Department of Microbiology and Molecular
Genetics, The University of Texas Health Science Center Medical
School, Houston, Texas 77030
FnrL, the homolog of the global anaerobic regulator Fnr, is
required for the induction of the photosynthetic apparatus in Rhodobacter sphaeroides 2.4.1. Thus, the precise role
of FnrL in photosynthesis (PS) gene expression and its
interaction(s) with other regulators of PS gene expression are of
considerable importance to our understanding of the regulatory
circuitry governing spectral complex formation. Using a CcoP and
FnrL double mutant strain, we obtained results which suggested
that FnrL is not involved in the transduction of the inhibitory signal,
by which PS gene expression is "silenced," emanating from the
cbb3 oxidase encoded by the ccoNOQP
operon under aerobic conditions. The dominant effect of the
ccoP mutation in the FnrL mutant strain with respect to spectral complex formation under aerobic conditions and restoration of
a PS-positive phenotype suggested that inactivation of the cbb3 oxidase to some extent bypasses the
requirement for FnrL in the formation of spectral complexes.
Additional analyses revealed that anaerobic induction of the
bchE, hemN, and hemZ genes, which are involved in the tetrapyrrole biosynthetic pathways, requires FnrL.
Thus, FnrL appears to be involved at multiple loci involved in the
regulation of PS gene expression. Additionally, bchE was also shown to be regulated by the PrrBA two-component system, in
conjunction with hemN and hemZ. These and other
results to be discussed permit us to more accurately describe the role
of FnrL as well as the interactions between the FnrL, PrrBA, and other
regulatory circuits in the regulation of PS gene expression.
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INTRODUCTION |
Rhodobacter
sphaeroides 2.4.1 is a purple nonsulfur photosynthetic
bacterium which displays a versatile metabolic lifestyle. It is able to
grow by aerobic and anaerobic respiration and photosynthetically in the
light under anaerobic conditions. The major determinant for the
synthesis of the photosynthetic apparatus is oxygen. When oxygen
tensions fall below a certain threshold value (~2.5%), the
photosynthetic apparatus is synthesized. In addition, the levels and
the ratios of the light-harvesting complexes are determined by the
incident light intensity.
Transcriptional regulation of the photosynthesis (PS) genes in R. sphaeroides involves the coordinate action of at least four major regulatory systems (the PrrBA two-component activation system, the AppA-PpsR antirepressor-repressor system, FnrL, and TspO) which are
responsible for redox sensing (25). Previous studies in our
laboratory have shown that mutations in the ccoNOQP operon encoding the cbb3 oxidase result in the
induction of PS gene expression under aerobic growth conditions
(18, 19, 28). This observation was interpreted to suggest
that a pathway involving the cbb3 oxidase in
response to O2 serves as an oxygen sensor which generates
an inhibitory signal for PS gene expression. This signal is mediated by
the PrrBA two-component regulatory system, which is a major switch
controlling PS gene expression (17).
However, we could not rule out the possibility that FnrL, at least in
part, is somehow involved in transmission of the inhibitory signal
emanating from the cbb3 oxidase, since
expression of the puc operon and hemA gene, which
are in part regulated by FnrL, is partially derepressed in the Cco
mutants under aerobic conditions (18, 27-29).
The construction of a CcoP and FnrL double mutant enabled us to answer
this question. In this study, we demonstrate that FnrL does not mediate
the derepression signal derived from the inactivation of the
cbb3 oxidase under aerobic conditions.
Furthermore, we do present evidence that the bchE,
hemN, and hemZ genes, which have previously been
identified as potential targets on the basis of sequence analyses of
their upstream regions for regulation by FnrL, are in fact regulated by
both FnrL and the PrrBA two-component system. These observations, as
well as earlier results, will be discussed in conjunction with the role
of FnrL and its relation to other regulatory circuits in controlling PS
gene expression.
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MATERIALS AND METHODS |
Strains, plasmids, and growth conditions.
