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J Bacteriol, February 1998, p. 699-704, Vol. 180, No. 3
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Reconstitution of an Active Magnesium Chelatase
Enzyme Complex from the bchI, -D, and
-H Gene Products of the Green Sulfur Bacterium
Chlorobium vibrioforme Expressed in
Escherichia coli
Bent L.
Petersen,1
Poul Erik
Jensen,2
Lucien C. D.
Gibson,2
Bjarne M.
Stummann,1
C. Neil
Hunter,2 and
Knud W.
Henningsen1,*
Department of Ecology and Molecular Biology,
Royal Veterinary and Agricultural University, DK-1871 Frederiksberg
C, Denmark,1 and
Krebs Institute for
Biomolecular Research and Robert Hill Institute for Photosynthesis,
Department of Molecular Biology and Biotechnology, University of
Sheffield, Sheffield S10 2TN, United Kingdom2
Received 2 July 1997/Accepted 21 November 1997
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ABSTRACT |
Magnesium-protoporphyrin chelatase, the first enzyme unique to the
(bacterio)chlorophyll-specific branch of the porphyrin biosynthetic
pathway, catalyzes the insertion of Mg2+ into
protoporphyrin IX. Three genes, designated bchI,
-D, and -H, from the strictly anaerobic and
obligately phototrophic green sulfur bacterium Chlorobium
vibrioforme show a significant level of homology to the magnesium
chelatase-encoding genes bchI, -D, and
-H and chlI, -D, and -H
of Rhodobacter sphaeroides and Synechocystis strain PCC6803, respectively. These three genes were expressed in
Escherichia coli; the subsequent purification of
overproduced BchI and -H proteins on an Ni2+-agarose
affinity column and denaturation of insoluble BchD protein in 6 M urea
were required for reconstitution of Mg-chelatase activity in vitro.
This work therefore establishes that the magnesium chelatase of
C. vibrioforme is similar to the magnesium chelatases of
the distantly related bacteria R. sphaeroides and
Synechocystis strain PCC6803 with respect to number of
subunits and ATP requirement. In addition, reconstitution of an active
heterologous magnesium chelatase enzyme complex was obtained by
combining the C. vibrioforme BchI and -D proteins and the
Synechocystis strain PCC6803 ChlH protein. Furthermore, two
versions, with respect to the N-terminal start of the bchI
gene product, were expressed in E. coli, yielding ca. 38- and ca. 42-kDa versions of the BchI protein, both of which proved to be
active. Western blot analysis of these proteins indicated that two
forms of BchI, corresponding to the 38- and the 42-kDa expressed
proteins, are also present in C. vibrioforme.
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INTRODUCTION |
In photosynthetic organisms, the
enzyme magnesium-protoporphyrin IX chelatase (Mg-chelatase) catalyzes
the insertion of Mg2+ into protoporphyrin IX, the first
step unique to the synthesis of (bacterio)chlorophyll. Situated at the
branch point of the heme- and (bacterio)chlorophyll-specific parts of
tetrapyrrole biosynthesis, Mg-chelatase is believed to have an
important regulatory role in channelling intermediates into the
(bacterio)chlorophyll-specific branch in response to conditions
determining photosynthetic growth. Recently, the genes bchI,
-D, and -H of the facultatively anaerobic bacterium Rhodobacter sphaeroides (3) and the
homologous genes chlI, -D, and -H of
the chlorophyll a-synthesizing cyanobacterium Synechocystis strain PCC6803 (5) have been
overexpressed in Escherichia coli. In each case
ATP-dependent reconstitution of activity has been achieved in vitro by
combining the BchI/ChlI (38 and 42 kDa), BchD/ChlD (60 and 74 kDa), and
BchH/ChlH (140 and 150 kDa) overexpressed gene products, thereby
demonstrating that the Mg-chelatase enzyme complexes of the two
bacteria are encoded by these genes.
