains and in the C-terminal portion of the
protein that was predicted to be cytoplasmic (Fig. 2). The
prrB-phoA gene fusions were made in vitro and
encode hybrid proteins in which the amino terminus corresponded to the
R. sphaeroides PrrB amino-terminal region and the
carboxyl-terminal portion corresponded to the E. coli
alkaline phosphatase lacking its signal peptide sequences. Plasmids
containing the different fusions were expressed in E. coli
DH5
-phe, and alkaline phosphatase activity was screened on 5-bromo-4-chloro-3-indolyl phosphate/LB plates.

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Fig. 1.
PrrB profile and prrB-phoA
fusions. Shown is the hydropathy profile of PrrB according
to the algorithm of Kyte and Doolittle (19), revealing the N-terminal
membrane-spanning domain and the C-terminal cytoplasmic domain.
Arrows 1-18 indicate the prrB-phoA and
prrB-lacZ fusion junctions. The putative six
transmembrane-spanning domains are indicated as black boxes.
The table indicates the restriction sites used to construct
prrB-phoA. Asterisks indicate sites that were
introduced in the prrB gene following site-directed
mutagenesis. Number signs indicate constructs with both
phoA and lacZ fusions. aa, amino
acids.
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Fig. 2.
Schematic model of the PrrB polypeptide of
R. sphaeroides. The model is based on the hydropathy
analyses and the experimental results obtained in this work. The six
putative transmembrane domains are numbered I-VI; the
periplasmic loops are designated PLI-PLIII; and the
cytoplasmic loops are designated CLI and CLII.
Positions of the fusion junctions are numbered 1-18 and are
shown with shaded circles. The asterisk indicates
the mutant position in strain PRRB78. Number signs indicate
constructs with both phoA and lacZ fusions. The
numbers of the positively charged residues in the cytoplasmic loops are
indicated. Per, periplasm; Cyt, cytoplasm.
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Most of the fusions in E. coli displayed a white color,
suggesting a cytoplasmic location of the PrrB junction region with the
PhoA moiety. Only fusions 3, 9, and 12-14 produced pale blue colonies
in E. coli, suggesting a periplasmic location for the PhoA
moiety in these fusions. These results were confirmed by measuring the
alkaline phosphatase activity in permeabilized cells (see Table III).
Only fusions 3, 9, and 12-14 displayed relatively high activities. It
is well established that the E. coli alkaline phosphatase is
active when it is exported to the periplasmic compartment and is
inactive or marginally active in the cytoplasmic compartment (25).
Based on these data, our results indicate that PrrB has three
periplasmic loops. Additionally, fusions 1 and 15-18 were white and
had low alkaline phosphatase activity, suggesting that both the N and C
termini are located in the cytoplasm. According to these results in
E. coli, the PrrB protein should possess six transmembrane
domains; this is consistent with the theoretical model. However, the
pale blue color observed in E. coli might indicate either a
low level of expression or instability of the hybrid PrrB-PhoA proteins.