The bacterial
strains and plasmids used in this study are listed in Table
1. R. sphaeroides 2.4.1 strains were grown at 30°C on Sistrom's medium A (SIS)
(3) containing succinate as the carbon source and
supplemented, as required, with the following antibiotics:
tetracycline, 1 µg/ml; kanamycin, 25 µg/ml; trimethoprim, 50 µg/ml; and streptomycin and spectinomycin, 50 µg/ml each.
Chemoheterotrophic cultures were grown either aerobically, sparged with
30% O2-69% N2-1% CO2, or
semiaerobically, sparged with 2% O2-97%
N2-1% CO2. Photosynthetic cultures were grown
at a low incident light intensity of 3 W/m2 in completely
filled screw-cap glass tubes. The tubes containing photosynthetic
cultures were rotated by using a rotary drum to keep cells suspended
and mixed. Photosynthetic growth of R. sphaeroides was
monitored with a Klett-Summerson colorimeter with a no. 66 filter.
Anaerobic growth with dimethyl sulfoxide (DMSO) as a terminal electron
acceptor was performed in SIS medium supplemented with 0.1% (wt/vol)
yeast extract in the presence of DMSO (0.5% vol/vol) in screw-cap
tubes in the dark. Escherichia coli strains were grown at
37°C on Luria-Bertani medium supplemented, when required, with
the following antibiotics: ampicillin, 50 µg/ml; kanamycin, 50 µg/ml; tetracycline, 20 µg/ml; and streptomycin and spectinomycin, 50 µg/ml each.
DNA manipulations and conjugation techniques.
Genomic DNA
from R. sphaeroides was isolated by the method of
Ausubel et al. (1). Standard protocols or the
manufacturer's instructions were followed for recombinant DNA
manipulations (20).
Plasmids were mobilized by either biparental or triparental matings
from E. coli strains into R. sphaeroides
strains as described elsewhere (5).
Construction of mutants and lacZ transcriptional
fusions.
For the construction of the CCOP1FNRL double mutant, the
ccoP gene was mutated in the FNRL background using plasmid
pUI2801, which had been used for the construction of the original CCOP1 mutant (18).
PrrBCA2 was constructed by crossing pJE2077 into R. sphaeroides 2.4.1 and selecting for the double-crossover
recombinant. Plasmid pJE2077 is a suicide vector in R. sphaeroides, and it contains the prr region, with a
2,069-bp deletion extending from a Tth111I site within
prrA to a BspEI site within prrB. In
addition, the 
Tp cassette from pUI1680 was cloned within these
boundaries as a selectable marker. The structure was verified by
Southern hybridization.
In order to construct the bchE::lacZ
transcriptional fusion, the bchE promoter region was
amplified with primers 5'-CTGTCGCTGCAGTCGCTTTATGTG-3' and
5'-CGGCCTTCTAGAGCGCGCCACACA-3' to generate a 318-bp product with PstI and XbaI restriction sites at both
ends. The PCR product was restricted with PstI and
XbaI and cloned into the promoterless lacZ vector
pCFL digested with the same enzymes, yielding plasmid pBCHE. The
sequence of the insert in pBCHE was confirmed by DNA sequencing.
To construct the hemZ::lacZ
transcriptional fusion, the 0.75-kb EcoRV-XbaI
fragment containing the promoter region of hemZ was cloned
into pCF1010 restricted with StuI and XbaI from
pJE3153 to give plasmid pJE3170.
Quantitative analysis of spectral complexes.
The harvested
cells were resuspended in ICM buffer (10 mM
KH2PO4/K2HPO4, 1 mM
EDTA [pH 7.2]) and disrupted by passage through a French pressure
cell (ca. 0.9-cm-diameter piston) at 90 MPa. Cell-free crude extracts
were obtained following centrifugation in a benchtop microcentrifuge at
13,000 rpm for 15 min at 4°C to remove unbroken cells and cell
debris. Spectra were recorded with a UV 1601PC spectrophotometer
(Shimadzu Corp., Columbia, Md.). The B800-850 and B875 complex levels
were determined by the method of Meinhardt et al. (12) from
the spectral data collected.