Several indications of functional properties of Mg-chelatase subunits
have been reported. Spectofluorometric analysis has shown that during
expression of the R. sphaeroides bchH gene in E. coli, protoporphyrin IX accumulates, probably because the
overexpressed BchH protein binds protoporphyrin IX (3). In
support of this assumption, Willows et al. (29) have
reported that purified R. sphaeroides monomeric BchH protein
binds protoporphyrin IX in an approximate molar ratio of 1:1
(29).
In pea (Pisum sativum) it has been demonstrated that two
crude protein fractions of broken chloroplasts confer Mg-chelatase activity when combined and that ATP is absolutely required for both
activation (seen as a 6-min lag period preceding activity) of the
proteins involved and the chelation of Mg2+ into
protoporphyrin IX (26-28). A similar lag period can be
eliminated by preincubation of purified R. sphaeroides BchI
protein with partially purified BchD protein in the presence of ATP and
Mg2+, suggesting that a ternary complex between BchI and -D
and Mg2+-ATP is formed during the activation
(29). A phosphate-binding motif
(GX4GKSX6A) common to a number of
ATP/GTP-binding proteins (13) is found near the N terminus
in the products of all genes homologous to the Rhodobacter
capsulatus bchI gene of prokaryotic (1, 5, 18) as well
as eukaryotic (6, 9, 14, 17) origin. The deduced amino acid
sequences of the proteins BchD of R. capsulatus
(1), BchD of Chlorobium vibrioforme
(19), and ChlD of Synechocystis strain PCC6803
(5) all contain a short and very distinct proline-rich
stretch, situated approximately in the middle, which may divide the
protein into two major domains. Furthermore, the N-terminal halves of
the three deduced "D" proteins display significant intraspecies
amino acid identity to the "I" proteins (5, 19),
suggesting that the ancestral D gene has arisen from a gene duplication
of the I gene.
We have previously reported that a ca. 15-kbp region (18,
19) of the genome of C. vibrioforme includes three
open reading frames (ORFs), designated bchI, -D,
and -H, encoding proteins with deduced molecular masses of
38, 67, and 145 kDa, respectively. The three ORFs display significant
homology to the Mg-chelatase-encoding genes of R. capsulatus
and Synechocystis strain PCC6803, and the three genes are
homologous to the putative Mg-chelatase-encoding genes of higher
plants.
In the present work the genes bchI, -D, and
-H of C. vibrioforme f. sp.
thiosulfatophilum NCIB 8327 have been expressed separately in E. coli, and evidence showing that the BchI, -D, and -H
polypeptides are required for reconstitution of Mg-chelatase activity
in vitro is presented. In addition, reconstitution of an active
heterologous Mg-chelatase enzyme complex consisting of the C. vibrioforme BchI and -D proteins and the Synechocystis
strain PCC6803 ChlH protein is demonstrated. Furthermore, two
constructs expressing a ca. 42- and a ca. 38-kDa version of the BchI
protein, which were both active in Mg-chelatase assays, have been made.
Western blot analysis of protein extracts of C. vibrioforme
indicates that two forms of the BchI protein, corresponding to the 42- and 38-kDa versions of the expressed BchI proteins, are indeed present
in C. vibrioforme.
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MATERIALS AND METHODS |
Construction of pET derivates of the C. vibrioforme
bchI, -D, and -H genes.
