J Biol Chem, Vol. 274, Issue 30, 21234-21243, July 23, 1999
A Novel Mechanism for the Regulation of Photosynthesis Gene
Expression by the TspO Outer Membrane Protein of Rhodobacter
sphaeroides 2.4.1*
Alexei A.
Yeliseev and
Samuel
Kaplan
From the Department of Microbiology and Molecular Genetics,
University of Texas Health Science Center at Houston, Medical School,
Houston, Texas 77225
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ABSTRACT |
A bacterial homolog of the mammalian
mitochondrial benzodiazepine receptor, the tryptophan-rich sensory
protein (TspO) has been previously demonstrated to negatively affect
the transcriptional expression of several photosynthesis genes of
Rhodobacter sphaeroides. To identify components of the
signal transduction pathway from the outer membrane-localized TspO to
the DNA-active transcription factor(s), we examined the involvement of
TspO in the regulation of tetrapyrrole metabolism in R. sphaeroides. By analyzing the tetrapyrrole pigments accumulated
by resting cell suspensions of R. sphaeroides, we
demonstrated that TspO negatively regulates the activity of
coproporphyrinogen III oxidase in this bacterium. hemN,
encoding one of the isoenzymes of coproporphyrinogen III oxidase of
R. sphaeroides, provided in trans to the wild
type strain, produced a TSPO1 mutant phenotype by abolishing the
negative effect of TspO on the transcription of the photosynthesis
genes, crtI and puc. It is proposed that TspO,
by regulating the exit of certain tetrapyrrole intermediates of the
heme/bacteriochlorophyll biosynthetic pathways in R. sphaeroides in response to the availability of molecular oxygen,
causes the accumulation of a biosynthetic intermediate that serves as a
corepressor for both specific pigment gene transcription and the
puc operon. The relationship between the bacterial TspO and
the mitochondrial peripheral benzodiazepine receptor is discussed.
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INTRODUCTION |
Rhodobacter sphaeroides is a facultative phototrophic
bacterium capable of growth both aerobically and anaerobically
(photosynthetically). Decreased oxygen tension induces the development
of the intracytoplasmic membrane system, which harbors all the
components of the photosynthetic apparatus. Several regulatory systems
have been identified in R. sphaeroides that govern the
synthesis and/or assembly of the components of the intracytoplasmic
membrane system (1). One of these, the tryptophan-rich sensory protein
(TspO) has been demonstrated to modulate the synthesis of the
structural polypeptides of the B800-850 photosynthetic antenna complex
as well as the photopigments, bacteriochlorophyll, and carotenoids, in
response to variations in oxygen tension and/or light intensity. TspO
has been shown to negatively affect the transcription of specific bacteriochlorophyll and carotenoid biosynthesis genes as well as the
puc operon under aerobic, semiaerobic, or photosynthetic conditions (2).
Using polyclonal antibodies raised against the purified protein, TspO
has been shown to reside in the outer membrane of R. sphaeroides, similar to the localization of its mammalian homolog pk18, a drug-binding component of the mitochondrial benzodiazepine receptor (MBR).1 Although the
precise biological role of MBR is not yet understood, this receptor was
shown to take part in the regulation of a number of metabolic
activities in mammalian cells including steroidogenesis, heme
biosynthesis, and mitochondrial respiratory control (3). A cDNA
clone of the rat pk18 gene, when expressed in R. sphaeroides TSPO1 cells, functionally substituted for the bacterial homolog, negatively affecting the expression of the crtI gene,
involved in carotenoid biosynthesis, and puc operon,
encoding the polypeptides of the peripheral light harvesting complex
(LH2). These results suggested that TspO is both evolutionarily and
functionally related to the mammalian MBR and indicated the possibility
that in both mammalian and bacterial cells these proteins are involved
in similar metabolic activities (4).
The cellular localization of TspO and its effect upon transcription of
a number of photosynthesis genes in R. sphaeroides pose a
question concerning the pathway and mechanism of signal transduction
from this outer membrane-localized receptor to the regulated DNA-bound
transcription factors. Useful information on the possible mechanism of
TspO activity is provided by studies of the mammalian MBR. Because MBR
binds with nanomolar affinity to a variety of benzodiazepines as well
as to the dicarboxylic porphyrins, it was proposed to be involved in
the regulation of the heme biosynthetic pathway, controlling the flow
of tetrapyrrole intermediates across the mitochondrial membrane (5-9).
As such we focused our attention on the pathways for tetrapyrrole
biosynthesis in R. sphaeroides. In the present study we
provide further insight into the mechanism of the biological activity
of this receptor by demonstrating that TspO appears to affect the
activity of hemN-encoded coproporphyrinogen III oxidase of
R. sphaeroides and thereby negatively regulates the
expression of specific photosynthesis genes by causing the accumulation
of a specific corepressor molecule.
Fig. 1 depicts the organization of the
heme biosynthetic pathway in mammalian cells. It has been proposed that
5-aminolevulinic acid (ALA), formed in the mitochondrion, is exported
to the cytoplasm where it is converted to coproporphyrinogen via
porphobilinogen (PBG) and uroporphyrinogen III (10). Coproporphyrinogen
III then re-enters the mitochondrion, where it is converted
subsequently to protoporphyrinogen IX, protoporphyrin IX, and heme.
