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(Received for publication, May 22, 1995; and in revised form, June 26, 1995) From the
The Rhodobacter sphaeroides 2.4.1 tryptophan-rich
sensory protein gene, tspO (formerly crtK, ORF160)
encodes a 17-kDa protein which has an unusually high content of
aromatic amino acids in general and of L-tryptophan in
particular. The TspO protein was localized to the outer membrane of
aerobically grown R. sphaeroides 2.4.1 by use of a polyclonal
antibody against the purified protein. This protein is present in
severalfold higher levels in photosynthetic as opposed to aerobic grown
cells. Although tspO lies within the crt gene
cluster, null mutations have an intact carotenoid biosynthetic pathway.
In the TSPO1 mutant there was an increased production of carotenoids
and bacteriochlorophyll relative to the wild type, particularly when
cells were grown aerobically or semiaerobically. When present in
trans the tspO gene restored ``normal'' pigment
production to TSPO1. The effect of the tspO gene on pigment
production was shown to take place at the level of gene expression.
Because the tspO gene product of R. sphaeroides 2.4.1
shows significant sequence homology and similarity to the
peripheral-type benzodoazepine receptor from mammalian sources,
TspO-specific antibodies when probed against liver and kidney
mitochondrial protein showed strong cross-reactivity. The role of TspO
in R. sphaeroides 2.4.1 and its relation to photosynthesis
gene expression are discussed.
Rhodobacter sphaeroides is a member of the
proteobacteria, and it is characterized by its metabolic versatility
including growth either chemoheterotrophically or
photoheterotrophically. A decrease in oxygen level results in the
induction of the photosynthetic membrane system designated the
intracytoplasmic membrane (ICM). ( Carotenoids (Crt) in
addition to bacteriochlorophyll (Bchl) are an important structural
component of the ICM. They take part in the entrapment and utilization
of light energy, and furthermore, have an important antioxidative
function during aerobic growth. The crt gene cluster of Rhodobacter capsulatus has been well characterized
by(2, 3) , and the genes and likely enzymatic
activities which they encode have been discussed. In R.
sphaeroides, these genes have been recently shown to occupy a
similar arrangement as in R. capsulatus(4) . According to the phenotype of a number of Crt mutants in R.
capsulatus(3) and in R. sphaeroides NCIB8253 (4) enzymatic activities have been assigned to seven of the
eight genes constituting this cluster but not to crtK, herein
designated tspO (tryptophan-rich sensory protein). Based on
the amino acid sequence deduced from the nucleotide sequence it was
suggested that TspO was an integral membrane protein. Most of the
enzymes of the carotenoid biosynthetic pathway were also shown to be
localized to the cell membrane, and it was proposed that TspO is a site
for docking of the enzymes involved in carotenoid
biosynthesis(3) . However, no evidence either for this or for
any other physiological activity associated with this protein has been
available, until now. Comparison of the deduced amino acid sequence
of the R. capsulatus TspO with that of adrenal peripheral-type
benzodiazepine receptor (PBR) revealed a high degree of homology
between the two proteins; of the 129 amino acid residues comprising 75%
of each of these proteins, there were approximately 35% identity and a
further 15% conservative replacements(5) . The PBR is
present in many types of mammalian tissues including kidney, liver,
brain, adrenal gland, testes, etc.(6) . It has been shown that
this 18-kDa protein is localized to the outer mitochondrial membrane
and is associated with an outer membrane, voltage-dependent anion
channel (VDAC) and adenine nucleotide carrier, which is an inner
mitochondrial membrane localized protein. The PBR binds with nanomolar
affinity to a variety of benzodiazepines as well as to dicarboxylic
porphyrins(7) . A number of metabolic activities have been
ascribed to the PBR including regulation of steroidogenesis and
participation in tetrapyrrole metabolism. However, the precise
physiological function of this mitochondrial membrane protein still
remains unclear. Here, we attempt to bring together this disparity
of observations by defining a physiologic role for TspO in R.
sphaeroides 2.4.1, describing its cellular localization, and
further showing that antibody produced against TspO cross reacts quite
specifically with a protein(s) of the rat mitochondrion.
Cell growth was monitored
turbidometrically with a Klett-Summerson colorimeter using a no. 66
filter. 1 KU is equivalent to 1
In
vitro alkaline phosphatase (APase) activity was determined
following the hydrolysis of o-nitrophenyl-phosphate as
described previously(14) .
Plasmids
carrying crtA::lacZ and crtI::lacZ transcriptional
fusions were constructed by the blunt end ligation of 0.13-kb PstI fragment of pUI3101, containing promoter region for
divergently transcribed genes, crtA and crtI, into
the XbaI site of pLX1 vector, Sm Plasmid pUI2715 carrying a translational fusion
of tspO and the glutathione S-transferase (GST) gene
was constructed by ligation of 0.7-kb BamHI-EcoRI
fragment of pUI1124, containing tspO, into the BamHI-EcoRI sites of pGEX-2TK vector (Pharmacia).