The primer
pairs, which include two possible start codons and the corresponding
stop codon of ORFs bchI, -D, and -H,
respectively, were designed as follows: PCVI
(5'-AACCAAGTGCATATGACCCAGACTG-3') or
PCVI2 (5'-GAAGAAGAGTCATATGGCATTCC-3')
and PCVI3 (5'-CTTGGCCAGATCTATTCCTACAAT-3'), PCVD (5'-CCGAAGTAGCCATATGATAGCAT-3')
and PCVD3' (5'-ATAAGAGGGGGATCCAGGTAATGA-3'), and PCVH
(5'-GGTTCTTAACCATATGTCAGTAG-3') and
PCVH3 (5'-ACGGTCAAGGATCCACTAATCGTC-3'), where
underlining designates the restriction sites NdeI,
BglII, and BamHI and boldface designates the ATG
start codon. These primer pairs were used in PCRs to amplify the ORFs
designated bchI, -I(2), -D, and
-H, respectively, where 2 designates the shorter of the two
ORFs (Fig. 1). Approximately 100 ng of
C. vibrioforme f. sp. thiosulfatophilum NCIB 8327 genomic DNA was subjected to amplification by PCR in a total volume of
100 µl containing 1× PCR buffer (Boehringer Mannheim), 1.8 mM
MgCl2, 0.2 mM four deoxynucleoside triphosphates, 0.2 pM
each primer, and 3.5 U of Expand High Fidelity PCR System (Boehringer
Mannheim). Cycle parameters were 97°C for 4 min (hot start) and then
30 cycles of 95°C for 1 min 30 s, 60°C for 1 min, and 70°C
for 2 min (4 min for bchH). PCR products were purified from
agarose gels and cloned, by standard procedures (2, 23), in
the pET15b vector (22), yielding the constructs
pET15b-bchI and -I(2), pET15b-bchD,
and pET15b-bchH (Fig. 1). The correctness of the 5' cloning
site for each of the different ORFs was verified by sequence analysis.
Sequencing reactions were carried out with the Thermo Sequenase core
sequencing kit with 7-deaza-dGTP and run on a Vistra 725 DNA sequenator
(Amersham Life Science). Insert-specific oligonucleotide primers were
labelled with the 5' oligonucleotide Texas red labelling kit (Amersham
Life Science) according to the manufacturer's instructions and used
together with the Texas red-labelled T7 primer (Amersham Life Science)
to sequence the 5' ends, containing the cloning site, of all
pET-derived constructs.
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FIG. 1.
N-terminal starts of the constructs designated
pET15b-bchI, -I(2), -D, and
-H. Underlining and boldface designate the possible ATG
start codon(s) and possible ribosome binding sequences, using the
consensus motif AGGAGG(N2-10)ATG (1). Note that
the stop codon (TGA) of the bchI ORF overlaps the start
codon of the bchD ORF. The underlined amino acids designate
conserved amino acids within the first eight and nine residues of the
N-terminal parts of the deduced BchI(2) and -D proteins.
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Expression and partial purification of gene products.
The
optimal temperature, with regard to yield of soluble protein, during
induction was determined in initial small-scale experiments (10-ml
volume) in accordance with pET System Manual (6th ed.) from Novagen
(data not shown). In these experiments induction at 30°C for
pET15b-bchI and pET15b-bchI(2), 18°C for
pET15b-bchD, and 37°C for pET-bchH was found to
be optimal (data not shown). However, the construct
pET15b-bchD was induced at 30°C and processed as described
below.
The pET15b-derived constructs were transformed into
E. coli
BL21(DE3) and grown at 25°C in Luria-Bertani medium containing
100 µg of ampicillin per ml in volumes of 0.4 liter for the
bchI-
and
-D-containing constructs and 3 liters
for the
bchH-containing
constructs until the
A600 of the cultures reached 0.6 to 0.8.
Gene
expression was induced by addition of
isopropyl-
-
D-thiogalactopyranoside
to the cultures at a
final concentration of 1 mM. One hour prior
to the time of induction,
5-aminolevulinic acid was added at a
final concentration of 0.2 mM to
cultures of pET15b-
bchH. After
6 to 8 h of induction at
temperatures found optimal in the small-scale
experiments, cells were
harvested by centrifugation at 5,000 ×
g for 15 min at
4°C, and the pellets were stored at
20°C until
use. His-tag
purification of overexpressed BchI, -I(2), and -H
proteins on an
Ni
2+-agarose affinity column was carried out according to
the pET
System Manual (6th ed.) from Novagen, with the exception that
the imidazole concentration in the elution buffer was 0.5 instead
of 1 M. One-milliliter fractions with high protein concentrations
were
pooled (typically 4 to 6 ml) and dialyzed twice against 3
liters of
Mg-chelatase buffer (50 mM Tricine [pH 7.9], 0.3 M glycerol)
containing 2 mM dithiothreitol at 4°C. BchH protein was additionally
dialyzed in the presence of 1 µM protoporphyrin IX and 2 mM
MgCl
2.