Although the mitochondrially localized biosynthesis of ALA and the
conversion of coproporphyrinogen to heme are well documented, little is
known about the mechanism of intracellular transport of
coproporphyrinogen. It has been proposed that MBR may play a role in
this process (7). Therefore, our studies on the TspO-mediated
regulation of tetrapyrrole biosynthesis in R. sphaeroides
may help to shed light on the function of this receptor in mammalian
cells.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains, Plasmids, Media, and Growth
Conditions--
Bacterial strains and plasmids used in this study are
described in Table I. 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 (11) as described previously (12). Aerobic cells were grown in
200-ml glass bottles filled with 100 ml of medium, under continuous
sparging with a mixture of gases 78% N2/20%
O2/2% CO2. Semiaerobic cells were grown by
sparging gas mixture of 96% N2/2% O2/2%
CO2. Photosynthetic cells were grown in front of a light
source at 10 W/m2, by continuous sparging with 98%
N2/2% CO2.
Cell growth was monitored turbidometrically with a Klett-Summerson
colorimeter using a number 66 filter. 1 Klett unit is equivalent to
1 × 107 cell/ml. To minimize tetracycline
photooxidation (13), liquid cultures of R. sphaeroides grown
photoheterotrophically in the presence of tetracycline were placed
behind a CS7-69 filter (620-1100 nm; Corning Glass Works, Corning,
NY). Strains of Escherichia coli were grown as described
previously (14). When appropriate, antibiotics were added to the
following final concentrations: ampicillin, 100 µg/ml; kanamycin, 25 µg/ml; spectinomycin, 50 µg/ml; streptomycin, 50 µg/ml; and
tetracycline, 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 (15).
-Galactosidase Activity in Cell Extracts--
R.
sphaeroides cultures were grown to a cell density of approximately
1.8 × 108 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 (15). All experiments involving -galactosidase
assay were performed at least twice, with results being reproducible to
within ± 15%.
Spectrophotometric Analysis--
R. sphaeroides cells
were harvested by centrifugation (15 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
with a Shimadzu UV-1601 spectrophotometer. Equivalent protein
concentrations of the cleared lysate were used when the spectral
profiles of different strains of R. sphaeroides were compared.
For the analysis of pyridine spectra, cells were harvested by
centrifugation and resuspended in alkaline pyridine (pyridine: 0.1 N NaOH, 2:1 v/v). After incubation for 10 min in the dark, cell lysates were cleared by centrifugation and analyzed
spectrophotometrically. Extinction coefficients for the porphyrins and
metalloporphyrins were used as reported previously (16).
Preparation of Resting Cell Suspensions--
R.
sphaeroides cells were grown either aerobically or
photosynthetically to a cell density of approximately 1.8 × 108 cells/ml, harvested by centrifugation (15 min,
10,000 × g, 10 °C), washed with 0.1 M
potassium phosphate buffer, pH 7.4, and resuspended in the same buffer
containing 80 µg/ml chloramphenicol to terminate protein synthesis.
HPLC Analysis of Tetrapyrrole Pigments--
HPLC analysis of the
tetrapyrrole pigments accumulated by resting cells of R. sphaeroides was performed according to the method described
previously (17). Cells were separated by centrifugation (15 min,
10,000 × g, 4 °C), and the pH level of the
supernatant was adjusted to 3.5 with 6 N HCl. Talc powder
was added (1 g/100 ml) to the supernatant, and the suspension was
vigorously shaken for 30 min. The talc was than collected by filtration
and washed with water, and the porphyrin pigments were eluted with a
mixture of acetone and 0.1 N HCL (9:1 v/v). Eluates were
dried under vacuum, and pigments were stored at 
80 °C prior to
further analysis. HPLC analysis of pigments was performed with a
Shimadzu SCL-10A HPLC system equipped with an SPD-M10AV diode array
detector. Pigments were separated on a SAS Hypersil (Keystone
Scientific Inc, Belleforte, PA) reversed-phase column (150 × 4.6 mm) using a gradient elution system as described (18). Porphyrin
pigments that accumulated in cells were extracted and analyzed
essentially as described in Ref. 19. Analysis of the magnesium and iron
content in the isolated metalloporphyrins was done using atomic
absorption spectroscopy at the Analytical Chemistry Center at The
University of Texas Medical School.
Analysis of Enzymatic Activities--
Analysis of the enzymatic
activities of ALA synthase, ALA dehydratase, and PBG deaminase was
conducted as described earlier (20-22).
Molecular Techniques--
Standard procedures were used for
plasmid isolation, restriction endonuclease digestion, isolation of DNA
fragments from gels, ligations, and other molecular biological
techniques (14, 23). DNA sequencing was performed 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.
