(Received for publication, February 10, 1995; and in revised form, June 13, 1995)
From the
Glutathione reductase (GR) was purified from the cyanobacterium Anabaena PCC 7120. A 3-kilobase genomic DNA fragment containing the coding sequence for the GR gene (gor) was identified and cloned by polymerase chain reaction based on sequences of selected peptides isolated from proteolyzed GR. The coding sequence encompassing 458 amino acid residues, as well as 360 base pairs of the 5`-flanking region and 430 base pairs of the 3`-flanking region, were determined. Genomic Southern analysis indicates that gor is a single-copy gene. A gor antisense RNA probe hybridized with a 1.4-kilobase transcript, suggesting that the gene is not part of an operon including additional genes. The deduced GR amino acid sequence shows 41 to 48% identity with those of human, Escherichia coli, Pseudomonas aeruginosa, pea, and Arabidopsis thaliana GR. The coding sequence of GR was overexpressed in a GR-deficient E. coli strain, SG5, and the recombinant protein was purified. Anabaena GR is NADPH-linked, but a Lys residue replaces an Arg residue involved in NADPH binding in GR from other species. In addition, Anabaena GR carries the GXGXXG ``fingerprint'' motif which otherwise characterizes NAD(H)-dependent enzymes. These differences may contribute to the lack of affinity for 2`,5`-ADP-Sepharose 4B of Anabaena GR. Three E. coli-type promoter sequences and a BifA/NtcA binding motif were found upstream of the open reading frame. The middle and the proximal promoters were shown to be active. However, the use of the middle promoter was dependent on the nitrogen source in the culture medium. Both GR activity and GR protein concentration increased in ammonium grown cultures in which both the middle and proximal promoters were used for transcriptional initiation. The BifA/NtcA-binding site overlaps the middle promoter sequence and may thus be involved in regulation of differential transcription.
Glutathione reductase (GR), ()which is a widespread
enzyme catalyzing the reduction of GSSG to GSH with NADPH as the
reducing cofactor, is necessary for maintaining high GSH/GSSG ratios in
cells(1) . GSH plays an important role in many cellular
functions, including protection against oxidative
stress(2, 3) . In particular, it is a key enzyme in
the glutathione-ascorbate cycle, which functions in peroxide scavenging
and protection against other oxidative processes(4) . A major
source of active oxygen species in green, chlorophyllous tissues is
derived from the photosynthetic machinery. Green tissues are therefore
particularly dependent on efficient scavenging mechanisms, since active
oxygen species are not only produced under stress but also under most
growth conditions. GR activities in leaves are higher than that in
non-photosynthetic tissues and increase with elevated concentrations of
oxygen(5) . Enzymes of the GSH-ascorbate pathway may also serve
an essential protective role in relation to nitrogen fixation, a
process catalyzed by the extremely oxygen-sensitive enzyme nitrogenase.
For instance, in nitrogen-fixing soybean root nodules, the activities
of all enzymes in the GSH-ascorbate pathway are elevated as compared to
those in non-fixing nodules; e.g. the GR activity is increased
about 4-fold(6) . Since diazotrophic cyanobacteria rely on a
plant-type oxygenic photosynthesis as well as nitrogen fixation for
survival, the risk of oxidative damage is particularly pronounced.
However, protective mechanisms operative in cyanobacteria have not been
fully elucidated.
GR has been purified from a few cyanobacterial strains(7, 8) . It shows similar kinetic properties to that of the chloroplast enzyme(9) . Furthermore, it has been suggested that in the cyanobacterium Gloeocapsa sp. LB795, GR together with other enzymes of the GSH-ascorbate pathway may serve to protect nitrogenase from being damaged during oxidative stress(10) .
The enzyme has been characterized from a large number of sources, e.g. eubacteria, fungi, plants, and human(11) . All the GRs isolated show remarkable similarity in molecular and kinetic properties, indicating high evolutionary conservation of the protein. X-ray crystallographic analysis of human GR at 1.54-Å resolution (12) and of Escherichia coli GR at 1.8-Å resolution (13) have been published.
In contrast to the large number of studies available on the enzymology of GR, the gene encoding this key enzyme (gor) has only been isolated from two prokaryotes, E. coli(14) and Pseudomonas aeruginosa(15) . GR cDNA has been obtained from two plants, pea (16) and Arabidopsis thaliana(17) , as well as from mouse and human cells(18) . However, the regulation of gor in response to oxidative stress has been reported only for E. coli and Salmonella typhimurium(19, 20) , in which OxyR (a transcriptional activator) regulates the overexpression of nine proteins, including GR. However, no evidence that OxyR interacts directly with the gor promoter region has been presented. Here, we report the isolation and characterization of the GR gene from a filamentous nitrogen-fixing cyanobacterium, Anabaena PCC 7120. Furthermore, we present data on the influence of the nitrogen source in the growth medium on the regulation of GR gene expression and propose a potential regulatory mechanism for GR.