Cell pellets containing BchD protein were dissolved in 10 ml of
Mg-chelatase buffer containing 1 mM dithiothreitol and disrupted
by
sonication in an ice bath. Insoluble protein and cellular debris
were
pelleted by centrifugation at 40,000 ×
g for 20 min at
4°C.
The supernatants were discarded, and the pellets, which by
phase-contrast
microscopy revealed the presence of inclusion bodies of
BchD,
were dissolved and incubated overnight at 4°C in Mg-chelatase
buffer containing 6 M urea (analytical grade) and 1 mM dithiothreitol.
Cellular debris was pelleted by centrifugation at 40,000 ×
g for
20 min at 4°C, and the protein concentration in the
supernatant
was typically 25 to 30 mg/ml. As judged from sodium dodecyl
sulfate
(SDS)-polyacrylamide gels, the BchI protein was purified to
homogeneity,
the BchI(2) and -H proteins were estimated to be >80 and
>10%
pure, respectively, and the urea-solubilized BchD protein was
estimated to be >80 to 90% pure (Fig.
2). The difference in N
termini between
the BchI and BchI(2) proteins seems to have a
decisive effect on the
purity of the BchI protein. Two contaminating
E. coli-derived proteins, seen as the ca. 80- and 85-kDa bands
on
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig.
2),
were
present after His-tag purification of the BchI(2) and -H
proteins.
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FIG. 2.
SDS-PAGE of His-tag-purified proteins. Lane 1, BchI(2)
(38 kDa); lane 2, BchI (42 kDa); lane 3, BchD (67 kDa); lane 4, BchH
(145 kDa). Approximately 0.5 µg of the His-tag-purified or
urea-dissolved proteins was loaded onto the gel. The two bands of ca.
80 and 85 kDa present in preparations of BchI(2) and -H proteins are
considered to be E. coli-derived contaminants.
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SDS-PAGE, protein concentration, and Mg-chelatase assays.
Protein concentrations were estimated with the Bio-Rad Protein Assay
reagent according to the manufacturer's instructions. SDS-PAGE was
carried out and porphyrin solutions were made as described by Laemmli
(10) and Gibson et al. (3), respectively. The
Mg-chelatase assay mixture contained 20 mM MgCl2, 4 mM ATP, 5 µM protoporphyrin IX, 20 mM phosphocreatine, 5 U of creatine kinase, 1.5 to 2 mM dithiothreitol, protein, and Mg-chelatase buffer
adjusted to a total volume of 100 µl. Assay mixtures containing ca.
30 µg of BchI and -D proteins and 60 to 70 µg of BchH protein, deduced from the purity on SDS-polyacrylamide gels following His-tag purification, were incubated at 30°C in the dark for 4 h, the reactions were stopped by addition of 1.9 ml of
acetone-H2O-32% NH4OH (80:20:1, vol/vol/vol)
and the mixtures were centrifuged at 15,000 × g for 5 min. The preparation and use of purified Synechocystis strain PCC6803 subunits will be described elsewhere (7). A fluorescence emission spectrum of the aqueous phase (excitation, 420 nm) of each assay mixture was obtained in a Kontron SFM 25 spectrofluorimeter and recorded at between 550 and 650 nm. The emission
at 595 nm was used as a measure of the Mg-protoporphyrin IX formed,
which was quantified by integration of the peak area from the
fluorescence intensity response of authentic Mg-protoporphyrin IX
(Porphyrin Products, Logan, Utah); this was found to be linear in the
range of 1 to 500 pmol.