Construction of Plasmids--
Plasmid pUI 2730 was constructed
by introducing a 1.6-kb HindIII-BamHI fragment of
pUI2080 containing hemN with the upstream promoter region
into the HindIII-BamHI sites of the broad host range expression vector pRK415. Plasmid pUI2731 was constructed by
introducing a 1.1-kb HindIII-Asp718 fragment of
pUI1124 containing tspO under PrrnB into the
pUI2080 digested with HindIII-Asp718. Plasmid
pUI2732 containing hemN and tspO under
PrrnB was constructed by ligating a 2.6-kb
XbaI-Asp718 fragment of pUI2731 into pRK415 digested with XbaI-Asp718. Plasmid pUI2751 was
constructed by introducing a 1.6-kb fragment containing polymerase
chain reaction-generated hemZ with its upstream promoter
region into the pBSIIKS+. pUI1958 was used as a template for the
polymerase chain reaction, with primers 5Z-PST,
5'-ATCTGCTGCAGCGTCGCGGAGTT-3', and 3Z-ECORV,
5'-GCTGATATCGGTGCCTCAGATC-3'. The 1.6-kb polymerase chain reaction
product was digested with PstI and EcoRV and
introduced into the PstI-EcoRV-digested pBSIIKS+. Plasmid pUI2752 containing hemZ with the upstream promoter
sequence was constructed by ligating a 1.6-kb
PstI-EcoRV fragment of pUI2751 into the
PstI-Ecl136II-digested pRK415. Plasmid pUI2761
containing the promoter region for hemN was constructed by
introducing a 0.5-kb BamHI-EcoRI fragment of
pUI2080 into the BamHI-EcoRI-digested pBSIIKS+.
Plasmid pUI2762 containing a hemN::lacZ
transcriptional fusion was constructed by introducing a 0.5-kb
NotI-EcoRV fragment of pUI2761 into the
NotI-StuI-digested pCF1010.
Materials--
Restriction endonucleases and nucleic
acid-modifying enzymes were purchased from New England Biolabs, Inc.
(Beverly, MA). Antibiotics, ALA, PBG,
5-bromo-4-chloro-3-indolyl--D-galactoside, o-nitrophenyl--D-galactopyranoside, and
vitamins were obtained from Sigma. Porphyrins and metalloporphyrins
were purchased from Porphyrin Products (Logan, UT).
[14C]Coproporphyrin was from Leeds Radioporphyrins
(Leeds, UK).
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RESULTS |
Accumulation of Porphyrin Intermediates in Wild Type and TSPO1
Cells--
R. sphaeroides cells accumulate only minute
amounts of free porphyrins when grown in minimal Sistrom's medium
aerobically in the dark or anaerobically (photosynthetically). Our
preliminary studies revealed no significant differences in the
accumulation of heme pigments by either the wild type or TSPO1 cells
grown under these conditions. We previously reported, however, elevated levels of bacteriochlorophyll in TSPO1 cells grown semiaerobically (2),
which could be accounted for by the increased transcription of the
respective bacteriochlorophyll biosynthesis genes in the TSPO1 cells.
Thus, to study the possible effect(s) of TspO on the early steps in the
biosynthesis of tetrapyrroles, common to both hemes and
bacteriochlorophylls, it was necessary to create conditions that would
result in an increased flow of the tetrapyrrole intermediates along the
pathway. This was achieved by addition of exogenous ALA, a common
biosynthetic precursor of all tetrapyrroles. It has been repeatedly
demonstrated in this
laboratory2 that addition of
exogenous ALA to growing R. sphaeroides 2.4.1 cells leads to
a marked inhibition of growth under either aerobic or anaerobic
conditions. Prolonged incubation of the cells in the presence of
exogenous ALA (concentrations in the range 0.02-1 mM) can
lead to the accumulation and enrichment of spontaneous mutations, many
of which are altered in pigment biosynthesis. To avoid these
undesirable effects of ALA, experiments were conducted using resting
cell suspensions of R. sphaeroides.
Wild type or TSPO1 cells were grown aerobically, collected by
centrifugation, and resuspended as described under "Experimental Procedures." Cells were challenged with 0.02-0.1 mM ALA
followed by incubation for 4-8 h. Following incubation, cells were
collected by centrifugation, and endogenous and exogenous tetrapyrrole
pigments were analyzed separately.
Differences between the wild type and TSPO1 mutant cells in the
accumulation of endogenous tetrapyrrole pigments are demonstrated by
the absorption spectra of the alkaline pyridine extracts. Tetrapyrroles accumulated in wild type cells exhibited an aethio-type spectrum with
the Soret maximum at 402-404 nm, characteristic of free porphyrins (Fig. 2). HPLC analysis of these pigments
demonstrated that they were represented by a mixture of uro-, copro-,
and protoporphyrin as well as some small amounts of hemes and other
metalloporphyrins (Results not shown). The pigments extracted from the
mutant TSPO1 cells produced a spectrum with a maximum at 423 nm with a
shoulder at 404 nm, as well as two other peaks of lower amplitude, 549 and 587 nm, characteristic of the magnesium-containing protoporphyrins or monomethyl ester of the magnesium-protoporphyrin. Oxidized (air) and
reduced (sodium dithionite) spectra of these pigments are identical to
that of an authentic magnesium-protoporphyrin (not shown). The
introduction of the tspO gene in trans
(approximately five copies) under a strong rrnB promoter
into the TSPO1 strain led to a marked decrease in the accumulation of
tetrapyrrole pigments from exogenously supplied ALA, well below the
levels observed for the wild type or TSPO1 mutant cells. The
inset in Fig. 2 shows the relative levels of TspO in the
outer membranes of the various cell types used in this study. The
dramatic decrease in the accumulation of tetrapyrrole pigments by the
TSPO1 (pUI2701) inversely correlates with the significant increase of
TspO levels in these cells. On the other hand, wild type cells
containing the "physiological" concentration of TspO were
characterized by relatively higher amounts of accumulated free
porphyrins than found in the TSPO1 cells (Fig. 2). Therefore, it
appears that depending upon the levels of normal TspO in the outer
membrane, it can either positively (when present at low levels) or
negatively (when present at very high levels) affect the accumulation
of tetrapyrroles in the cells. But what is clear is that in the absence
of TspO, cells accumulate considerably less porphyrins than found in
the wild type.