N-terminal
amino acid sequence of the R. sphaeroides 2.4.1 major outer
membrane protein was performed at the analytical chemistry center, UT
Medical School, on an Applied Biosystems 477 Protein Sequencer using a
procedure described(21) . Protein was electroblotted onto
polyvinylidene difluoride membrane, stained with Coomassie Blue, and
sequenced directly.
Antibodies against TspO were
affinity purified using the modification of a protocol
described(22) . A preparative SDS-PAGE was loaded with
approximately 0.8 mg of protein extract containing TspO and subjected
to electrophoresis. Proteins were electrophoretically transferred to a
nitrocellulose membrane; the protein band corresponding to TspO was
located, removed from the membrane, and blocked 1.5 h in 5%BSA in TBST.
After washing three times for 15 min each in TBST, the nitrocellulose
strip was incubated overnight at 4 °C in a solution containing 1 ml
of serum and 5 ml of TBST. The nitrocellulose strip was washed three
times for 15 min each in TBST, and antibodies were eluted with 100
mM glycine, pH 2.2 (five times, 1 ml each). The glycine
solution was neutralized with 1 M Tris pH 9.0, and the
resulting solution was characterized for antibody reactivity against R. sphaeroides 2.4.1 membrane proteins. Antibodies against
the major outer membrane protein were prepared by C. Deal and S.
Kaplan(23) .
Proteins incorporating the radiolabeled drug were
estimated using SDS-PAGE electrophoresis in 12.5% gel(24) . The
gel was then treated with EN
Figure 1:
Alignment of amino acid sequences of
the mammalian peripheral-type benzodiazepine receptors with R.
sphaeroides and R. capsulatus TspO proteins. The
sequences are as follows: HsPkbS, human; MmPkbS,
mouse; RnPkbS, rat; BtPkbS, bovine mitochondrial
peripheral-type benzodiazepine receptors; RcCrtK, R.
capsulatus CrtK; RsNCIB, R. sphaeroides TspO
from wild type strain NCIB8253, and RsTspO, R. sphaeroides 2.4.1 TspO. In bold are amino acid residues conserved in
all seven sequences.
Using the cartridge encoding kanamycin resistance from pUC4K, a tspO disruption in pUI1108 was constructed (Table 1).
This construction was subsequently used to generate the pSUP202-derived
vector pUI1110. One Km
Figure 2:
Absorption spectra of R. sphaeroides 2.4.1 membrane preparations. Cells were grown
photoheterotrophically in the light at 10 W/m
To
assess the abundance of the photopigments (Crt and Bchl), these were
extracted from 2.4.1 and TSPO1 cells grown at various light intensities
or in the presence of Me
Because the low oxygen tension used in
these experiments is probably a more accurate reflection of what
actually takes place in nature, we followed the dynamics of pigment
accumulation in aerobic cultures shifted to lower oxygen tensions. As
is evident from Fig. 3, TSPO1 responded to oxygen deprivation
more rapidly than the wild type: significant differential increases in
both Crt and Bchl accumulation were observed within 1.5 h after the
cultures were shifted to low oxygen. After approximately 15 h of
growth, the accumulation of both Crt and Bchl by the wild type was
equivalent to that of TSPO1. At the cell densities reached after this
time, there is virtually no free oxygen in the culture.
Figure 3:
Pigment accumulation by R. sphaeroides 2.4.1 and TSPO1 cells. Cells were grown aerobically at
30%O
Expression of the bchF::lacZ transcriptional fusion was also higher in TSPO1 cells than in
2.4.1 grown either aerobically or semiaerobically. Expression of the puc operon encoding the structural polypeptides for
B800-850 spectral complex was found to be 2-3-fold higher
in TSPO1 cells grown aerobically or semiaerobically when compared to
wild type. On the other hand, there were no differences in the
expression of a puf::lacZ transcriptional fusion
between wild type and TSPO1 grown under any of these conditions (data
not shown).
Fig. 4A represents an SDS-PAGE of TspO-GST expressed
in E. coli and stained with Coomassie Blue. The band of 44 kDa (lane 2) disappeared following treatment with thrombin and was
replaced by two new bands (lane 3) corresponding to GST (27
kDa) and TspO (17 kDa). Using antibodies raised against TspO (Fig. 4B) revealed one immunoactive band in
preparations treated with thrombin (lane 2). In the sample of
GST-TspO (Fig. 4B, lane 1), both the fusion
protein and several bands, apparently corresponding to products of
nonspecific cleavage, were detected.
Figure 4:
Overexpression of the TspO-GST fusion
protein in E. coli.A, Coomassie Brilliant
Blue-stained SDS-PAGE. TspO-GST fusion protein was overexpressed in E. coli JM109. Cells were disrupted by French Press,
fractionated, and the membrane fraction containing TspO-GST was
solubilized with 1% lauroyl sarcosine as described under
``Experimental Procedures.'' Lane 1, molecular
weight standards; lane 2, solubilized TspO-GST; lane
3, solubilized extract treated with thrombin; lane 4,
affinity purified fusion TspO-GST treated with thrombin. B,
immunoblot analysis of TspO-GST and TspO. Immunoblot was performed
using affinity purified antibodies raised against TspO. Lane
1, solubilized membrane fraction of E. coli containing
TspO-GST fusion protein; lane 2, the same sample treated with
thrombin.