Growth of C. vibrioforme and Western blot analysis.
C. vibrioforme f. sp. thiosulfatophilum NCIB 8327 was grown in 100-ml completely filled screw-capped bottles containing
the medium of Sirevåg and Ormerod (25) as described by
Rieble et al. (21) under incandescent tungsten lamps at a
light intensity of ca. 5,000 lx at 25°C. Cell cultures in the
exponential phase were harvested after reaching an optical density at
650 nm of approximately 0.7, and cells of the stationary phase were
harvested 2 to 4 h after having reached an optical density at 650 nm of ca. 1.2. For protein extraction, 50 ml of cell culture was
pelleted by centrifugation at 10,000 × g for 10 min at
4°C, and the pellets were stored at 70°C until use. The pellets
were dissolved in 1 ml of 50 mM Tris-HCl-2 mM EDTA (pH 8.0)-0.01%
Triton X-100, lysozyme at a final concentration of 0.1 mg/ml was added,
and the mixture was incubated for 30 min at 30°C and sonicated
vigorously in an ice bath until clearing of the suspension. Protein was
precipitated by the addition of 5 volumes of
acetone-H2O-32% NH4OH (80:20:1, vol/vol/vol)
mixed twice with 1 volume of hexane in order to remove bacteriochlorophylls. This suspension was mixed vigorously and incubated at 4°C for 1 h, and protein was pelleted by
centrifugation at 10,000 × g for 10 min at 4°C.
Western blot analysis was done in accordance with the instructions
supplied with the ECL Western blotting analysis kit (Amersham Life
Science), with the exception that the blocking time was 2 h.
Primary antibodies were raised in rabbit against purified
Synechocystis strain PCC6803 ChlI, -D, and -H proteins and
used at a final dilution of 1:10,000 (ChlI) or 1:5,000.
HPLC analysis of pigments.
For analysis of porphyrin
content, 200 µl of the assay mixture was extracted with 1.0 ml of
80% acetone containing 0.1 M NH3, mixed with 400 µl of
hexane, and centrifuged at 20,000 × g for 3 min. The
acetone phase was analyzed for porphyrins by high-pressure liquid
chromatography (HPLC) with a Beckman Ultrasphere octyldecyl silane
column (150 by 4.6 mm). The column was eluted with a 10-min linear
gradient from 15% solvent A to 100% solvent B at 1 ml/min. Solvent A
contained 0.05% (vol/vol) triethylamine in water; solvent B contained
100% acetonitrile. Porphyrins were detected with a Waters in-line
fluorescence detector. Excitation at 420 ± 5 nm and emission at
595 ± 5 nm were used to detect Mg-protoporphyrin IX.
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RESULTS |
Features of the C. vibrioforme bchI and -D
ORFs.
In Chlorobium, a number of genes have no
Shine-Dalgarno (SD) motifs at the more typical locations just upstream
of the start codon (11, 12, 15, 20, 30), and putative
intragenic SDs have been suggested to exist within 70 bp of the start
codons of the atp2
and the putative hemACD
operons (24), a phenomenon which has also been observed in
other bacteria (for instance, the archaea [31]). The
existence of more than one possible translation start codon and
intragenic SD-like sequences found near the N-terminal end of the
bchI ORFs complicates the prediction of the in vivo translation start codon (Fig. 1). The overlapping stop and start codons
of the bchI and -D ORFs, respectively, indicate
that the bchD ORF is the one translated in C. vibrioforme, and alignment of the deduced BchI(2) and -D proteins
suggests that these proteins are the ones translated in C. vibrioforme (19), a notion which is supported by the
presence of three nested SD motifs found just upstream of the
bchI(2) ORF. However, a lysine-, alanine-, and threonine
rich-stretch of 44 amino acids is found upstream and in frame with the
bchI(2) ORF yielding the bchI ORF (38 and 42 kDa
from the deduced amino acid sequence, respectively). Residues 3 to 33 of BchI are able to form a coiled coil structure, and short coiled
coils are known to serve as dimerization domains in several families of
proteins. Interestingly, Willows et al. (29) have reported
that purified R. sphaeroides BchI protein is dimeric. Thus,
two constructs corresponding to the bchI and -I(2) ORFs, respectively, were made (Fig. 1).