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Fig. 2.
Accumulation of tetrapyrrole pigments by
R. sphaeroides cells. Absorbance spectra in
alkaline pyridine are shown. Cells were grown aerobically as described
under "Experimental Procedures," resuspended in 0.1 M
potassium-phosphate buffer pH 7.5, containing chloramphenicol, and
incubated aerobically on a shaker (100-ml flasks filled with 20 ml of
medium, 300 rpm) in the presence of 0.2 mM ALA for 6 h
at 30 °C. Cells were collected by centrifugation, washed twice with
the potassium phosphate buffer, and resuspended in alkaline pyridine.
Inset, Western blot of the cell extracts from R. sphaeroides 2.4.1, TSPO1 and TSPO1 (pUI2701). Proteins were
separated on a 15% SDS-polyacrylamide gel electrophoresis,
electroblotted onto nitrocellulose membranes, and probed with
antibodies against TspO. 40 µg of protein was applied per lane.
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HPLC analysis of the pigments accumulated in the culture supernatant
demonstrated that wild type cells excreted predominantly uroporphyrin
III and I as well as the decarboxylation products (Fig.
3). The total amount of porphyrin
pigments excreted from the mutant cells was 30-35% of that of the
wild type cells. The mutant TSPO1 cells excreted almost exclusively
coprpoporphyrin (III and I), as well as magnesium-coproporphyrin.
Magnesium-coproporphyrin was identified by HPLC and by electronic
absorbance spectra. The presence of magnesium in this metalloporphyrin
was supported by the results of atomic absorption analysis (results not
shown). Mutant TSPO1 cells harboring the tspO gene in
trans under the rrnB promoter accumulated significantly
lower amounts of porphyrin pigments (~7-8% of that of the wild type
accumulation) in the culture supernatants, consistent with the
decreased accumulation of endogenous tetrapyrroles in these cells. A
similar effect of the tspO gene in trans was
observed when pUI2701 was introduced in the wild type cells (results
not shown). Exogenous pigments were represented by
magnesium-coproporphyrin, coproporphyrin, as well as other
decarboxylation products of uroporphyrinogen. Therefore it appears that
the presence of increased levels of normal TspO in the outer membrane
inversely affects the ability of R. sphaeroides cells to
excrete porphyrin pigments.
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Fig. 3.
HPLC analysis of the pigments excreted from
R. sphaeroides cells incubated in the presence of 0.2 mM ALA. Pigments were extracted and analyzed as
described under "Experimental Procedures."
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On the other hand, wild type cells excreted significantly more
porphyrins into the supernatant than the cells devoid of TspO. These
results correlate with the differential accumulation of tetrapyrroles
in R. sphaeroides cells, depending on the levels of TspO in
the outer membrane (Fig. 2). The results suggest that when TspO is
present in cells in concentrations not exceeding certain physiological
levels, this protein positively affects the efflux of tetrapyrroles
from the cells. When the tspO gene is provided in multicopy
and expressed from the strong rrnB promoter, the levels of
this protein in the outer membrane increase dramatically (our estimates
show about an 30-50-fold increase over the levels observed in the wild
type cells). When present at such high concentrations, TspO inversely
affects the efflux of tetrapyrroles and behaves more like a TspO mutant
strain. Such an effect could be explained by the presence of excess
TspO engaging in "interactions" that are not conducive to normal
TspO function.
It had been demonstrated previously that deprivation of methionine
limits bacteriochlorophyll synthesis in R. sphaeroides, because the methylation of magnesium-protoporphyrin involves
S-adenosylmethionine as a methyl donor (24, 25). In wild
type R. sphaeroides, limitation of methionine can be
achieved by the addition of threonine, which inhibits its formation at
the stage of homoserine dehydrogenase (26). We therefore studied the
effect of threonine upon the accumulation of tetrapyrroles by the wild
type and TSPO1 cells. The results summarized in Fig.
4 indicated that threonine had no
appreciable effect on the wild type 2.4.1 cells upon the accumulation of porphyrins from exogenously supplied ALA. However, when threonine was added to a suspension of TSPO1 cells, challenged with ALA, the
composition of the accumulated tetrapyrroles in these cells was
severely affected, as judged by the absorption spectra of the pyridine
extract. The total absorption increased 2.5-fold, and the peak maximum
shifted from 423 to 409 nm with a shoulder at 406 nm, indicating a
decrease in the accumulation of magnesium-protoporphyrin and an
increase in the amounts of free porphyrins, namely protoporphyrin IX,
which is characterized with an absorption maximum at 409 nm in alkaline
pyridine.
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Fig. 4.
Effect of threonine on accumulation of
porphyrins by R. sphaeroides cells. Spectra in
alkaline pyridine are shown. Cells were grown as described in the
legend to Fig. 2 and incubated with 0.2 mM ALA, with 5 mM threonine, or with 0.2 mM ALA + 5 mM threonine.