Crt and Bchl analyses.
For extraction of photopigments, 0.5 ml of crude cell extract was mixed with 1.5 ml of acetone-methanol
(7:2, vol/vol), followed by vigorous vortexing for 1 min. After
centrifugation in a benchtop microcentrifuge at 13,000 rpm for 15 min,
the supernatant was used for quantitation of carotenoid (Crt) and
bacteriochlorophyll (Bchl) as described previously (3).
-Galactosidase assays and protein determinations.
-Galactosidase assays were performed on crude cell extracts as
described elsewhere (19). Protein concentration was
determined by the bicinchoninic acid protein assay (Pierce, Rockford,
Ill.) using bovine serum albumin as the standard protein.
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RESULTS AND DISCUSSION |
Formation of spectral complexes in the CCOP1FNRL double mutant
under aerobic conditions.
In order to address the question of
whether the signal emanating from the cbb3
oxidase involves the participation of the FnrL protein, a double mutant
carrying mutations in both fnrL and ccoP was
constructed (strain CCOP1FNRL). When grown on SIS agar plates aerobically, the double mutant formed colonies with red pigmentation, the extent of which was intermediate between the appearance of the wild
type and the high levels of pigmentation of the CCOP1 mutant
(18). To confirm that the increased pigmentation of the CCOP1FNRL mutant under aerobic conditions was due to the presence of photosynthetic spectral complexes, crude extracts from
cultures of this mutant strain grown aerobically were prepared, and the levels of the light-harvesting complexes (B800-850 and B875) as well as
the photopigments Bchl and Crt were determined
spectrophotometrically (Table 2).
For comparison, crude extracts were also prepared from the wild-type,
CCOP1, and FNRL strains grown under the same conditions. As expected,
the wild-type and FNRL mutant strains produced virtually no
light-harvesting complexes under aerobic conditions. In contrast, the
CCOP1FNRL mutant strain exhibited 50 and 64% of the levels of the
B800-850 and B875 complexes, respectively, relative to the levels which
were detected in the CCOP1 mutant. In agreement with these results, the
cellular levels of the photopigments Bchl and Crt also increased in
both CCOP1 and CCOP1FNRL strains compared with those levels in the
wild-type and FNRL strains. These data demonstrated that the mutation
in ccoP led to the oxygen-insensitive formation of the
photosynthetic complexes even in the FnrL-minus background. It should
be recalled that an FnrL mutant strain of R. sphaeroides
cannot produce spectral complexes even under inducing conditions such
as 2% oxygen (26).
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TABLE 2.
Levels of spectral complexes and photopigments in
R. sphaeroides strains grown under
aerobic conditionsa
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The reduced levels of spectral complexes in the CCOP1FNRL
mutant grown aerobically compared with those in the CCOP1 mutant grown under the same conditions suggested that FnrL of R. sphaeroides is "active" under aerobic conditions. This had
been predicted previously after the observation that R. sphaeroides FnrL contains the two residues which, when changed
in E. coli Fnr, enable the latter protein to be active under
aerobic conditions of growth, which is not the normal physiological
situation (27). This finding is in agreement with a similar
conclusion which was reached under different experimental conditions by
Zeilstra-Ryalls and Kaplan (27, 29). Mouncey and Kaplan
(14) reported that FnrL is a positive regulator of genes
encoding the cbb3 oxidase. The fact that the
FNRL mutant is incapable of spectral complex formation under aerobic
conditions indicates that sufficient cbb3
oxidase is produced in order to be able to repress spectral complex
formation in this mutant under the same conditions.
FnrL is not involved in the cbb3-mediated
signal transduction pathway under aerobic conditions.