Reconstitution of activity in vitro and properties of the
Mg-chelatase subunits.
Activity from crude extracts of
overexpressed R. sphaeroides BchI, -D, and -H subunits and
Synechocystis strain PCC6803 ChlI, -D, and -H subunits was
routinely obtained, in accordance with results of Gibson et al.
(3) and Jensen et al. (5). However, in vitro
reconstitution of activity from crude extracts of the three C. vibrioforme subunits could not be achieved. As a consequence of
this, the overexpressed His-tagged BchI, -I(2), and -H proteins were
purified on an Ni2+-agarose affinity column, and insoluble
BchD proteins, mainly as inclusion bodies, were solubilized in 6 M
urea. ATP-dependent Mg-chelatase activity was obtained when
His-tag-purified BchI or -I(2), BchH, and urea-denatured BchD proteins
were combined (Fig. 3), thereby
establishing that the C. vibrioforme Mg-chelatase is similar
to the Mg-chelatases of R. sphaeroides and
Synechocystis strain PCC6803 with respect to the number of
subunits and the ATP requirement. In assays with 30 µg of BchI or
-I(2), 30 µg of BchD, and 60 to 70 µg of BchH, ca. 15 pmol of
Mg-protoporphyrin IX was formed after 4 h. The continued formation
of Mg-protoporphyrin IX during the relatively long incubation time
suggests that a large fraction of one or more of the three subunits was
initially inactive and was subsequently activated during the incubation period. When the BchH protein was replaced by an approximately equal
amount of His-tag-purified Synechocystis strain PCC6803 ChlH
protein, activity was increased twofold. Thus, the BchH protein was
found to be limiting in assays with the other two C. vibrioforme subunits. Several possibilities might explain this:
(i) only a minor fraction of BchH protein is folded into an active
form, (ii) the contaminating ca. 80- and 85-kDa E. coli-derived proteins bind to or form aggregates with BchH or have
some other inhibitory effect on activity, or (iii) the
Mg-protoporphyrin IX formed is not released or is only slowly released
from the BchH subunit, thereby limiting the amount of active protein
over time. The bchI, -I(2), -D, and
-H ORFs have been reamplified by PCR and recloned in pET15b
(see Materials and Methods), and identical results with respect to
protein expression and Mg-chelatase activity were obtained when
expressed proteins from three clones derived from each PCR were
assayed. Thus, it seems unlikely that PCR-derived errors could have
caused the limited activity of especially the BchH protein.
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FIG. 3.
(A) Fluorescence emission spectra from assays
demonstrating that the C. vibrioforme subunits BchI/I(2),
BchD, and BchH and ATP are required for in vitro reconstitution of
Mg-chelatase activity. Trace 1, BchI plus BchD; trace 2, BchI plus
BchH; trace 3, BchD plus BchH; traces 4 and 5, BchI plus BchD plus
BchH; trace 6, BchI(2) plus BchD plus BchH; trace 7, BchI plus BchD
plus ChlH. Assay conditions were as described in Materials and Methods,
with the exception that ATP was omitted in the assay shown as trace 4. The characteristic fluorescence maxima of protoporphyrin IX and
Mg-protoporphyrin IX are 633 and 595 nm, respectively. (B) HPLC
identification of the product formed in Mg-chelatase assays. Trace 1, authentic Mg-protoporphyrin IX; trace 2, product formed from the assay
shown as trace 5 in panel A. tR, retention time.