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These observations were additionally supported by HPLC analysis of the
tetrapyrrole pigments extracted from the cells (Table II). Uroporphyrin and its decarboxylation
products (hepta-, hexa-, and penta-carboxylic porphyrins) constituted
the majority of the tetrapyrrole pigments accumulated by wild type
cells. These cells also contained small amounts of coproporphyrin III
and magnesium-containing porphyrins. In contrast, mutant TSPO1 cells
accumulated predominantly early intermediates of the
bacteriochlorophyll branch of the pathway, magnesium-protoporphyrin and
magnesium-protoporphyrin monomethyl ester. The addition of threonine to
the TSPO1 cells challenged with 0.2 mM ALA led to the
decreased proportion of magnesium-containing porphyrins and increased
amounts of protoporphyrin and early decarboxylation products of
uroporphyrinogen III.
To obtain further insight into the mechanism of TspO involvement into
tetrapyrrole metabolism, we studied the accumulation of porphyrins and
metalloporphyrins in R. sphaeroides cells incubated in the
presence of PBG, the next (after ALA) common intermediate of the
tetrapyrrole biosynthetic pathway. When wild type cells were incubated
aerobically with exogenous PBG, they accumulated predominantly free
porphyrins (uroporphyrin and its decarboxylation products), whereas
TSPO1 cells accumulated predominantly early intermediates in the
bacteriochlorophyll branch, namely magnesium-protoporphyrin and its
monomethyl ester. These results are similar to what was observed in the
experiments with ALA (data not presented).
In a control experiment, free porphyrins, namely coproporphyrin III and
uroporphyrin III, which are products of the oxidation of their
respective porphyrinogens and are not a physiologic intermediates of
the tetrapyrrole biosynthesis pathway, were used in an incubation medium containing suspensions of resting R. sphaeroides
cells. No significant increase in the accumulation of intracellular
tetrapyrrole pools was observed. When cells were incubated in the
presence of labeled [14C]coproporphyrin III, only trace
amounts of cell-associated radioactivity could be detected. Thus, it
appears, that even if coproporphyrin III is taken up by R. sphaeroides cells, it is not reduced to coproporphyrinogen and
hence is not converted into protoporphyrinogen, protoporphyrin, and its
metal-containing derivatives.
Effect of Oxygen on Tetrapyrrole Pigment Production--
To gain
further insight into the mechanism of TspO involvement into the
metabolism of tetrapyrroles, we examined the effect of oxygen upon the
production of tetrapyrrole pigments, because it was previously shown
(2) that the accumulation of Bchl in TSPO1 appeared maximal under
conditions of low aeration. When aerobically grown cells (78%
N2/20% O2/2% CO2) were incubated under conditions of limited aeration (96% N2/2%
O2/2% CO2) at low cell density in the presence
of ALA, no differences in accumulation of the tetrapyrrole pigments
between the wild type and TSPO1 cells were observed (Fig.
5). Decreased oxygen tensions led to a
sharp decrease in the accumulation of intermediates in the
bacteriochlorophyll biosynthesis pathway, namely,
magnesium-protoporphyrin and magnesium-protoporphyrin monomethyl ester
by TSPO1 cells. Similarly, decreased oxygen availability led to a sharp
decrease in the accumulation of free porphyrins by the wild type cells.
Thus, it appears that the limitation of oxygen negatively affects the
flux of precursors through the tetrapyrrole pathway in resting cell
suspensions of both wild type and mutant TSPO1. One of the possible
explanations of this phenomenon is that anaerobic (or oxygen-limiting)
conditions prevented O2-dependent heme
breakdown, allowing the build up of increased concentrations of heme,
which feed back inhibited early steps of the pathway (25, 27). Another
possibility is that limited oxygen availability slows down one or
several steps of the bacteriochlorophyll branch, promoting the
accumulation of intermediates that inhibit early steps. Therefore using
this experimental approach, it was not possible to determine whether a
TspO-mediated signaling pathway responds directly to variations in
oxygen availability.
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Fig. 5.
Effect of oxygen on accumulation of
tetrapyrrole pigments by R. sphaeroides cells.
Spectra in alkaline pyridine. Cells were grown either aerobically (78%
N2/20% O2/2% CO2) or
semiaerobically (96% N2/2% O2/2%
CO2), incubated, and processed as described in the legend
to Fig. 2.
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Photosynthetic Cells--
When photosynthetically grown cells were
incubated aerobically in the presence of exogenous ALA, wild type cells
accumulated predominantly free porphyrins, as judged by the absorbance
spectrum in alkaline pyridine (Fig. 6).
Under these same conditions TSPO1 cells accumulated pigments,
characterized by two distinct maximums, at 401 (free porphyrins) and
414 nm (metal-containing porphyrins). These results indicated a shift
in the ratio between the early and late intermediates of the
tetrapyrrole pathway in the TSPO1 mutant cells, similar to what was
observed for aerobic grown cells. Similar to aerobic grown cells, the
accumulation of tetrapyrrole pigments by the photosynthetic grown wild
type or TSPO1 cells, incubated in the presence of ALA under limited
oxygen availability, were significantly decreased (results not
shown).
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Fig. 6.
Accumulation of tetrapyrrole pigments by
photosynthetically grown cells. Spectra in alkaline pyridine are
shown. R. sphaeroides cells were grown photosynthetically at
10 W/m2, collected by centrifugation, and resuspended in
0.1 M potassium phosphate buffer, pH 7.5, containing
chloramphenicol. Cell suspensions were incubated aerobically on a
shaker in the presence of 0.2 mM ALA. Pigments extracted as
described in the legend to Fig. 2.