Previous
studies revealed that mutations of the cco operon in
R. sphaeroides lead to the aerobic derepression of not
only those PS genes regulated by the PrrBA two-component system, such as puc and puf, but also hemA, which
encodes the 5-aminolevulinic acid (ALA) synthase and which is, at least
in part, under the control of FnrL (6, 18, 28). An
interpretation of these results raised the possibility that FnrL is an
additional regulator which communicates with the
cbb3 oxidase. To address this question, we
compared the promoter activities of genes which are both regulated by
FnrL and derepressed by mutations in the cco operon under
aerobic conditions. These experiments were performed with the
wild-type, CCOP1, and CCOP1FNRL mutant strains grown under aerobic
conditions. We speculated that if FnrL mediates the derepression signal
resulting from inactivation of the cbb3 oxidase
under aerobic conditions, then the derepression of PS gene expression
observed in the CCOP1 mutant strain should be abolished in the
CCOP1FNRL mutant. As shown in Table 3,
the puc operon and hemA gene were strongly derepressed under aerobic conditions in the CCOP1 mutant by factors of
2.8- and 7.2-fold, respectively, compared with the wild type. The
disruption of fnrL in the background of the CCOP1 mutant
resulted in a further 1.4- and 2.3-fold increase in puc and
hemA promoter activities, respectively, compared with the
CCOP1 mutant. This result suggested that FnrL does not mediate the
derepression signal resulting from inactivation of the
cbb3 oxidase under aerobic conditions, which is
in good agreement with the oxygen-insensitive formation of the spectral
complexes in the CCOP1FNRL mutant under aerobic conditions. The higher
promoter activity of the hemA gene in the CCOP1FNRL mutant
grown under aerobic conditions than in the CCOP1 mutant can be
explained by the fact that the transcription start point of
hemA under aerobic conditions lies within the predicted FnrL
binding motif (16), and therefore suggests that FnrL might be a repressor of hemA under aerobic conditions. In the case
of increased puc operon expression, the FnrL binding
sequence overlaps the integration host factor binding sequence, and
removal of FnrL could permit enhanced activation of puc
operon expression by integration host factor (11). These
results raise an additional question. If hemA and
puc operon expression is increased in the CCOP1FNRL mutant background, why do we not see at least the level of spectral complexes in the double mutant as observed for the CCOP1 mutant? One
possible answer is that there might be some PS genes which are
regulated by FnrL and which are not derepressed by the inactivation of
the cbb3 oxidase under aerobic conditions.
Thus, these gene products might be limiting factors for spectral
complex formation.
Regulation of the bchE gene.
A putative
FnrL-binding motif is located 53 nucleotides upstream of the
translational start codon of the bchE gene, whose product
catalyzes the conversion of Mg-protoporphyrin monomethyl ester to
divinyl-protochlorophyllide in the Bchl biosynthetic pathway. The
presence of an FnrL-binding motif upstream of the bchE gene
prompted us to examine whether this gene is regulated by FnrL. To
address this question, we assayed the promoter activity of
bchE in the FnrL-minus background. Since the FNRL mutant is unable to grow under anaerobic conditions, we performed an oxygen shift experiment in which cultures grown with 30% oxygen were shifted to 2% oxygen (Fig. 1). At
the time intervals indicated, samples were harvested, and their
-galactosidase activities were determined. In the wild-type strain,
the levels of bchE::lacZ expression increased
steadily and appreciably after the cultures were shifted to 2% oxygen.
In contrast, the promoter activity of bchE in the FNRL
mutant showed a marginal increase up to 4 h after the shift to 2%
oxygen. This indicated a clear requirement for FnrL for the induction
of bchE expression in response to lowering of the oxygen
tension.
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FIG. 1.
Induction kinetics of bchE expression from
the bchE::lacZ transcriptional fusion plasmid
pBCHE in the wild-type strain ( ) and FNRL mutant strain ( )
following a shift from 30 to 2% oxygen (indicated by the vertical
arrow). Strains were grown under 30% O2 to an optical
density at 600 nm (OD600) of 0.15 to 0.2, and the partial
pressure of O2 was reduced to 2%. Cultures were sampled at
the times indicated, and crude extracts were assayed for
-galactosidase activity.