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With respect to the BchD subunit, activity was achieved only when
highly concentrated insoluble BchD protein was solubilized
in 6 M urea
and added as the last component to the assay, yielding
a final urea
concentration of 60 mM. Refolding of urea-denatured
BchD protein is
likely to occur during the formation of the putative
ternary complex
between the BchI and -D subunits and Mg
2+-ATP, as proposed
for the Mg-chelatase of
R. sphaeroides (
29).
Alignment of the deduced N-terminal amino acid sequences of the "I"
and "D" genes from
R. capsulatus (
1),
C. vibrioforme (
19), and
Synechocystis
strain PCC6803 (
5) suggests that
the
C. vibrioforme BchI(2) and -D proteins delineate a homologous
core of
the aligned proteins (Fig.
4). When
assayed under identical
conditions together with BchD and -H proteins,
the BchI and -I(2)
proteins were found to be approximately equally
active (Fig.
3).
It can thus be concluded that the BchI(2) protein is
sufficient
for obtaining activity and that the 44-amino-acid N-terminal
part
of the BchI protein does not have a negative effect on activity.
In order to gain further information on the start of the
bchI ORF in
C. vibrioforme, total protein of
C. vibrioforme was subjected
to Western blot analysis.
Surprisingly, the antibody raised against
the
Synechocystis
strain PCC6803 ChlI subunit reacted specifically
with two bands, which
appeared to comigrate along with the His-tag-purified
BchI and -I(2)
proteins (Fig.
5). This suggests that the
native
BchI protein might be present as two alternative forms. While
the 42-kDa form seems to dominate in cells in the exponential
phase,
the ca. 38-kDa protein is most abundant in cells in the
stationary
phase. This might indicate either that the ca. 42-kDa
protein is the
one which is translated and subsequently processed
to yield the ca.
38-kDa protein or that the two proteins are translated
separately.
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FIG. 4.
Alignment of the N termini of the I and D deduced amino
acid sequences from C. vibrioforme (Cv),
Synechocystis strain PCC6803 (SP), and R. capsulatus (Rc). Amino acids conserved in four or more
of the aligned sequences are marked by reverse shading, and asterisks
designate the phosphate-binding motif
(GX4GKSX6A) present in the three I sequences.
With respect to the N termini, note that the C. vibrioforme
BchI(2) and -D deduced proteins seem to delineate a homologous core of
the aligned proteins.
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FIG. 5.
C. vibrioforme proteins cross-reacting with
antibodies directed against purified Synechocystis strain
PCC6803 ChlH (lane 1), -D (lane 2), and -I (lanes 3 to 6) proteins.
Lanes 1 to 3, ca. 10 µg of total protein of C. vibrioforme
in exponential phase; lane 4, ca. 10 µg of total protein of C. vibrioforme in stationary phase; lanes 5 and 6, ca. 0.05 µg of
His-tag-purified BchI (42-kDa) and -I(2) (38-kDa) proteins,
respectively (cf. Fig. 2). While the 42-kDa-like protein is dominant in
exponential phase cells, the 38-kDa-like protein is most prominent in
cells in the stationary phase.
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Finally, all combinations of the
C. vibrioforme and
Synechocystis strain PCC6803 subunits were assayed.
Reconstitution of
an active heterologous enzyme complex was achieved
only when the
C. vibrioforme BchI/I(2) and -D proteins and
the
Synechocystis strain PCC6803 ChlH protein were combined
(Fig.
3) or when the
Synechocystis strain PCC6803 ChlI and
-D proteins and the
C. vibrioforme BchH protein were
combined, with the latter of the combinations
giving rise to only trace
amounts of activity (data not shown).
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DISCUSSION |
We have, through reconstitution of Mg-chelatase activity in vitro,
demonstrated that the Mg-chelatase of C. vibrioforme is composed of three subunits encoded by the genes bchI,
-D, and -H. The Mg-chelatase of C. vibrioforme is similar to the Mg-chelatases of the distantly
related bacteria Synechocystis strain PCC6803 (5)
and R. sphaeroides (3) with respect to the number
and sizes of the subunits as well as the absolute requirement for ATP.