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In summary, the results of experiments on the accumulation of porphyrin
pigments from exogenously supplied ALA indicated that the flow of
tetrapyrrole intermediates along the heme/bacteriochlorophyll pathway
is negatively regulated by TspO at a step preceding the formation of
protoporphyrin IX. In all instances we see that the presence of the
normal tspO in either single or extra copy significantly affects the levels of accumulation and the kind of intermediate accumulated when compared with mutant TSPO1. Thus, TspO appears to
regulate the flow and qualitative nature of the intermediates in the
pathway. In R. sphaeroides cells, in addition to the heme and bacteriochlorophyll branches, there are also siroheme and vitamin
B12 branches of the tetrapyrrole biosynthetic pathway. Thus, the interplay between these various branches is difficult to
predict and to assess. However, the effect of TspO on these pathways
seems clear. Two enzymes in R. sphaeroides,
coproporphyrinogen III oxidase and protoporphyrinogen oxidase,
catalyzing, respectively, the oxidative decarboxylation of
coproporphyrinogen III into protoporphyrinogen IX and oxidation of
protoporphyrinogen IX into protoporphyrin, are involved in the
conversion of coproporphyrinogen III into protoporphyrin IX (28). The
oxidation of protoporphyrinogen into protoporphyrin can also proceed
nonenzymatically in the presence of molecular oxygen. Because wild type
cells of R. sphaeroides accumulate predominantly
uroporphyrin and its decarboxylation products but only small amounts of
protoporphyrin when incubated in the presence of ALA aerobically, it
seemed likely that the activity of coproporphyrinogen III oxidase and
not protoporphyrinogen oxidase was affected in the TSPO1 mutant strain.
Therefore this reaction appears to be rate-limiting in the pathway and
may serve as an important step at which control over the flow of
intermediates along the heme/bacteriochlorophyll branches can be
exerted. The activities of several enzymes of the
heme/bacteriochlorophyll pathway have been tested in cell extracts
prepared from the aerobic grown R. sphaeroides wild type or
TSPO1 cells. There were no significant differences in the activities of
ALA-synthase and ALA-dehydratase in the TSPO1 cells when compared with
the wild type cells. These data are summarized in Table
III.
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[in this window]
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|
Table III
Activities of tetrapyrrole biosynthetic enzymes in cell-free extracts
of R. sphaeroides 2.4.1 (wild type) and TSPO1
Cells were grown aerobically and collected by centrifugation, and
enzyme activity was determined as described under "Experimental
Procedures."
|
|
Effect of TspO on the Transcription of hemN/hemZ in R. sphaeroides--
In an attempt to identify the possible target for
TspO, we studied the effect of TspO upon the transcription of the two
genes encoding isoenzymes of the "oxygen-independent"
coproporphyrinogen III oxidase. Transcriptional fusions of the promoter
regions for hemN and hemZ and a promoterless
-galactosidase were constructed and introduced in trans
into the wild type and TSPO1 mutant cells of R. sphaeroides
(Fig. 7). Under all growth conditions
tested, the activity of hemN::lacZ and
hemZ::lacZ did not vary significantly between the
wild type and TSPO1 cells. The transcription of hemN was
about 2-3-fold higher than that of hemZ under conditions of low aeration or under photosynthetic conditions.
hemN::lacZ was also expressed at high levels
(~50% of maximal activity) under aerobic conditions, whereas
hemZ::lacZ exhibited only basal levels of activity
under these conditions.
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Fig. 7.
Transcriptional activity of hemN
and hemZ. Cells were grown aerobically
(20% O2, 2% CO2, 78% N2),
semiaerobically (2% O2, 2% CO2, 96%
N2), or photosynthetically 10 W/m2 (2%
CO2, 98% N2), and transcriptional activity was
analyzed as described under "Experimental Procedures."
|
|
Effect of hemN/hemZ in Multicopy on the Transcription of
Photosynthesis Genes--
From the above discussed results we
concluded that TspO was somehow involved in the flow and/or regulation
of tetrapyrrole biosynthesis in R. sphaeroides. To gain
insight into the possible mechanism of this regulation, we introduced
the genes encoding the coproporphyrinogen III oxidase isoenzymes in
multicopy (approximately 4-6 copies/cell) in the wild type and TSPO1
cells of R. sphaeroides and assessed their effect upon the
transcription of TspO targeted photosynthesis genes (2). The results of
these experiments are summarized in Fig.
8. The expression of either the
crtI or puc genes in TSPO1 cells was not affected
by the presence of additional copies of hemN. However, the
introduction of extra copies of hemN in trans into wild type
cells led to a significant increase in expression of the reporters
crtI::lacZ and puc::lacZ to
levels observed for the expression of these same constructs in
TSPO1cells. The introduction of hemZ in trans resulted in
only a slight increase in expression of these reporter genes under
aerobic growth conditions (Table IV).
Therefore, it appears that the presence of the tspO mutation
affects the expression of the targeted photosynthesis genes by somehow
"altering" the activity of the hemN-encoded
coproporphyrinogen III oxidase. When both tspO and
hemN were provided in extra copies (pUI2731) to wild type
cells, expression of puc and crtI decreased to
the levels observed for the wild type cells, containing vector alone.