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A determination of bchE promoter activity using the
bchE::lacZ transcriptional fusion revealed
that expression of bchE was substantially increased under
aerobic conditions (30% O2) in the CCOP1 and CCOP1FNRL
mutants compared with that in the wild type (Table 3). The derepression
of bchE in the CCOP1 mutant under aerobic conditions also
indicated the involvement of the PrrBA two-component system in the
regulation of bchE. To examine this possibility, we measured
the promoter activities of bchE in the PrrCBA-minus
background (PRRBCA2 mutant) as well as in the wild-type strain. Under
30% oxygen conditions, both wild-type and mutant strains exhibited
basal levels of promoter activity (Fig.
2). In the wild type grown under
dark-DMSO conditions, the expression of bchE was induced by
a factor of 72 compared to that found in the same strain grown
aerobically. In contrast, the promoter activity increased only sixfold
in the PRRBCA2 mutant strain grown under dark-DMSO conditions. Taken
together, these results clearly suggest that bchE is
regulated by both FnrL and the PrrBA two-component system. They are
both needed for the full induction of bchE under oxygen-limiting conditions, although either of them alone can induce
the gene only partially. The approximately sixfold induction of
bchE under dark-DMSO conditions in the PrrBCA-minus
background suggests that FnrL might produce an approximately sixfold
increase in bchE gene expression under the same conditions.
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FIG. 2.
-Galactosidase activities in R. sphaeroides strains containing the
bchE::lacZ transcriptional fusion plasmid
pBCHE. The wild-type (2.4.1) and prrCBA-minus (PRR) strains
were grown aerobically (30% oxygen, black bars) to an
OD600 of 0.3 to 0.4 or anaerobically in the dark with 0.5%
DMSO (gray bars). All values are the averages of two independent
determinations. Vertical bars represent the standard deviations from
the mean.
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Regulation of the hemZ and hemN genes.
Sequence analyses have also revealed the presence of FnrL binding
sequences 35 and 34 bp upstream of the presumed translational start
codons of the hemN and hemZ genes, respectively,
encoding isoenzyme forms of the coproporphyrinogen III oxidase involved in the tetrapyrrole biosynthetic pathway. Previous work by Yeliseev and
Kaplan (24) revealed that both genes are derepressed under semiaerobic and anaerobic conditions and that the hemN gene
appears to encode a form of the enzyme that may be active aerobically. To examine the role of FnrL in the regulation of hemZ and
hemN, the corresponding transcriptional lacZ
fusions were introduced into the wild-type strain 2.4.1 as well as the
FNRL mutant strain. The strains were grown either under 30% oxygen
conditions or under 2% oxygen conditions, in which many of the genes
known to be under the control of FnrL appear to be fully induced.
The expression of hemZ is strictly regulated by FnrL (Fig.
3). Under 30% oxygen conditions, the
promoter activity of hemZ is at basal levels in both the
wild-type and FNRL mutant strains. A 23-fold increase in expression of
hemZ was observed in the wild type grown under 2% oxygen
conditions, whereas the gene was not induced in the FNRL mutant grown
under the same conditions, which is indicative of the essential role of
FnrL in the induction of hemZ. These results are in
agreement with those of Yeliseev and Kaplan (24), suggesting
that hemZ encodes an anaerobic coproporphyrinogen III
oxidase. Furthermore, the PrrBA two-component system is in part
responsible for the induction of this gene under anaerobic conditions
(J. M. Eraso and S. Kaplan, unpublished data). Although the
hemZ gene is positively regulated by the PrrBA two-component system, derepression of the gene under 30% oxygen conditions by the
inactivation of the cbb3 oxidase was not
observed in the FnrL-minus background, which confirms the absolute
requirement for FnrL for the expression of hemZ (Table 4).
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FIG. 3.
-Galactosidase activities in R. sphaeroides strains containing either the
hemZ::lacZ or
hemN::lacZ transcriptional fusion plasmid.