Chlorobium, being a strictly anaerobic and obligate
phototrophic organism, is believed to resemble some of the earliest
life forms present in an anaerobic environment. The finding that the
Mg-chelatases of C. vibrioforme, R. sphaeroides,
and Synechocystis strain PCC6803 are encoded by three
homologous genes, together with genetic and biochemical data which
strongly imply that the higher-plant Mg-chelatase is also encoded by
three genes (4, 6, 8), makes the three-gene scheme of the
Mg-chelatase a ubiquitous feature of probably all photosynthetic
organisms. Thus, the ATP-requiring chelation of Mg2+ into
protoporphyrin IX seems to be a reaction which imposes considerable constraints on the enzyme.
Two interesting features of the C. vibrioforme Mg-chelatase
have been found. First, Western blot analysis indicates the existence of two forms of the BchI protein, which probably correspond to the
overexpressed BchI and -I(2) proteins, which were found to be equally
active when assayed in vitro. The probable presence of the two BchI
forms in C. vibrioforme might relate to the finding that
anesthetic gases, such as N2O, ethylene, and acetylene, in C. vibrioforme have been found to inhibit the formation of
chlorosomes and bacteriochlorophyll d, which is primarily
found in the chlorosomes, without affecting the synthesis of
bacteriochlorophyll a, which is associated primarily with
the cell membrane and its photosynthetic reaction centers
(16). Inhibited cells were found to accumulate primarily
Mg-protoporphyrin IX monomethyl ester, the substrate of the enzyme
Mg-protoporphyrin IX monomethyl ester cyclase, and it was suggested
that bacteriochlorophylls a and d could be
differently affected because of compartmentalization of their
biosynthetic apparatus (16). Second, in vitro reconstitution
of an active heterologous Mg-chelatase enzyme complex between C. vibrioforme and Synechocystis strain PCC6803 subunits
has been demonstrated for the first time, and it was found that
reconstitution of activity required that the I and D subunits be
derived from the same species. The Synechocystis strain
PCC6803 ChlH protein has recently been shown to act as a carrier of the
substrate protoporphyrin IX, and the interaction of ChlH-protoporphyrin
IX with the presumed ternary complex between ChlI and -D and
Mg2+-ATP was found to be of a transient nature, at least in
vitro (7). The finding that the H subunit from one species
can interact with I and D subunits from another species supports the
role of the H subunit as a substrate carrier and also suggests
involvement of weak or unspecific protein-protein interactions between
H and the I-D-Mg2+-ATP complex.
Recloning of the BchH ORF in an alternative expression system and
further purification of the subunits have to be carried out before a
more comprehensive characterization of the enzyme complex will be
possible. Verification of the existence of the two forms of the BchI
protein and their localization in C. vibrioforme is in
progress.
| |
ACKNOWLEDGMENTS |
Inoculum stocks of C. vibrioforme f. sp.
thiosulfatophilum NCIB 8327 were kindly provided by H. Scheller, Department of Plant Biology, Royal Veterinary and
Agricultural University, Frederiksberg, Denmark.
This work has been supported by grants from the Danish Agricultural and
Veterinary Research Council to Knud W. Henningsen (no. 9601206 and
13-5005) and Poul Erik Jensen (no. 9600928) and from Købmand Sven
Hansen og hustru Ina Hansens Fond, Sorø, Denmark. C. Neil Hunter and
Lucien C. D. Gibson gratefully acknowledge financial support from
the BBSRC, United Kingdom. We are grateful to Ulla Rasmussen and
Kirsten Henriksen for excellent technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Ecology and Molecular Biology, Royal Veterinary and Agricultural
University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
Phone: 45 35 28 26 08. Fax: 45 35 28 26 06. E-mail:
blp{at}kvl.dk.
| |
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J Bacteriol, February 1998, p. 699-704, Vol. 180, No. 3
0021-9193/98/$04.00+0
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Abstract
Introduction