Membrane fractions of R.
sphaeroides 2.4.1 and TSPO1 were separated by SDS-PAGE and probed
with TspO-specific antibody. In Fig. 5, one band corresponding
to a protein of 17 kDa was visible in the outer membrane preparations
from 2.4.1 cells grown semiaerobically (lane 1) or
photoheterotrophically (lane 3). No immunoactive band could be
detected in TSPO1 (lanes 4 and 5). TspO expressed in E. coli from the construct pUI2715 (Fig. 5, lane
6) migrated more slowly in SDS-PAGE than its homologue from the
membrane fraction derived from R. sphaeroides 2.4.1. This
difference correlated with the presence of an additional 13 amino acid
residues in the genetically engineered protein expressed in E.
coli.
Figure 5:
Immunoblot analysis of the TspO protein in R. sphaeroides 2.4.1. Cells of R. sphaeroides 2.4.1 and TspO1 were grown semiaerobically or
photoheterotrophically at 10 W/m
To gain additional insight into the question of how TspO
acts as a sensory transducer, membranes from both aerobic and
photosynthetic grown R. sphaeroides 2.4.1 cells were reacted
with anti-TspO antibodies following separation on SDS-PAGE (Fig. 5, lanes 1 and 3). Membranes from
aerobic grown cells showed low levels of TspO in agreement with the
APase results. However, the bulk of the reactive species migrated with
an apparent size of
Figure 6:
Absorption spectra of membrane
preparations from R. sphaeroides 2.4.1 cells grown in presence
of flurazepam. A, R.sphaeroides 2.4.1; B, R. sphaeroides TSPO1. Cells were grown
photoheterotrophically in the light at 10 W/m
To obtain additional insight into the possible target, we used
[N-methyl-
Figure 7:
Identification of the major outer membrane
protein as a [
Previous work from this laboratory revealed
that the major outer membrane protein or porin from R. sphaeroides 2.4.1 migrates at approximately 47 kDa(23) . An antibody
raised against this polypeptide was found to react with the same
protein binding the labeled drug (Fig. 7C), thus
identifying it as the major outer membrane protein. We determined the
N-terminal sequence of this polypeptide (EISFSGYAAE) and found it was
44% identical and a further 30% similar to that of the previously
reported porin of R. capsulatus(28) .
Figure 8:
Immunoblot analysis of mitochondrial
proteins. Preparations of rat kidney and liver mitochondria (gift of
Dr. M. McEnery) were solubilized in sample buffer, run on a 12%
SDS-PAGE, and blotted onto nitrocellulose membranes. Proteins were
probed with antibodies raised against TSPO. Lanes 1 and 2, preparations from kidney mitochondria solubilized at 90 and
37 °C, respectively. Lanes 3 and 4, preparations
from liver mitochondria, solubilized at 90 and 37 °C. Lane
5, preparation of overexpressed in E. coli TspO-GST
protein treated with thrombin.
The similarity between the tspO gene cloned from
bacterial sources (R. sphaeroides 2.4.1 and NCIB8253, R.
capsulatus), and PBRs from various mammalian tissues raises
questions of the physiological function(s) and evolutionary
relationship between these diverse polypeptides. A number of
physiological activities have been ascribed to the PBRs which are
localized in the outer mitochondrial membrane, such as the regulation
of steroidogenesis and their involvement in porphyrin transport across
the mitochondrial membrane. It was proposed that the PBR in association
with two proteins VDAC and adenine nucleotide carrier comprises a
porphyrin transport site at the junction of the two mitochondrial
membranes(6) . We observed that several benzodiazepines,
choosen primarily because of their aqueous solubility, and which are
known to bind with high affinity to PBRs, affected the biosynthesis of
the B800-850 antenna complex in R. sphaeroides 2.4.1.
When present in concentrations 0.02-0.1 mM, flurazepam
suppressed complex formation by 50-95% in wild type R.
sphaeroides 2.4.1 grown photoheterotrophically. However, the same
effect was also observed with TSPO1. Further studies using a Comparison of the nucleotide sequence of tspO from R. sphaeroides 2.4.1 with that of R. sphaeroides NCIB8253 (4) , revealed 99.7% identity, with three
nucleotides absent from the NCIB8253 sequence in the positions between
nucleotides 4095 and 4096 (G); 4124 and 4125 (G), and 4146 and 4147
(C), relative to the sequence from 2.4.1. The resulting frameshifts led
to a decreased similarity between the two derived polypeptides (91%
identity). All nonidentical amino acid residues were found to be
localized to the region of the frameshift between positions 62 and 86.
The codon usage assessment program indicated five rarely used codons in
the DNA sequence from NCIB8253 compared to 2.4.1. However, strains
2.4.1 and NCIB8253 are considered to be similar if not identical (29) on a basis of available sequencing data and AseI
DNA digestion patterns. Disruption of the R.
sphaeroides' tspO by insertion of a kanamycin
resistance gene did not lead to any significant change in the phenotype
of cells grown either aerobically or photosynthetically, except for
some small alteration in the ratio between B800-850 and B875
complexes. However, a profound phenotypic difference was observed
between the wild type and TSPO1 during transition from aerobic to
anaerobic growth. TSPO1 also produced substantially increased levels of
both Crt and Bchl relative to the wild type. Studies showed that there
was a 3-5-fold effect of TspO on expression of crtA, crtI and bchF in the mutant when compared to the wild type.