The wild-type (2.4.1) and fnrL-minus (FNRL) strains were
grown aerobically (30% oxygen, black bars) to an
OD600 of 0.3 to 0.4 or semiaerobically (2% oxygen, gray
bars) to an OD600 of 0.4 to 0.5. All values provided are
the averages of two independent determinations. Vertical bars represent
the standard deviations from the mean.
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As shown in Fig. 3, these same experiments were performed with the
hemN::lacZ fusion. When the promoter activity
of hemN was compared in strains grown under 2% oxygen
conditions with that found in the strains grown under 30% oxygen, a
ninefold induction of hemN expression was observed in the
wild-type strain grown under 2% oxygen conditions. Only a threefold
induction was found in the FNRL mutant grown under the same
conditions. The promoter activity found in the FNRL mutant grown under
2% oxygen conditions amounted to ~25% of that present in the
wild-type strain grown under the same conditions. Furthermore, a
significant level of hemN promoter activity was detected in
both strains even under 30% oxygen conditions compared with that of
hemZ. This finding is also in agreement with that of
Yeliseev and Kaplan, suggesting that hemN is both an
aerobically and anaerobically active enzyme (24). Taken
together, these results indicated that hemN is also positively regulated by FnrL under oxygen-limiting conditions and that
the gene is less stringently regulated by FnrL than is hemZ.
The threefold induction of hemN under 2% oxygen
conditions in the FnrL-minus background suggests that another
regulatory system(s) is involved in the regulation of the gene. In
fact, the anaerobic induction of hemN in part requires
the PrrBA two-component system (Eraso and Kaplan, unpublished
data). Furthermore, the hemN gene is derepressed under 30%
O2 conditions in the CCOP1FNRL mutant, again
indicating that FnrL is not essential for the expression of the
gene (Table 4).
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TABLE 4.
Promoter activities of hemZ and hemN
in R. sphaeroides FNRL and CCOP1FNRL mutants grown
under aerobic
conditionsa
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We can probably rule out the regulation of hemN by the PpsR
repressor, since there is no PpsR binding sequence upstream of the
hemN gene.
Regulation of the tetrapyrrole biosynthetic pathway.
As
outlined in Fig. 4, both the Bchl and
heme biosynthetic pathways have the same intermediates from ALA through
protoporphyrin IX, where they branch (22). Hemes are
absolutely required for both aerobic and anaerobic growth of R. sphaeroides. In contrast, the synthesis of Bchl should be
inhibited in cells grown under high-oxygen conditions, since free Bchl
in the presence of light and O2 produces toxic free
radicals and the expression of the puf and puc
operons is minimal under these conditions. In fact, the levels of
detectable Bchl found in R. sphaeroides grown under high-oxygen conditions is minimal. Therefore, the "opening" of the
pathway from protoporphyrin IX to Bchl a must be stringently controlled and exquisitely responsive to the levels of O2
as well as light present in the environment. The results presented here and those published earlier (25) indicate that R. sphaeroides has developed overlapping regulatory circuits to
enable such controls (Fig. 4). R. sphaeroides has two
isoenzymes for the synthesis of ALA (HemA and HemT) (15, 16)
and two coproporphyrinogen III oxidases (HemN and HemZ) (4,
27), which have been identified as points of FnrL regulation in
the tetrapyrrole biosynthetic pathway prior to protoporphyrin IX. The
hemT product was shown to be fully functional under both
aerobic and anaerobic conditions in the absence of HemA (in the
HemA-minus mutant), and hemT seems not to be regulated by
FnrL due to the lack of an observed FnrL-binding motif in the control
region (15, 16).
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FIG. 4.
