(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). ()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) .
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 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) .
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/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.
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 ENHANCE (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.
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 KmTc
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.
Figure 2:
Absorption spectra of R. sphaeroides 2.4.1 membrane preparations. Cells were grown
photoheterotrophically in the light at 10 W/m. Samples of
equal protein concentration (1 mg/ml) were examined as described under
``Experimental Procedures.''
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 MeSO 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.
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/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%.
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 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.
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 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.
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 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.
To obtain additional insight into the possible target, we used
[N-methyl-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.
Figure 7:
Identification of the major outer membrane
protein as a [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.
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 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.
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) 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/Me
SO, 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.
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 ()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.
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 level, the likely response regulator
with which it interacts and to further investigate the functional and
structural relationships between TspO and the mammalian PBR.