Transcription of the puc operon was similarly affected in the
mutant strain under semiaerobic conditions. Introduction of tspO in trans into TSPO1 mutant or wild type resulted in a
severe reduction in pigment accumulation to below the wild type level,
which was most evident in cells grown under semiaerobic or aerobic
conditions. Differences in expression of both crt and bch genes between mutant and wild type cells were also
detected when cells were grown photoheterotrophically at medium light
intensity (10 W/m We have previously described the existence of an
additional oxygen sensing system in R. sphaeroides 2.4.1, the
PrrA system which is typical of a two-component regulatory system and
where a decrease in oxygen tension results in an activation of
photosynthesis gene expression(30) . The TspO system appears to
involve the negative regulation of photosynthesis gene expression.
Whether or not TspO acts through the regulatory protein PpsR remains to
be determined(31, 32) . However, the possibility
exists that a small ligand molecule could be involved in this
regulatory pathway as revealed by the effect of benzodiazepines. Thus, R. sphaeroides 2.4.1 appears to have at least two systems able
to sense the level of oxygen in the environment. Very recent work ( Antibodies raised against TspO specifically recognize this protein
in the outer membrane fraction of R. sphaeroides 2.4.1 cells.
Membrane localization of this protein was also supported by the results
of the TspO-alkaline phosphatase-fusion analysis which showed high
level of APase when conjugated to the 52 N-terminal amino acid portion
of TspO. It is known that mammalian PBR interacts with VDAC in the
outer mitochondrial membrane and adenine nucleotide carrier, localized
to the inner mitochondrial membrane. VDAC is a porin-type protein
apparently involved in porphyrin transport across the membrane. It is
therefore possible to speculate that in R. sphaeroides 2.4.1
TspO interacts with the major outer membrane protein, which is shown to
bind porphyrins and benzodiazepines with high affinity, thus affecting
biosynthesis and/or assembly of the photosynthetic antenna complexes
through an as yet unknown mechanism. Analysis of the amino acid
sequence of TspO does not predict any potential DNA-binding domains in
this polypeptide. Therefore, the pathway and mechanism of signal
transmission from the membrane-localized TspO to the DNA-bound
transcriptional effector is not clear. It would seem reasonable to
assume that there is in addition to TspO an inner membrane-localized
component of this pathway. The nature of this hypothetical protein is
unknown. Nor do we know how this protein might interact with the
repressor component of this pathway. We do know, however, that this
pathway does not directly affect puf operon expression, but
may do so indirectly through Crt and Bchl availability. Therefore, in
future studies we plan to determine the mechanism(s) by which TspO
senses changes in O
Volume 270,
Number 36,
Issue of September 08, pp. 21167-21175, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)The ICM contains all of
the components necessary to convert light energy into chemical energy
during phototrophic growth. The ICM is also gratuitously synthesized by R. sphaeroides during anaerobic growth in the dark in the
presence of an alternative electron acceptor, such as dimethyl
sulfoxide (Me
SO). While ICM synthesis and composition are
known to be tightly regulated, the molecular mechanisms which govern
the biosynthesis and assembly of the ICM are only beginning to yield to
molecular genetic analysis(1) .
Bacterial Strains, Plasmids, Media, and Growth
Conditions
Bacterial strains and plasmids used in this study are
described in Table 1. Cultures of R. sphaeroides 2.4.1
and its derivatives were grown in Sistrom's minimal medium A
containing 0.4% succinate as a carbon source (8) as described
previously(9, 10) .
10
cell/ml. To
minimize antibiotic photooxidation(11) , liquid cultures of R. sphaeroides grown photoheterotrophically in the presence of
tetracycline were placed behind a CS 7-69 filter (620-1100
nm; Corning Glass Works, Corning, NY). Strains of Escherichia coli were grown as described previously(12) . When appropriate,
antibiotics were added to the following final concentrations:
ampicillin (Ap), 100 µg/ml; kanamycin (Km), 25 µg/ml;
spectinomycin (Sp), 50 µg/ml; streptomycin (Sm), 50 µg/ml; and
tetracycline (Tc), 10 µg/ml for E. coli, 1 µg/ml for R. sphaeroides. Conjugal matings between E. coli and R. sphaeroides were performed as described by Moore and
Kaplan(13) .