With these encouraging, but preliminary results in hand, we proceeded
to follow the expression of these same constructs in R. sphaeroides. Plasmids were mobilized into R. sphaeroides strain PRRB1 from E. coli, and exconjugants
were selected on plates containing the appropriate antibiotic plus
5-bromo-4-chloro-3-indolyl phosphate to directly screen for cells
capable of expressing the fusion proteins. R. sphaeroides
lacks significant endogenous alkaline phosphatase activity; and
therefore, alkaline phosphatase activities detected in the exconjugants
should result from the expression of the hybrid proteins. Seven fusion
strains displayed a blue color, suggesting a periplasmic location of
the alkaline phosphatase moiety, and 11 were pink, suggesting
cytoplasmic or membrane localization of the alkaline phosphatase. In
general, the alkaline phosphatase activities in R. sphaeroides were
20-100-fold higher than the corresponding
activities in E. coli (see Table III, part A). As expected
from the phenotypes of these strains on plates, colonies with a blue
phenotype presented high activity values. In addition to fusions 3, 9, and 12-14, which produced pale blue colonies in E. coli,
fusions 4 and 5 also displayed a blue color and high alkaline
phosphatase activities in R. sphaeroides. Fusion 4 was expected to be blue despite the fact that it gave white colonies in
E. coli since it was located only one amino acid from fusion 3, which produced a blue phenotype. The reason for this difference could be that this fusion is very unstable or poorly exported in
E. coli. For fusion 5, the predicted PrrB topology suggested a transmembrane location for the fusion; however, the fusion gave a
blue phenotype. On the other hand, fusion 6 (predicted to be cytoplasmic) also displayed slightly high alkaline phosphatase activity. When control values are subtracted from all the values recorded for each fusion protein, those showing high levels of PhoA
activity (including fusion 5) were on the average 20-fold greater than
the average value obtained for those showing low levels of activity. In
both E. coli and R. sphaeroides, the
prrB-phoA fusions that produced blue phenotypes were located
only in the amino-terminal region of the PrrB protein, and no fusion in
the carboxyl-terminal end of PrrB yielded fusions possessing a blue phenotype.
Analyses of prrB-lacZ Fusions--
Because of the elevated
alkaline phosphatase activity obtained for fusion 5 in the second
transmembrane-spanning domain, we decided to investigate further the
topology of the protein in this region by constructing lacZ
fusions in this region of the prrB gene (Table
II).
-Galactosidase activity was
determined in both E. coli and R. sphaeroides
cells (Table III, part B). In both
organisms, fusions 3# and 9# (where # indicates constructs with both
phoA and lacZ fusions; predicted to be in the
periplasm) and fusion 8# exhibited very low
-galactosidase activity,
whereas fusion 6# exhibited the highest activity, consistent with a
cytoplasmic exposure of the fusion. Fusion 5# exhibited a low but more
intermediate activity. These results, taken together with the alkaline
phosphatase activity for the same fusions, support the model proposed
in Fig. 2. The slightly high alkaline phosphatase and
-galactosidase activities obtained with fusion 5 can possibly be explained by the
inability of the fusions to completely reenter the cytoplasm. This
problem has been reported in other topological studies using these
approaches (26-29). Furthermore, we analyzed the structure of each
transmembrane-spanning domain using a computer program (30) to predict
their secondary structure. According to this analysis, five
transmembrane-spanning domains (I and III-VI) should have a pronounced
-helical structure. However, the program was unable to predict the
structure for the second transmembrane-spanning domain (II; containing
fusion 5). This transmembrane-spanning domain may possess a structure
that permits this region to "move in or out" of the membrane
depending upon the nature of the fusion protein, and this may explain
the relatively high enzymatic activities observed with fusions in this
region (see "Discussion"). In conclusion, results obtained with
PrrB-PhoA and PrrB-LacZ fusion proteins expressed in either E. coli or R. sphaeroides are in accordance with the
computer-predicted topology, are generally consistent, and suggest the
same relative model for PrrB polypeptide topology.
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Table II
DNA sequences of the primers used in site-directed mutagenesis
The primers used to engineer the new blunt-ended restriction sites at
the 5'-end of the prrB gene are listed below. The
substituted nucleotides are indicated in lowercase, and the generated
restriction sites are in boldface. Five of the mutations resulted in
amino acid substitutions; the nature and the position of these
substitutions are indicated.