Figure 1:
Map of
restriction endonuclease recognition sites of the gor region
of Anabaena PCC 7120 genomic DNA and strategy for isolation,
cloning, and sequencing of the gor gene. The arrows at the top show the direction and location of oligonucleotide
primers in relation to the gor gene below. In the restriction
map, the heavy black line represents the the region coding for
GR. The HindIII linker sequence is indicated by (&cjs2109;).
Only restriction sites for HindIII, TaqI, XbaI, and RsaI are shown. Genomic DNA and
size-fractionated DNA prepared from Anabaena PCC 7120 were
used as templates for PCR, as described under ``Experimental
Procedures,'' to amplify the gor gene region as three
fragments: A (254 bp; degenerate primers P,
P
and P
), B (532 bp; anchor PCR primer
L
and specific primers P
and P
),
and C (
2.3 kb; specific primers P
, P
and anchor PCR primer L
). Some of the primers used
had a recognition sequence for restriction enzymes at their 5` ends to
facilitate subsequent cloning of the PCR-generated fragments into pGEM
3Zf(+) for sequencing. In the sequencing strategy scheme, the arrows show the direction and approximate extent of each
sequencing reaction. Arrows originating from vertical bars indicate sequence information obtained from DNA fragments
subcloned into pGEM 3Zf(+) vector and primed with the universal or
reverse vector primers. Arrows originating from a dot indicate sequence information obtained by the use of
oligonucleotide primers complementary to cloned fragment
sequences.
Figure 2: SDS-polyacrylamide gel electrophoresis of GR purified from Anabaena PCC 7120. Protein standards (lane 1) and 0.35 µg of purified enzyme (lane 2) were subjected to SDS-polyacrlamide gel electrophoresis. Both lanes were stained with Coomassie Brilliant Blue. The arrow indicates the position of the GR subunit with a molecular mass of 50 kDa.
About 50 µg of the purified GR protein was digested with a lysine-specific protease, and the resulting peptides were separated and isolated by reversed phase high performance liquid chromatography. Several of the peptides obtained in homogeneous form were subjected to sequence analysis. Based on sequence similarities to GRs from other organisms, the relative positions of three internal peptides was determined. These peptides were suitable for the design of degenerate primers used in the isolation of the gor gene.
Figure 3:
Southern blot analysis of Anabaena PCC 7120 genomic DNA. Total Anabaena DNA was digested
with EcoRI, DraI, and HindIII as well as
with HindIII together with DraI. The digests were
electrophoresed, transferred to a nylon membrane, and hybridized with P-``antisense'' RNA probe synthesized using the
riboprobe system. Positions of DNA M
standards are
indicated on the left. The sizes of fragments hybridizing with the gor probe are given on the right.
Figure 4:
Nucleotide and deduced amino acid
sequence of the Anabaena PCC 7120 gor gene. The
deduced amino acid sequence in single- letter code is shown
below the nucleotide sequence; the amino acid sequences determined
directly by analysis of proteolytic peptides are underlined. A
potential ribosome-binding site and three putative -10 and
-35 sites are indicated by underlining of the nucleotide
sequence. A potential binding site for BifA/NtcA,
TGT(N)ACA, is boxed, and its highly conserved
sequences TGT and ACA are marked by asterisks (
)
above. The vertical arrows indicate transcription start sites.
The potential transcription terminators (two inverted repeat
structures) are indicated by facing half-arrows beneath.
Three
putative canonical E. coli-type promoters were found within
100 nucleotides upstream of the translation start codon (Fig. 5A). At least two of them can be used for
transcriptional initiation as demonstrated below. Furthermore, a
putative BifA/NtcA binding site with the consensus sequence motif TGT(N)ACA(30) , was found
upstream of the proximal promoter and overlapping with the middle
promoter. The two DNA binding factors BifA and NtcA have been
identified in Anabaena PCC 7120 and in the unicellular strain Synechococcus PCC 7942,
respectively(31, 32) . Both belong to the cyclic AMP
receptor protein family of prokaryotic regulatory
proteins(33) . NtcA apparently regulates nitrogen assimilation,
while the function of BifA is not clear. The binding site sequence
noted in front of the gor gene, TGTTGACAACTGACA (-70 to
-56), is comparable to sequences identified as BifA binding
sequences upstream of the glnA, xisA, and rbcL genes in Anabaena PCC 7120 (Fig. 5B)(30) . It shares particularly high
sequence similarity with the proximal BifA-binding site upstream of rbcL, even within the non-conserved central part. Examination
of the noncoding sequence in the 3` region of the gor gene
revealed two putative prokaryotic terminators, i.e. two
inverted repeats at positions 1405-1480 and 1529-1575. No
open reading frame was found within 430 bp of the 3`-flanking region.