R. sphaeroides cultures were grown to a
cell density of approximately 1.8 
-Galactosidase and Alkaline Phosphatase Activity in
Cell Extracts 10
cells/ml, and
chloramphenicol was added to a final concentration of 80 µg/ml to
terminate protein synthesis. Assay of -galactosidase activity in
cell extracts was performed as described previously(13) . All
experiments involving -galactosidase assay were performed at least
twice, with results being reproducible to within ±15%.Cell Fractionation
Subcellular fractions of R.
sphaeroides 2.4.1 were prepared by the method of Weiss (15) as modified by Tai and Kaplan(16) . The protease
inhibitor phenylmethylsulfonyl fluoride was added to cell extracts at a
final concentration of 1 mM. Protein concentration was
determined using the Pierce reagent (Pierce); bovine serum albumin was
the standard.Carotenoid and Bacteriochlorophyll
Determination
Pigments were extracted from R. sphaeroides 2.4.1 cells and quantitated according to procedures
described(8) . HPLC analysis of pigments was done on a Waters
HPLC system equipped with a model 411 absorbance detector. Suitable
pigment separation was achieved with a Spherisorb ODS2 (Rainin
Instruments Company, Emeryville, CA) reversed-phase column (25
0.46 cm) using a gradient elution system described(17) .
Spectrophotometric Analysis
R. sphaeroides 2.4.1 cells were harvested by centrifugation (10 min, 10,000
g, 4 °C) and disrupted by sonication for 5 min
(50% duty cycle). Unbroken cells and cell debris were removed by
centrifugation (30 min, 30,000
g, 4 °C).
Absorption spectra were analyzed on an SLM DW2000 double-beam UV-VIS-IR
spectrophotometer (SLM-Aminco Instruments, Urbana, IL). Equivalent
protein concentrations of the cleared lysate were used when the
spectral profiles of different strains of R. sphaeroides were
compared. The extinction coefficients reported by Meinhardt et al.(18) were used to calculate the levels of individual
spectral complexes.
Molecular Techniques
Standard procedures were used
for plasmid isolation, restriction endonuclease digestion, isolation of
DNA fragments from gels, ligation, and other molecular biological
techniques(12, 19) . Sequencing was done with 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.DNA Sequence Analysis
Sequence analyses were
performed with the computer programs DNA Strider (Institut de Recherche
Foundamentale, Commissariat a l'Energie Atomique, Paris, France).
Programs FASTA and TFASTA were used to screen EMBL and GenBank data
bases for sequence similarities. These programs use algorithms based on
that of Lipman and Pearson(20) . The program Peptidestructure
was used to predict the secondary structure of TspO.Construction of Plasmids
Plasmid pUI2701 was
constructed by introducing of 1.1-kb KpnI fragment of pUI1124
containing tspO under PrrnB into the KpnI
site of the broad host range expression vector pRK415.
/Sp,
in both orientations. The presence and orientation of the insert were
verified by sequencing from the 5` end of the lacZ toward the
promoter region. Plasmid containing crtA::lacZ fusion was
designated pUI2711, and plasmid carrying crtI::lacZ was
designated pUI2712.SDS-PAGE, Western Blot Analysis, and N-terminal Amino
Acid Sequencing
SDS-PAGE and Western blots were performed as
described (12) . Cultures were grown and cells broken as
described(9) . Western blots were probed with antibodies
against the major outer membrane protein or against TspO.Expression and Partial Purification of TspO-GST Fusion
Protein
TspO-GST fusion protein was expressed in E. coli JM109 from pUI2715 after induction with 100 mM -isopropyl-thiogalactoside for 3 h. Cells were disrupted by
French Press and fractionated by centrifugation (10 min, 20,000 g, 1 h, 150,000
g). Fractions were subjected
to SDS-PAGE, and the TspO-GST protein was found to be localized to the
membrane fraction. Membranes were solubilized with 1% N-lauroylsarcosine in phosphate-buffered saline (140 mM NaCl, 2.7 mM KCl, 10.1 mM Na
HPO
, 1.8 mM KHPO, pH 7.3) and subsequently dialyzed
against 0.05% N-lauroylsarcosine in the same buffer. The
resulting solution was treated with thrombin (10 units of thrombin/mg
protein) at 25 °C overnight. Cleavage products were run at 15%
SDS-PAGE, and a strip of gel corresponding to the 17-kDa protein (TspO)
was removed and used as an antigen for raising antibodies against TspO. Antibodies
Antiserum against TspO was obtained
from Cocalico Biologicals (Cocalico Biologicals, Inc., Reamstown, PA)
using standard procedures. The TspO isolated from SDS-PAGE was used to
immunize two white New Zealand rabbits. The immune and preimmune sera
were characterized for their reactivity against total E. coli and R. sphaeroides 2.4.1 protein and membrane protein.
For immunological detection of transferred proteins, antisera were
diluted in TBST (20 mM Tris, pH 7.4, 500 mM NaCl,
0.05% Tween 20) plus 5% bovine serum albumin and incubated with protein
transferred to nitrocellulose filters for 1.5 h at room temperature.
After extensive washing in TBST and incubation with anti-rabbit
alkaline phosphatase secondary antibodies (1:30,000) for 1 h at room
temperature, the blot was developed by using nitroblue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate.Photoafinity Labeling of the R. sphaeroides 2.4.1 Major
Outer Membrane Protein
Membrane samples (20 µl) containing
50 µg/ml protein in 50 mM Tris-HCl buffer, pH 7.5, were
preincubated with
[N-methyl-
H]flunitrazepam (2
nM) for 30 min and then irradiated for 15 min by a Black-Ray
ultraviolet lamp (Ultraviolet products, San Gabriel, CA). The samples
were then removed from under the lamp and excess of unlabeled
flunitrazepam (10 µM) was added in order to displace any
remaining [H]flunitrazepam not covalently bound
to protein.HANCE (DuPont Biotechnology
Systems, Boston, MA) according to the procedure recommended by the
manufacturer and exposed to Kodak film (Eastman Kodak Co.) for 1.5 mo
at -80 °C.Materials
Restriction endonucleases and nucleic
acid-modifying enzymes were purchased from New England Biolabs, Inc.