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Table III
Alkaline phosphatase (part A) and -galactosidase (part B) activities
In part A are shown the prrB-phoA fusion plasmids and the
corresponding alkaline phosphatase activity in E. coli and
R. sphaeroides. Fusion numbers correspond to fusions in Fig.
1; C corresponds to the control. Alkaline phosphatase activity was
determined as described (21). Assays were performed on permeabilized
cells, and the specific activity is expressed as alkaline phosphatase
units/absorbance unit of cells at 600 nm. For a better
comparison, the alkaline phosphatase activity values in the controls (3 in E. coli and 89 in R. sphaeroides) were
subtracted from the values recorded for each fusion. All values are the
averages of two independent determinations, and S.D. values were
<10%. In part B are shown the prrB-lacZ fusion plasmids
and the corresponding -galactosidase activity in E. coli
and R. sphaeroides. Fusion numbers correspond to fusions in
Fig. 1; C corresponds to the control. Assays were performed on
permeabilized cells, and the activity is expressed as -galactosidase
units/absorbance unit of cells at 600 nm. For a better
comparison, the -galactosidase activity values in the controls (0.1 in E. coli and 0.3 in R. sphaeroides) were
subtracted from the values recorded for each fusion. All values are the
averages of two independent determinations, and S.D. values were
<10%.
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Immunoblot Analyses--
To ensure that the prrB-phoA
fusions expressed in E. coli and R. sphaeroides
were of relatively equal abundance, we performed Western blot analyses
with an anti-alkaline phosphatase antibody on all of the strains
described here. In E. coli, for all the fusion proteins
constructed, only an immunoband corresponding to the mature alkaline
phosphatase (43 kDa) was revealed, but no bands of the predicted size
corresponding to the hybrid proteins were observed (data not shown).
These results show that although the fusions were expressed in E. coli, the hybrid proteins are unstable and rapidly degraded. This
could explain the very pale blue color of the colonies and the low
alkaline phosphatase activities observed in E. coli. In
R. sphaeroides, immunoblot analyses showed that the hybrid
proteins were expressed, but only PhoA fusions that were predicted to
be in the periplasm (fusions 3, 4, 9, and 12-14) and fusion 5 could be
detected (Fig. 3). Its seems that the
periplasmic fusions are more stable, probably because of the dimerization of the alkaline phosphatase that occurs only in the periplasm. These results correlated well with the high alkaline phosphatase activity detected in these periplasmic fusions (Table III).
Hybrid proteins from fusions that are proposed to be in the cytoplasm
could not be detected; probably because of the improper folding of the
alkaline phosphatase in the cytoplasm, they were degraded, yielding
free alkaline phosphatase and giving a low alkaline phosphatase
activity. Other cross-reacting bands were detected, and they could
correspond to degradation products; the band at 43 kDa could correspond
to the free alkaline phosphatase. Hybrid PhoA proteins are often
unstable and rapidly degraded; such instability of fusion proteins has
been observed previously for different phoA fusions (28,
29, 31-34)

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Fig. 3.
Western blot analysis of the PrrB-PhoA fusion
proteins from the constructs listed in Fig. 1. Identical amounts
of total protein (40 µg) were run on a 12% SDS-polyacrylamide gel,
and the hybrid proteins were detected with the anti-PhoA antibody (see
"Experimental Procedures"). The first lane corresponds
to the strain containing the pSOP1 plasmid; the numbers at the top of
each lane correspond to the fusions listed in Table I and in Fig. 2.
Arrows show the bands that could be detected only in the
periplasmic fusions. Molecular mass markers (in kilodaltons) are
indicated to the right of the blots.
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Topological Model for PrrB--
Based upon the results presented
in this study together with the theoretical predictions, the PrrB
protein can be divided into two distinct domains: the amino-terminal
domain from residues 1 to ~183 comprises the membrane-spanning domain
probably containing the as yet unknown input module responsible for
sensing the hypothetical redox signal, and the carboxyl-terminal domain
from residues ~184 to 462 forms the cytoplasmic domain comprising the
transmitter module responsible for communicating with the response
regulator PrrA. In the membrane-spanning domain, the data best fit six
transmembrane helices (I-VI) that are well defined. With both the
amino-terminal end and the carboxyl-terminal portion of the protein
located in the cytoplasm (Fig. 2), the protein possesses three
periplasmic loops and two cytoplasmic loops. In addition, computer
analysis suggests the presence of an amphiphilic
-helix
(Ala251-Ala292) in proximity to the
His221 presumed to be the phosphorylation site (2, 6).