Bchl and heme biosynthetic pathways. The
relevant genes, the regulation of which is either established or
predicted on the basis of sequence analyses, are depicted. The question
mark to the right of a regulator symbol indicates that regulation by
the corresponding regulator is inferred from sequence analyses and is
therefore not proven experimentally. The Bchl biosynthetic branch is
shown in a yellow box. To the left of the yellow box, the regulation of
four inferred operons which are involved in Bchl biosynthesis is
represented. The red and blue arrows represent induction under
oxygen-limiting conditions (semiaerobic or anaerobic conditions) and
repression under high-oxygen conditions, respectively. The outer
membrane TspO protein affects the expression of PpsR target genes by
acting on HemN posttranscriptionally (24). At the bottom,
the regulation of the puc and puf operons as well
as the puhA gene, which encode the structural polypeptides
of the reaction center and light-harvesting complexes, is presented.
Abbreviations: MV, monovinyl; DV, divinyl; CoA, coenzyme A.
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As shown above, the hemN gene is transcribed at quite
significant levels under aerobic conditions, and although clearly under FnrL control, it is less stringently regulated by FnrL than is hemZ. This observation helps to explain the "less
stringent" regulation of heme biosynthesis than of Bchl. In addition,
hemA, hemZ, and hemN are also
regulated by the PrrBA system, and from the data provided here, we can
provide some quantitative measures as to the contributions of each of
these systems. Thus, for the first time we begin to describe an
overlapping regulatory network in the control of PS gene expression in
R. sphaeroides.
With regard to the regulation of the Bchl biosynthetic branch, at least
one additional regulatory point was reported in R. sphaeroides. The repressor PpsR represses the bchF
gene, whose product catalyzes the conversion of chlorophyllide
a to 2-hydroxyethyl bacteriochlorophyllide a
under aerobic conditions (oxidized conditions) (7). In
addition, sequence analyses of the PS gene cluster of R. sphaeroides, whose sequence was recently determined, revealed that there are PpsR binding sequences 16 bp upstream of the
bchE gene as well as 72 and 98 bp upstream of the
bchC genes (2). The genes involved in the Bchl
biosynthetic branch appear to be clustered into four transcriptional
units (bchFNBHLM, bchEJG, bchCXYZ, and
bchLDO), which also suggests that regulation of the bchC, bchE, bchL, and bchF
genes governs the expression of the corresponding downstream genes.
Considering the data presented above, we conclude that the stringent
control of the biosynthesis of Bchl appears to be achieved throughout
the Bchl biosynthetic branch by the coordinate action of the regulators
FnrL, AppA-PpsR, and the PrrBA two-component activation system without
interrupting the flow of tetrapyrrole intermediates to heme, depending
on the availability of oxygen, light intensity, and/or the cellular
redox state (Fig. 4).
Inactivation of the cbb3 oxidase bypasses
the requirement for FnrL in the formation of spectral complexes.
Spectral complex formation under inducing conditions, such as 2%
oxygen, is severely affected in the FNRL mutant of R. sphaeroides, indicating that FnrL is an essential regulator in
the formation of spectral complexes (26). However, the
oxygen-insensitive formation of spectral complexes in the CCOP1FNRL
double mutant also suggested that the requirement for FnrL in spectral
complex formation can be at least partially alleviated by the
inactivation of the cbb3 oxidase. This finding
raised the question of whether the CCOP1FNRL mutant is able to grow
photosynthetically. Zeilstra-Ryalls and Kaplan (27) have
reported that the FNRL mutant can grow neither photosynthetically nor
anaerobically with DMSO as a terminal electron acceptor for anaerobic
respiration. Surprisingly, we found that the CCOP1FNRL double mutant
regains the ability to grow, albeit slowly, under photosynthetic
conditions. In liquid medium in completely filled screw-cap glass
tubes, a continuous increase in cell density was observed for the
CCOP1FNRL mutant after approximately the same lag phase as for the wild
type when the medium was inoculated with aerobically grown cells,
although its growth rate was slower than that of the wild-type and
CCOP1 mutant strains (Fig. 5). As
expected, the FNRL mutant cannot grow photosynthetically.
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FIG. 5.