(Beverly, MA). Antibiotics, ONPG, X-Gal, X-P, and vitamins were
obtained from Sigma. Unlabeled benzodiazepines were purchased from
Research Biochemicals International (Natick, MA).
[N-methyl-H]Flunitrazepam was from
Amersham (Amersham Corp.). All other chemicals were of reagent grade
purity and were used without further purification.
Cloning, DNA Sequence Determination, and Inactivation
of the Chromosomal tspO
The 2.1-kb SstI fragment of
pAS204 which complemented the mutation(s) in mutant CL(25) was cloned into pBSIIKS+ in both orientations
relative to vector sequences. The DNA sequence of both strands was
determined using overlapping clones covering the 2.1-kb insert. (
)Computer analysis (26) suggested that two open
reading frames (ORFs) which were likely to encode proteins were present
within the fragment. Comparison of the DNA sequences of these ORFs to
computer data base sequence libraries using software described under
``Experimental Procedures'' indicated significant homology to
the DNA sequence of the crtB and crtK genes of R.
capsulatus. The ORF, homologous to crtK, was 480
nucleotides in length (Fig. 1) and shared 60% sequence identity
with the R. capsulatus gene. There is 99.7% identity between
the nucleotide sequences encoding tspO from R. sphaeroides 2.4.1 and from R. sphaeroides NCIB8253(4) .
Tc
transconjugant was
designated TSPO1 after Southern hybridization confirmed the replacement
of the wild type tspO by the tspO::Km
construction from pUI1110. The absence of vector sequences in the
mutant was confirmed when a radioactively labeled pSUP202 probe failed
to hybridize to genomic DNA from the mutant.Analysis of the Deduced Amino Acid Sequence for
tspO
48% identity was observed between the aligned residues for
the deduced amino acid sequences of the R. sphaeroides 2.4.1 tspO and R. capsulatus crtK gene, with an additional
23% of the aligned residues being similar (Fig. 1). Seven
tryptophan residues were conserved in both sequences. Data base
searches revealed TspO was 35% identical to the human peripheral-type
benzodiazepine receptor with an additional 21% of the aligned residues
being similar (Fig. 1). TspO was also 57% similar (34%
identical) to bovine PBR, 55% similar (33% identical) to mouse PBR, and
54% similar (33% identical) to the PBR from rat adrenal mitochondria.
Eight of the 12 tryptophan residues conserved in the four mammalian
PBRs are also conserved in the R. sphaeroides 2.4.1 TspO
sequence. The 158-amino-acid TspO was predicted to have a molecular
mass of 17 kDa.Production of Pigments and Spectral Complexes by TSPO1
Grown Anaerobically
In order to approach the role of the TspO
protein a number of experiments with the mutant TSPO1 harboring a
disruption of tspO by the insertion of the Km gene
were undertaken. No difference in growth rates between wild type and
TSPO1 under a variety of cultivation conditions was observed (data not
shown). The mutant strain was found to produce normal levels of ICM and
all photosynthetic complexes during anaerobic growth in the dark in the
presence of MeSO. Spectral analysis of the membranes from
dark/MeSO grown cultures gave the same absorbance peaks for
wild type and mutant cells. The only differences observed were in
membrane preparations from TSPO1 cells grown photoheterotrophically at
either 10 or 3 W/m, which gave absorbance spectra slightly
different from that of the wild type (Fig. 2). The
B800-850/B875 ratio was 30-40% lower for TSPO1 in
comparison to wild type (Table 2). What is clear is that the
primary effect resulting in the altered ratio is the increased levels
of B875 complex relative to the B800-850 complex in TSPO1.
. Samples of
equal protein concentration (1 mg/ml) were examined as described under
``Experimental Procedures.''
SO in the dark. Quantitation of the
total Crt and total Bchl extracted from mutant and wild type cells did
not reveal any significant difference. HPLC analysis of the composition
of the Crt demonstrated that the major carotenoids (spheroidene,
spheroidenone, and neurosporene) were accumulated by the wild type and
mutant strain in essentially the same amounts.Production of Pigments under Semiaerobic
Conditions
During chemoheterotrophic growth in the presence of
oxygen, mutant TSPO1 cells were more intensively colored than the wild
type cells. Semiaerobic cultures of R. sphaeroides 2.4.1 and
TSPO1 were grown to follow the accumulation of Crt and Bchl by cells
cultivated under conditions of limited oxygen availability (Table 3). As is evident, cells of TSPO1 accumulated severalfold
more Crt and Bchl than those of wild type. Plasmid pUI2701, containing tspO under control of the strong rrnB promoter(27) , was used to study the effect of tspO in trans on pigment accumulation. When present in TSPO1,
pUI2701 led to substantial decreases in both Bchl and Crt accumulation
to below wild type levels.