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DISCUSSION |
The process of transmembrane signaling is of major importance in
understanding signal transduction pathways in biological systems,
specifically bacteria. Many studies have reported that the structural
features of the proteins are implicated in membrane signaling and that
alterations in signaling can induce conformational changes in response
to environmental stimuli that can serve to transmit the signal
intracellularly (10). Conformational and topological information for
membrane-localized signal transduction proteins should be very helpful
in targeting protein domains and/or segments that might play some role
in the signaling process. Therefore, we have chosen to begin an
examination of the topology of the PrrB protein, a sensor kinase from
R. sphaeroides involved in signaling and gene regulation in
response to changes in oxygen tension (1, 2). In the oxygen signal
transduction pathway of R. sphaeroides, other membrane
proteins such as cytochrome oxidase cbb3, RdxB,
and perhaps the PrrC polypeptide appear to be part of the signal
transduction pathway that might interact directly or indirectly with
PrrB (2, 12). We have recently shown that within the
cbb3 terminal oxidase, it is likely that the Q
polypeptide is responsible for transmitting the signal from cbb3 to PrrB (35). In this initial study, we
have begun to investigate the membrane topology of the PrrB protein,
which we believe will help in a more precise understanding of the
oxygen signal transduction pathway involving this protein.
The R. capsulatus PrrB homologue (RegB) shows a high degree
of similarity to the R. sphaeroides PrrB protein (2, 6); thus, one can anticipate that conserved structural features for both
proteins should be informative as to their structure/function interrelationship. Mosley et al. (6) proposed a model in
which RegB has five hydrophobic segments that could constitute a
membrane-spanning domain. In such a model, either the N or C terminus
should be periplasmic. The hydropathy computer analyses of PrrB
revealed six hydrophobic segments with lengths between 18 and 20 residues; all of these were located in the first 190 amino acids of the amino terminus of the protein. However, experimental evidence in
support of either model was lacking. The RegS protein from Bradyrhizobium japonicum, a homologue of PrrB/RegB, does not
show clear evidence of membrane-spanning domains (29).
To determine a structural model of PrrB, the topology of this protein
was studied by generating fusions to the alkaline phosphatase PhoA and
-galactosidase LacZ moieties derived from E. coli. The topology of the fusion proteins was inferred from the level of alkaline
phosphatase and
-galactosidase activities and was more strictly
refined through the results in R. sphaeroides. The
experimental data obtained from the characterization of the 18 PrrB-PhoA and five PrrB-LacZ fusion proteins gave a topological model
that fits well with our hydropathy computer analyses. According to our
results, both the N and C termini are located in the cytoplasm,
supporting the presence of six transmembrane segments. Using this
model, we identified three loops facing the periplasm, which is
inconsistent with a model of five transmembrane segments (6). According to the model for PrrB presented here, the PrrB protein comprises two
distinct domains with both amino and carboxyl termini located in the
cytoplasm: (i) the anchor, containing the membrane-spanning domain, is
composed of six transmembrane helices, three periplasm loops, and two
cytoplasmic loops and (ii) the catalytic region forming the cytoplasmic
domain. This topology is also in general agreement with the "positive
inside" rule, as most of the positively charged residues are located
in the cytoplasmic loops (36).
Computer analyses showed that the C terminus contains an amphiphilic
-helix located in proximity to His221. This
-helix
could serve either as a transmembrane span or as a site for interaction
with other proteins. In the first case, we would expect that the C
terminus should be on the periplasmic side of the membrane, but our
PhoA fusion analyses strongly support a cytoplasmic C-terminal
location. Thus, this amphiphilic domain could serve other structural
functions such as interaction with other proteins.
Most of the hybrid PrrB-PhoA proteins were unstable and probably
degraded in E. coli. This phenomenon has been reported
previously for different phoA fusions (28, 34, 37).