Figure 5: Aligment of promoter sequences and BifA/NtcA binding sequences. A, comparison of the three putative gor promoter sequences (two of them were demonstrated to be used) to E. coli promoter consensus shown at the top. Upper-case letters indicate nucleotides strongly conserved; lower-case letters indicate nucleotides conserved but less frequently. Boxes enclose the sequences that best approximate the E. coli consensus in the -35, -10 regions and the transcriptional start sites. B, putative BifA/NtcA binding sequences of Anabaena PCC 7120 gor, rbcL, xisA, and glnA gene are aligned 5` to 3` with respect to the open reading frame. Numbers indicate the first and last nucleotides in the sequence and are relative to the translation start site set as +1. A consensus BifA/NtcA binding sequence is also shown.
Both nucleotide and amino acid sequences of GR from Anabaena PCC 7120 showed high similarity to GR from other sources (Fig. 6, Table 3). As expected, the GR family signature at
amino acid residues 55-67, which is responsible for forming the
redox-active disulfide bridge between Cys and Cys
(numbering of human GR, (12) , omitting Met
)
is highly conserved. Two arginine residues (Arg
and
Arg
) required for binding of the 2`-phosphate group of
NADPH are also conserved in all five proteins, the only exception being
in Anabaena GR, in which Arg
is replaced by
lysine. This replacement may be contributing to the lack of binding to
the 2`,5`-ADP Sepharose 4B affinity chromatography matrix noted in the
attempts to purify both native and recombinant Anabaena GR.
Figure 6: Amino acid sequence alignment of the Anabaena PCC 7120 GR with sequences of GR from other species. Residues that are identical or similar in all six sequences are marked by open squares beneath. Gaps have been introduced to give better alignments (indicated by dots). Double dots below the sequences indicate regions of residues important for GSSG binding. The region surrounding the redox-active disulfide bridge is boxed. The fingerprint motif of NADPH binding is doubly underlined. The Arg residues involved in NADPH binding are indicated by *, but the second of these Arg residues is replaced by Lys in Anabaena GR. Numbers of Anabaena and human GR residues are given on the right side of the sequences. Abbreviations and sources of sequences: A. thaliana, Arabidopsis thaliana(17) ; pea, Pisum sativum L.(16) ; Ps. aeruginosa, Pseudomonas aeruginosa(15) ; E. coli, Escherichia coli(14) ; human(18) .
Figure 7: Origins of gor transcription. Lanes 1-3 represent the reverse transcriptase products of GR mRNA of Anabaena PCC 7120 cells grown on ammonium or nitrate or cultured under nitrogen fixing conditions, respectively. Lanes C, A, G, and T represent the results of sequence reactions in the region encompassing the promoter. The transcriptional start sites are indicated by arrows.
RNA preparations from nitrate-grown and
from N-grown cultures were also used for Northern blot
analysis. In both cases, a single RNA species of 1.4 kb corresponding
roughly to the coding length required for the gor gene (Fig. 8) was detected. This result together with the sequencing
data suggest that the gor transcripts are not linked to
transcripts of other genes and that the gene is not part of an operon.
Figure 8: Northern blot analysis of Anabaena PCC 7120 gor transcripts. About 8 µg of total RNA isolated from nitrate grown cultures (lane 1) and from nitrogen-fixing cultures (lane 2) was electrophoresed, blotted, and hybridized with RNA probes. In both lanes, the predominant band corresponds to a message size of 1.4 kb.
Figure 9:
Western
blot analysis of GR expression in Anabaena PCC 7120 grown on
various nitrogen sources. Extracts from cells that were grown in medium
under nitrogen-fixing condition (N), or in media containing
nitrate (NO
) or ammonium
(NH
), i.e. non-nitrogen-fixing
conditions; were electrophoresed on a SDS-polyacrylamide gel. Proteins
were blotted onto nitrocellulose, and GR was detected as an
immunocomplex with anti-GR antibodies and visualized by
chemiluminescence.
We here present the first complete DNA sequence determined for a gene encoding glutathione reductase from a photosynthetic prokaryote, the cyanobacterium Anabaena PCC 7120. The enzyme is of pivotal importance in the scavenging of reactive oxygen species. The significance of the cyanobacterial enzyme is particularly obvious, since cyanobacteria suffer electron leakage and consequent oxygen radical production not only during photosynthesis but also during nitrogen fixation.