/68%N/2%CO to an optical
density of 20 KU and shifted to semiaerobic conditions
(3%O/95%N/2%CO) at the time point
indicated by the arrow. Pigments were extracted and quantified
as described under ``Experimental Procedures.'' Carotenoid
accumulation by R. sphaeroides 2.4.1 (
) and TSPO1
(). Bacteriochlorophyll accumulation by R. sphaeroides 2.4.1 (
) and TSPO1 (). Each point represents a mean
of three experiments with deviations being less then ±
15%.
Analysis of puc, puf, crt, and bch Gene
Transcription
Because of the differences in Crt and Bchl
accumulation described above, we tested the possibility that the TspO
might be directly or indirectly involved in the transcriptional control
of the biosynthesis of these photopigments. Therefore, transcriptional lacZ fusions with the promoter region for the divergently
transcribed carotenoid biosynthesis genes crtA and crtI were constructed (see Table 1) and introduced into both R. sphaeroides 2.4.1 and TSPO1 cells. As is evidenced by Table 4, TSPO1 cells exibited 2-5-fold higher expression of
both crtA and crtI than wild type cells. Introduction
of tspO in trans on pUI2701 into TSPO1 cells lowered crt gene expression. In cells grown photoheterotrophically at
10 W/m the absence of TspO led to a 3-fold increase in crtA expression; the effect on crtI was smaller. The
expression of crtA and crtI were the same in both
mutant and 2.4.1 cells grown in the dark in the presence of
MeSO.
Characterization of tspO::phoA Gene Fusions
Due to
the significant homology between TspO and the membrane-localized
mammalian PBRs (Fig. 1) it was anticipated that TspO would be
membrane-localized in vivo. The hydrophobicity plot of TspO
indicated the possible presence of at least four membrane-spanning
domains. Therefore, two translational gene fusions were constructed
between the N-terminal portion of tspO and the genetically
engineered E. coli alkaline phosphatase (APase) gene phoA which is missing the DNA sequence encoding the signal
peptide(13) . The fusion containing the 52
N-terminal amino acids of TspO (pUI1180) exhibited APase activity in R. sphaeroides 2.4.1 cells grown both aerobically or
photosynthetically (300 and 7500 units of specific activity,
respectively). A second fusion following the 89 N-terminal amino acids
(pUI1182) was not active under either set of conditions. Thus, these
results are consistent with the conclusion that TspO resides within the
cell membranes.Immunochemical Detection of TspO
In order to
obtain antibodies against this polypeptide, we cloned tspO under the highly regulated tac promoter into the
expression vector pGEX-2TK (Pharmacia) at the BamHI-EcoRI sites, and the construction was
designated pUI2715. The protein was then overexpressed in E. coli as a fusion with GST after induction with
isopropyl--D-thiogalactoside. TspO-GST constituted more
than 10-15% of the cellular protein as evidenced by SDS-PAGE. The
fusion protein was purified and antibodies against TspO obtained as
described under ``Experimental Procedures.''
and membrane preparations
obtained as described under ``Experimental Procedures.''
Proteins were separated in SDS-PAGE (15%), electroeluted onto
nitrocellulose membranes, and probed with antibodies raised against
TspO. A, immunoblot analysis. Lanes 1 and 3,
outer membrane preparations from semiaerobically and photosynthetically
grown R. sphaeroides 2.4.1 cells, respectively; lane
2, inner membrane preparation from semiaerobically grown 2.4.1; lanes 4 and 5, outer membrane and inner membrane
preparations from semiaerobically grown TSPO1 cells; lane 6,
preparation of overexpressed in E. coli TSPO-GST cleaved with
thrombin; lane 7, molecular weight marker. B,
SDS-PAGE gel stained with Coomassie Blue. Lanes are the same as in A.
36 kDa. On the other hand, the level of TspO
in photosynthetic grown cells was considerably increased, but the
immunoactive species had a size of 17 kDa.
Experiments with Benzodiazepines
R.
sphaeroides 2.4.1 was cultured either photoheterotrophically (10
W/m light intensity) or in dark/MeSO in the
presence of varying concentrations of the benzodiazepines flurazepam or
flunitrazepam. The growth rate was not affected by either drug in the
range of concentrations from 0 to 80 µg/ml. However, when the
formation of spectral complexes was examined (Fig. 6A),
we observed that the amount of the B800-850 complex was reduced
by 40-45% when cells were incubated in the presence of 50
µg/ml of flunitrazepam, and the amount of B875 complex remained the
same as in control cells grown without the drug. An even stronger
effect was observed in the presence of flurazepam which inhibited the
accumulation of the B800-850 complex by 90-95% (Fig. 6A). The formation of the B875 complex was
decreased by 60-65% at this concentration of flurazepam. However,
the same effects were observed in experiments with mutant strain TSPO1 (Fig. 6B), thus eliminating the possible involvement of
TspO in the benzodiazepine sensitivity of R. sphaeroides 2.4.1
in the
presence of various concentrations of flurazepam, as indicated, and
membrane fractions obtained as described under ``Experimental
Procedures.'' Samples of equal protein concentrations (1 mg/ml)
were examined.