Expression levels and/or stability of the PrrB-PhoA fusion proteins in
R. sphaeroides was substantially higher than in E. coli, perhaps because of codon usage and/or the correct folding of
PrrB within the membrane environment of R. sphaeroides; this
was previously reported for PhoA fusions with other proteins from
R. sphaeroides and R. capsulatus (14, 17). On the
other hand, the cytoplasmic fusions were more unstable than the
periplasmic fusions, probably for the reason that in the cytoplasm,
these are unable to fold correctly and are degraded, whereas in the
periplasm, dimerization of PhoA prevents its rapid degradation. The
alkaline phosphatase activities obtained in E. coli
correlated mostly with the data obtained in R. sphaeroides, allowing a clearer assignment as to the periplasmic location of the
fusion junction of the hybrid proteins. The only exception to this
topological assignment was fusion 5 and, to a much smaller extent,
fusion 6; although these junctions were predicted to be in the
transmembrane domain and the cytoplasm, respectively, we recorded high
alkaline phosphatase activity in R. sphaeroides. A similar
effect was obtained for fusion 5 when the phoA gene was
replaced by the lacZ gene to create a LacZ fusion. We
believe that because this fusion junction occurs near the middle of the second transmembrane domain where reentry of the chimeric protein from
the periplasm takes place, improper folding of the chimera results in
exposure of the PhoA moiety to the periplasm, where PhoA dimerization
stabilizes the protein, and hence, activity is observed. The same
situation could prevail with the
-galactosidase fusion; the protein
should be retained within the membrane and not completely exposed to
the cytoplasm, giving rise to low but discernible
-galactosidase
activity. We should point out that in this transmembrane-spanning
domain, a cysteine residue is present, and this residue may alter the
protein structure of the chimera in case of dimerization. ,However when
considered in toto, the LacZ fusion results confirm and
strengthen the PhoA fusion studies.
With regard to the general structural features of the PrrB protein,
sequence analyses revealed homologies to other histidine kinases only
in its cytoplasmic region containing the previously designated H, N,
G1, F, and G2 boxes (2, 6), which are involved in phosphorylation of
the response regulator. In addition, a sequence presenting an
intriguing homology to the EAA conserved motif can be identified. Such
a motif seems to play an important role in periplasmic transporters by
interacting with hydrophilic components like ATPases (38). The role of
such a motif (if any) in PrrB is still unknown; its location at the end
of the amphiphilic
-helix may suggest a possible interaction of this
portion of the protein with other cytoplasmic factors such as PrrA.
This hypothesis will be tested by site-directed mutagenesis. The amino
terminus does not show any homology to any other protein sequences to
date. One can ask what the function of this portion of the protein is.
Based on the membrane topological model of PrrB presented in this paper
and that best fits all of the data and on previous results presented by
Eraso and Kaplan (2) on the analysis of the PRRB78 mutant (in which a
substitution of leucine 78 with a proline in the first cytoplasmic loop
dramatically affects photosynthesis gene expression by turning
expression on in the presence of O2), we can assume that
the amino terminus of the protein, in addition to functioning as an
anchor to the cell membrane, may also serve as a functional domain
involved in the oxygen transduction signal pathway. The apparent
absence of an
-helical structure for the second membrane span
together with the L78P substitution in the cytoplasmic loop following
this membrane span may suggest that this region of the protein responds
to a redox signal by altering conformation, which is alternatively
transmitted to the cytoplasmically localized enzymatic portion of the
protein. Since PrrB seems to receive a redox signal of some sort
involving electron transfer complexes such cytochrome oxidase
cbb3 (11, 12), it is thus tempting to speculate
that PrrB may interact with membrane and periplasmic electron carriers
or other proteins via this transmembrane portion of the polypeptide.
Site-directed mutagenesis of the PrrB amino terminus is currently being
carried out to test these assumptions, and our preliminary results
support the conclusions reached here.