Having obtained the nucleotide sequence, the amino acid sequence of the Anabaena enzyme could also be deduced. Both at the amino acid level and at nucleic acid level, GR of Anabaena PCC 7120 exhibits higher similarity to GR from plants than to those from bacteria (Table 3). In addition, codons which are low in G+C content are preferentially used (Table 2). This is in contrast to the situation in Pseudomonas and E. coli, where a marked bias in favor of either G or C residues at the third position of each codon has been noted(34) . These results support the endosymbiont theory, stating that cyanobacteria were forerunners of higher plant chloroplasts(35, 36) .
Catalytically important
residues responsible for the redox reaction or involved in the binding
of GSSG and NADPH are highly conserved in all six GR sequences
examined. These include the redox active cysteines (Cys,
Cys
; Fig. 6: numbering based on the human sequence)
and flanking amino acids. Also, Arg
and Arg
of human GR, involved in NADPH binding, are conserved in five out
of six GRs, the exception being Anabaena GR, in which Lys
replaces the second Arg
. Computer modeling indicates that
such a replacement gives rise to a longer distance between NADPH and
the NADPH-binding residues of the enzyme. Thus, the poor affinity of GR
from Anabaena for 2`,5`-ADP Sepharose may partly be caused by
this replacement. Furthermore, most redox enzymes using NADP(H) contain
a highly conserved GXGXXA ``fingerprint''
motif in the NADP(H)-binding domain. However, in NAD(H)-dependent
enzymes the alanine residue is almost universally replaced by
glycine(37) . Noticeably, the Anabaena GR carries the
GXGXXG sequence motif similar to that in
NAD(H)-dependent enzymes. However, kinetic studies revealed that the
enzyme still has about a 40-fold higher catalytic efficiency with NADPH
than with NADH (data not shown). Therefore, our result suggests that
the difference Ala/Gly
in the fingerprint motif is not
the sole determinant of the GR coenzyme specificity (cf. 38).
Although the GR sequence is highly conserved in all six species examined, the regulation of the gene expression may be different. For instance, GR isoenzymes seem to be products of a multigene family in soybean root nodule (39) and in red spruce(40) . In contrast, the multiple forms of GR identified in other photosynthetic organisms such as pea (16, 41) and Arabidopsis thaliana(17) indicate the existence of post-translational regulatory mechanisms, as only a single-copy gor gene has been detected. The Anabaena PCC 7120 gor gene examined here, as well as those from E. coli and P. aeroginosa, are also likely to be single-copy genes.
Comparisons of the promoter regions of gor from E. coli and P. aeroginosa reveals one -35 and one -10 element upstream of the E. coli GR coding sequence and one element similar to the E. coli -10 consensus region upstream of P. aeroginosa gor. In contrast, three E. coli-type promoters were detected upstream the Anabaena PCC 7120 gor gene. Moreover, we could demonstrate that two of the promoters, the middle and the proximal promoters, can be used alternatively or in combination during growth, depending on the nitrogen source used. The proximal promoter is used under all growth conditions, while in the ammonium-grown culture, the middle promoter is also used. Therefore, the high GR expression seen in ammonium-grown cultures probably reflects the dual transcriptional initiation in cultures using ammonium as the sole nitrogen source.
The putative
BifA/NtcA-binding site detected upstream of the proximal promoter is
partly overlapping with the middle promoter. Previous studies have
shown that BifA may bind to upstream sequences of genes which have
diverse functions in Anabaena PCC
7120(31, 32) . Depending on the position of its
binding site with respect to different promoters, it has been proposed
that BifA may act as an activator or a repressor. Its regulatory role
may be related to nitrate assimilation, as well as to other unknown
functions. In Anabaena PCC 7120, the location of the binding
position suggests that BifA probably is capable of repressing gor gene expression from the middle promoter, i.e. when grown
on NO and N
, since BifA
binding would interfere with the binding of RNA polymerase. However,
since the function of BifA is not completely known, it is difficult to
predict how and why such a binding factor would be involved in
transcriptional regulation of a gene whose main function is to produce
an enzyme contributing to a system involved in scavenging reactive
oxygen species. In general, the mechanisms by which multiple
transcription start sites of gene promoters are controlled remain
poorly understood. It cannot be excluded that other factors are
responsible for the differential transcription noted, e.g. high concentrations of ammonium may be toxic to
cyanobacteria(42) . Therefore, the up-regulation of GR through
multiple transcription start sites may be a response to stress rather
than to the nitrogen status. In order to clarify this point, the
regulation of GR under various external conditions will be examined in
nitrogen-fixing as well as non-nitrogen-fixing cyanobacteria.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X89712[GenBank].