H]flunitrazepam as a means of
following the binding of this drug to cellular proteins. Cells of
either R. sphaeroides 2.4.1 or TSPO1 grown aerobically or
photoheterotrophically were fractionated according to procedures
described under ``Experimental Procedures.'' These
preparations were preincubated with H-labeled flunitrazepam
and UV-cross-linked. SDS-PAGE fractionation of proteins and subsequent
radioautography revealed only one major radioactive band in the outer
membrane preparation corresponding to a protein of approximately 47 kDa (Fig. 7, A and B). No specific binding of
[H]flunitrazepam was detected in either the inner
membrane or cytoplasmic proteins. One potential difficulty in detecting
benzodiazepine binding to TspO is the very low level of TspO relative
to the major outer membrane protein in R. sphaeroides 2.4.1;
binding to TspO could be masked, and then special precautions might
have to be exercised.
H]flunitrazepam binding species. A, Coomassie Brilliant Blue-stained SDS-PAGE. R.
sphaeroides 2.4.1 and TSPO1 cells were grown semiaerobically and
fractionated as described under ``Experimental Procedures.'' Lanes 1 and 4, outer membrane preparations; lanes
2 and 5, inner membrane preparations; lanes 3 and 6, cytoplasmic fraction from 2.4.1 and TSPO1 cells,
respectively. B, radioautogram of R. sphaeroides 2.4.1 proteins labeled with
[H]flunitrazepam. Lanes are the same as in A. C, immunoblot analysis of major outer membrane
protein. Proteins from fractionated cells of R. sphaeroides 2.4.1 were resolved by SDS-PAGE and probed with antibodies raised
against major outer membrane protein (porin). Lanes 1 and 4 are the same as in A and B.
Reaction of Rat Mitochondria with Antibodies to
TspO
When we probed preparations of either kidney or liver, rat
mitochondria with antibodies raised against TspO, a strong and highly
specific cross-reactivity was observed with three protein species from
kidney and with two protein species from liver mitochondria (Fig. 8). In both liver and kidney, immunoactive bands at
approximately 30 and 50 kDa were present. In preparation from the
kidney mitochondrion additional bands of approximately 35 and 55 kDa
were observed; however, these bands were absent from the liver
preparation. The relative intensities of immunoactive bands were much
higher in preparations from kidney mitochondria which correlates with
the observed 2-3-fold higher level of the benzodiazepine binding
protein in kidney. ()
H-labeled structural analog of flurazepam, flunitrazepam,
demonstrated this drug specifically bound to the major outer membrane
protein of R. sphaeroides 2.4.1, whether or not TspO is
present. It was recently shown that the R. capsulatus major
outer membrane protein, or porin, binds with high affinity some of the
tetrapyrrole intermediates in Bchl a biosynthesis(28) . One possibility is that benzodiazepines
when present in micromolar concentrations compete for
tetrapyrrole-binding sites. However, why the biosynthesis of the
B800-850 complex is inhibited to a greater extent than that of
the B875 complex still remains unclear. These results may reflect
differences in the assembly pathways for each of these macromolecular
complexes.) thus indicating that TspO can exert its
effect not only under aerobic conditions, but also during
photosynthetic growth. However, mutant cells grown
photoheterotrophically at high light intensities (100 W/m)
or in dark/MeSO, showed no differences in crt,
bch, or puc gene expression when compared to the wild
type. Therefore, TspO because of its cellular location and its effect
on gene expression in response to oxygen or light behaves as a sensor
of these environmental stimuli. Both of these stimuli could act through
a common element, e.g. redox, or the protein could directly
sense changes in both oxygen and light by some unknown mechanism.
Reinforcing this latter interpretation is the cellular localization of
TspO, i.e. in the outer cell membrane; therefore, whatever
this protein detects it probably exists outside the cell, and changes
in redox would be less likely. Further, the presence of the apparently
dimeric form of TspO in aerobic grown cells and the monomeric form in
anaerobic grown cells could be indicative of a mechanism by which TspO
functions.)also confirms the presence of the Fnr system of
aerobic/anaerobic control in R. sphaeroides 2.4.1. In
addition, the TspO system also appears to be able to sense differences
in light intensity. We do not know the extent of overlap on
photosynthesis gene expression between these regulatory systems. level, the likely response regulator
with which it interacts and to further investigate the functional and
structural relationships between TspO and the mammalian PBR.
)SO, dimethyl sulfoxide; GST, glutathione S-transferase; PhoA, alkaline phosphatase; VDAC,
voltage-dependent anion channel; HPLC, high performance liquid
chromatography; kb, kilobase(s); PAGE, polyacrylamide gel
electrophoresis; ORFs, open reading frames(s).)))
We thank Dr. M. Wood for early work on tspO,
Drs. J. Eraso, M. Gomelsky, M. Sabaty, and L. Gong for providing
strains and plasmids, and Dr. M. McEnery for the preparations of rat
mitochondria.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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