In TSPO1 (pUI2731) the tspO gene is expressed from the strong rrnB promoter; therefore, TspO was present in the
outer membranes of these cells at a much higher levels than in wild type R. sphaeroides (inset in Fig. 2).
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Fig. 8.
Effect of hemN in extra copy
on the transcription of crtI and
puc. Cells were grown aerobically, and the
transcriptional activity of crtI and puc analyzed
as described under "Experimental Procedures."
|
|
View this table:
[in this window]
[in a new window]
|
Table IV
Effect of hemZ in extra copy on transcription of crtI and puc
Cells were grown aerobically, and transcription activity was analyzed
as described under "Experimental Procedures."
|
|
To analyze the specificity of the effect provided by hemN in
trans in wild type cells, we measured the transcriptional activity of the puf operon, which is not affected in TSPO1 (2). The activity of the puf::lacZ transcriptional fusion
was virtually identical in cells harboring pUI2730 (hemN) or
an empty vector: 459 ± 22 Miller units for 2.4.1 (pRK415);
476 ± 34 Miller units for 2.4.1 (pUI2730); 507 ± 46 Miller
units for TSPO1 (pRK415); and 498 ± 54 Miller units for TSPO1
(pUI2730). These results indicate that the effect of hemN is
quite specific, because only those photosynthesis genes that are
affected in the TSPO1 mutant are also affected by the presence
hemN in trans in the wild type.
Effect of hemN in Multicopy upon the Accumulation of Tetrapyrrole
Pigments by Resting Cells--
To determine whether the presence of
hemN in multicopy affects the accumulation of tetrapyrrole
pigments by aerobic cells, we repeated our studies on the accumulation
of porphyrins in the presence of exogeneous ALA. The results are
summarized at Fig. 9. The presence of
extrachromosomal copies of hemN in the wild type cells led
to a marked decrease in the amounts of tetrapyrrole pigments
accumulated in these cells. The pigments accumulated were mainly free
protoporphyrin, as well as magnesium-protoporphyrin and its monomethyl
ester. The presence of hemN in extra copy had no significant
affect upon the accumulation of porphyrins by the TSPO1 cells. These
results provide additional evidence that TspO negatively affects the
activity of coproporphyrinogen III oxidase.
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|
Fig. 9.
Effect of hemN in extra copy
on the accumulation of tetrapyrrole pigments by R. sphaeroides cells. Cells were grown aerobically and
incubated in the presence of 0.2 mM ALA. The spectra were
obtained as described in the legend to Fig. 2.
|
|
| |
DISCUSSION |
We have previously demonstrated a close evolutionary and
functional relationship between the TspO of R. sphaeroides
and the mammalian mitochondrial benzodiazepine receptor (4). Because of
this relationship we proposed that these proteins are involved in
similar metabolic activities in both bacterial and mammalian cells.
Because MBR has been reported to bind, with high affinity to
protoporphyrin IX, one of the tetrapyrrole intermediates of the heme
biosynthetic pathway, it was speculated that this receptor might be
involved in the regulation of tetrapyrrole metabolism in the mammalian
mitochondrion (6-8, 29). In the present manuscript we present
additional experimental support for this hypothesis by demonstrating
that TspO negatively "regulates" the activity of the
hemN-encoded coproporphyrinogen III oxidase of R. sphaeroides, an antepenultimate enzyme of the heme biosynthetic
pathway. The introduction of hemN in trans in the wild type
R. sphaeroides led to the significant increase in the
expression of the TspO-targeted photosynthesis genes to levels observed
for the TSPO1 mutant cells. Unlike hemN, the presence of
hemZ in trans in the wild type cells did not result in any
appreciable increase of expression of the reporter genes, which is
consistent with the observation that hemZ is expressed at
only very low levels under aerobic growth conditions.
Studies of the regulation of hemN and hemZ
expression in R. sphaeroides wild type and TSPO1 cells (Fig.
7) indicated the highest level of expression of both genes under
semiaerobic (2% O2) growth conditions. However, unlike
hemZ, the expression of which was at basal levels in aerobic
(20% O2) cells, hemN was found to be expressed
at a significant level under these same conditions. Therefore, although
both HemN and HemZ are considered to be "anaerobic" or
oxygen-independent oxidases, significant levels of expression of
hemN under aerobic conditions suggests the physiological
importance of HemN under these conditions. Our results suggesting that
HemN of R. sphaeroides is active both under aerobic and
anaerobic conditions are in line with the recent observation (30) that
hemN of Alcaligenes eutrophus was able to
complement a Salmonella tiphimurium double hemF
hemN mutation under both aerobic and anaerobic growth conditions. Interestingly, the authors (30) demonstrated that hemN is
highly expressed in A. eutrophus cells grown anaerobically
on nitrate and only weakly expressed in aerobic cells. Negative
regulation of the hemN expression by oxygen was also
observed in E. coli (31) and Pseudomonas
aeruginosa (32). On the other hand, no obvious oxygen regulation
was observed for the Bacillus subtilis hemN gene (33).