Growth characteristics of the wild-type and mutant
strains under photosynthetic conditions. Photosynthetic cultivations
were performed at low light intensity (3 W/m2) in
completely filled screw-cap glass tubes to minimize the repression
effect of PpsR. Cultures were inoculated with aerobically grown cells.
The levels of spectral complexes in the strains grown under the same
conditions to an OD600 of 0.3 to 0.4 are given above the
growth curves for B800-850 and (B875). OD600 = (Klett
unit + 3.06)/102.04.
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In order to examine whether the fnrL mutation affects
spectral complex formation under photosynthetic conditions, the
levels of the spectral complexes were determined with the
wild-type and mutant strains grown photosynthetically (3 W/m2). As shown in Fig. 5, the levels of B800-850 complex
were substantially decreased in the CCOP1FNRL mutant compared with
those found in the CCOP1 mutant and the wild type. The CCOP1FNRL
mutant exhibited only 58% of the B800-850 levels detected in the CCOP1
mutant under low light conditions, while the levels of B875 remained
relatively fixed in all three strains. The reduced levels of the
spectral complexes in the CCOP1FNRL mutant can account for the slow
growth of this mutant under photosynthetic conditions.
The CCOP1FNRL mutant is unable to grow anaerobically with DMSO in the
dark, as is the case for the FNRL mutant. When immunoblot analysis was
performed with an antibody against DorA, the catalytic subunit of DMSO
reductase, from Rhodobacter capsulatus, only a trace amount
of DorA was detected in both FNRL and CCOP1FNRL mutant strains grown
semiaerobically in the presence of 0.5% (vol/vol) DMSO, while a large
induction of DMSO reductase expression was observed in the wild-type
and CCOP1 mutant strains grown under the same conditions (data not
shown). This result confirmed earlier studies in this laboratory
(13, 26) that the fnrL mutation impairs the
expression of DMSO reductase, and unlike PS gene expression, the
inactivation of the cbb3 oxidase cannot bypass
the requirement for FnrL in the induction of the genes encoding DMSO
reductase, as is the case for hemZ.
A plausible explanation for the partial bypass of the FnrL requirement
in spectral complex formation and photosynthetic growth by the
inactivation of the cbb3 oxidase can now be
formulated. It is based on the observation that many genes involved in
Bchl biosynthesis or in the formation of the spectral complexes are coregulated by both FnrL and the PrrBA two-component system, and the
complete absence of the cbb3-generated
inhibitory signal provides for the full activation of the PrrBA system
(17) and thereby partially compensates for the lack of FnrL.
This rationale is supported by the data shown here and can be applied
to gene regulation of the hemA, hemN, and
bchE genes as well as the puc operon.
Recently Yeliseev and Kaplan (24) reported that the
overexpression of hemN in R. sphaeroides
leads to the aerobic derepression of genes which are regulated by the
AppA/PpsR antirepressor/repressor system and the outer membrane protein
TspO negatively controls the activity of coproporphyrinogen III oxidase
posttranscriptionally. Since hemN is positively regulated by
both FnrL and PrrBA, these regulators can indirectly affect the
expression of PpsR target genes. These observations imply that the
increased expression of hemN as the result of the combined
effect of the removal of the cbb3-generated
inhibitory signal (in the CCOP1 and CCOP1FNRL mutants) can bring about
the partial alleviation of PpsR repression. Thus, the data obtained
here and elsewhere (29) suggest that FnrL control of PS gene
expression is additive and cooperative, acting together with other
regulatory circuits in R. sphaeroides. This network of
interacting regulatory circuits may permit R. sphaeroides to more finely "tune" PS gene expression to a
wider array of environmental condition than R. capsulatus,
which does not require FnrL for PS gene expression (26).
Therefore, for the first time we are able to bring together, at least
preliminarily, all of the regulatory pathways (Prr, FnrL, AppA/PpsR,
and TspO) operating in R. sphaeroides to control PS gene
expression into a coherent whole.
We thank M. Choudhary for providing sequence information on the PS gene
cluster of R. sphaeroides 2.4.1.
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Abstract
Introduction