It has been demonstrated (34) that R. sphaeroides possesses
both aerobic (oxygen-dependent) and anaerobic
(oxygen-independent) coproporphyrinogen III oxidase activities. At
present it is not clear the extent of the contribution of
hemN to the total coproporphyrinogen oxidase activity under
aerobic conditions in R. sphaeroides 2.4.1 cells. If a
third, specific "aerobic" enzyme (hemF) does exist, one
would predict its activity to be negatively affected by TspO. However,
no experimental support for this hypothesis is available.
The results presented here further indicate a close interaction of the
two biosynthetic pathways in R. sphaeroides, namely tetrapyrroles and carotenoids. The overexpression of hemN
encoding a biosynthetic enzyme of the tetrapyrrole biosynthetic pathway resulted in altering the transcriptional activity of at least one of
the genes of the carotenoid biosynthesis pathway. The apparent increase
in the activity of HemN, resulting from hemN in extra copy,
which catalyzes one of the rate-limiting steps of tetrapyrrole biosynthesis, is interpreted as resulting in an apparent elevated flow
of the intermediates along the pathway. Therefore the mechanism by or
through which the signal from elevated coproporphyrinogen III oxidase
activity is transmitted to the DNA-active transcriptional factor(s)
seems to include some small, nonprotein component or components. This
component is likely to be an intermediate or intermediates of the heme
branch or an early intermediate of the bacteriochlorophyll branch of
the pathway (although we cannot exclude the B12 and
siroheme branches from consideration). We cannot exclude the
possibility that the specificity of this signaling system is restricted
to some particular tetrapyrrole molecule or for that matter to
coproporphyrinogen III oxidase itself. It may be that this regulatory
system is sensing not merely the presence or absence of any particular
component but rather responds to the alteration in the "general
balance" between early and late intermediates of the various branches
of the pathway. How all four of these branches might interact goes
beyond the scope of the present study. Nevertheless, it is clear that
TspO is one of the regulators that controls this balance and as such
results in the likely accumulation of some co-repressor. Because the
genes that are regulated by the TspO system are identical to those
controlled by the PpsR repressor-AppA antirepressor system (1), it is likely that this "co-repressor" acts together with PpsR. However, we cannot exclude the possibility that an activator of AppA activity would behave as a co-repressor of PpsR function.
The results presented here indicate that TspO acts as a negative
regulator of hemN, one of the isoenzymes of
coproporphyrinogen III oxidase in R. sphaeroides. Although
the exact mechanism of this regulation is still not understood, our
results indicate that TspO can control the exit of porphyrins
(porphyrinogens) from the cell. At present it is not known whether TspO
can act as an outer membrane channel specific for certain porphyrins or whether it functions in association with other outer membrane transport
systems, which might not necessarily be specific to the porphyrins,
i.e. major outer membrane protein (porin). This later
possibility is strengthened by the observation that the mammalian
homolog of TspO, MBR, may form a complex with the mitochondrial porin
(29). On the other hand, the outer membrane porin of Rhodobacter capsulatus has been demonstrated to participate in the transport of tetrapyrrole intermediates in the bacteriochlorophyll branch (35).
We have earlier demonstrated that the R. sphaeroides porin specifically binds 3H-labeled benzodiazepines (2). Thus by
controlling the efflux of a critical small molecule or intermediate in
the tetrapyrrole biosynthetic pathway, TspO could be
post-translationally regulating the activity of HemN, which might
produce a co-repressor necessary for transcriptional control. The fact
that the excess of TspO in the outer membrane, when tspO is
expressed in trans, is phenotypically similar to the TSPO1
mutant suggests that TspO could be interacting with another membrane
component and thus disturbing the normal physiologic effect of TspO
when overexpressed.
The results presented also clearly indicate the interaction between two
major biosynthetic pathways in R. sphaeroides: namely tetrapyrroles and carotenoids. These results demonstrate that in
addition to the other regulators of transcription of the photosynthesis genes (PpsR/AppA, Prr, and FnrL), the intermediate product(s) of one of
the biosynthetic pathways (porphyrins) can affect the expression of
genes, encoding enzymes of the other pathway (carotenoids) as well as
structural polypeptides for the major Bchl/Crt binding protein, namely
LH II. This pathway cross-talk allows for the coordinated regulation of
the biosynthesis of the components of intracytoplasmic membrane system
on the one hand and ensures the removal of potentially harmful cell
metabolites (porphyrins) on the other. It remains to be determined what
is the nature of the environmental stimulus to which TspO responds and
the mechanism by which it controls the transport of tetrapyrrole
pigments. To this end, recent studies in our laboratory on mutant forms
of TspO and their roles in tetrapyrrole accumulation have begun to shed
light on this process.3
| |
FOOTNOTES |
*
This work was supported by U. S. Public Health Service
Grant GM15590 (to S. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Microbiology
and Molecular Genetics, University of Texas Health Science Center at
Houston, P. O. Box 20708, Houston, TX 77225. Tel.: 713-500-5502; Fax:
713-500-5499; E-mail: skaplan@utmmg.med.uth.tmc.edu.
2
J. Zeilstra-Ryalls, A. Yeliseev, and S. Kaplan,
unpublished data.
3
A. Yeliseev and S. Kaplan, submitted for publication.
| |
ABBREVIATIONS |
The abbreviations used are:
MBR, mitochondrial
benzodiazepine receptor;
HPLC, high performance liquid chromatography;
kb, kilobase(s);
ALA, aminolevulinic acid;
PBG, porphobilinogen.
| |
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ABSTRACT
INTRODUCTION
