Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, F.
Right arrow Articles by Mannervik, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, F.
Right arrow Articles by Mannervik, B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Volume 270, Number 39, Issue of September 29, pp. 22882-22889, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning, Sequencing, and Regulation of the Glutathione Reductase Gene from the Cyanobacterium Anabaena PCC 7120 (*)

(Received for publication, February 10, 1995; and in revised form, June 13, 1995)

Fanyi Jiang (1),  Ulf Hellman (2),  Grazyna E. Sroga (3),  Birgitta Bergman (3),  Bengt Mannervik (1)(§)

From the  (1)Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, S-751 23 Uppsala, the (2)Ludwig Institute for Cancer Research, Biomedical Center, Box 595, S-751 24 Uppsala, and the (3)Department of Botany, Stockholm University, S-106 91 Stockholm, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.

INTRODUCTION

Glutathione reductase (GR), (^1)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.

EXPERIMENTAL PROCEDURES

Cyanobacterial Culture Conditions

Anabaena sp. strain PCC 7120 was grown photo-autotrophically at 25 °C with an illumination of approximately 100 µE s m in BG11 medium(21) , typically containing 20 mM NaNO(3) as the nitrogen source (nitrate-grown). Alternatively, the nitrate was removed from the medium to induce nitrogen-fixing conditions (N(2)-grown), or substituted with 3 mM (NH(4))(2)SO(4) (ammonium-grown). All cultures were bubbled with air.

GR Purification and Sequence Analysis of Peptides from Proteolyzed GR

Anabaena PCC 7120 (N(2)-grown) was harvested at late exponential growth phase. The GR protein was purified to homogeneity by a five-step procedure (7) . Enzyme activity assays were performed as described(22) . The purified protein was reduced with dithiothreitol and alkylated with 4-vinylpyridine, followed by desalting on a Fast Desalting PC 3.2/10 column. Peptides obtained from GR by digestion with Achromobacter lyticus protease I (WARO Pure Chemicals Industries Ltd. Osaka, Japan) were separated by reversed phase liquid chromatography (SMART system, Pharmacia Biotech, Uppsala, Sweden) on a µRPC C2/C18 SC 2.1/10 column. The amino acid sequences were determined using a gas-phase sequencer (Applied Biosystems, model 477A) fitted with an on-line PTH-derivative analyzer (model 120A).

Isolation of the GR Genomic DNA Fragments by PCR

Three degenerate oligonucleotide primers P(1), P(2), and P(3) (cf.Table 1) were synthesized based on the amino acid sequences of the internal peptides AIAEN, FDEDI, and ISGRAT, respectively. Initially, 500 ng of Anabaena PCC 7120 genomic DNA was amplifed for eight cycles at an annealing temperature of 37 °C by using primers P(1) and P(2). The product obtained was then reamplified for 30 cycles at an annealing temperature of 55 °C by using primers P(1) and P(3). The second PCR product of about 250 bp was purifed from an agarose gel and cloned into the vector pGEM 3Zf(+) (Promega). The identity of the second product, denoted fragment A, was determined by sequencing. Based on the nucleotide sequence of fragment A, four specific primers were synthesized (primers P(4), P(5), P(6), and P(7); cf.Table 1). A HindIII adaptor comprised of two complementary oligonucleotides with HindIII protruding ends (L(H), L; cf.Table 1) was designed and synthesized for attachment to HindIII restriction fragments. These were used to isolate the up and downstream regions of the gor gene. Following Southern blot analysis, a portion of 3-kb HindIII fragments supposed to contain the GR coding region was purified from an agarose gel, ligated with HindIII adaptors, and used as a template for PCR. Both the upstream fragment B and the downstream fragment C (cf.Fig. 1) were obtained by a two-step PCR amplification using the following cycle parameters: 94 °C for 1.5 min, 55 °C for 2 min, and 72 °C for 2 min, the procedure being repeated for 30 cycles. For isolation of fragment B, primer P(4) and adaptor primer L(H) were used for the first-step PCR, and primers P(5) and L(H) for amplification of the first-step PCR products. For isolation of fragment C, primers P(6) and L(H) were used for the first-step PCR, and primers P(7) and L(H) were used for amplification of first-step PCR products. All PCR amplifications were performed in 50-µl reaction volumes containing template DNA (about 500 ng), primers (20 pmol of each), dNTPs (200 µM each), and Taq DNA polymerase (2.5 units, Boehringer Mannheim Biochemica) in the buffer supplied with the enzyme.

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(1), P(2) and P(3)), B (532 bp; anchor PCR primer L(H) and specific primers P(4) and P(5)), and C (2.3 kb; specific primers P(6), P(7) and anchor PCR primer L(H)). 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.


Preparations of DNA, RNA, and Blotting Analysis

DNA and RNA from Anabaena PCC 7120 were prepared as described(23) . For Southern blot analysis 15 µg of genomic DNA was used for each restriction digestion. For Northern blot analysis 8 µg of RNA was used. The DNA and RNA fragments were separated electrophoretically and transferred to nylon sheets (Hybond N; Amersham Corp.). Both hybridizations were carried out at 58 °C for 15 h in 50% formamide, 5 times SSC, 0.5% SDS, 1 times Denhardt, 1 mM EDTA, using P-labeled antisense RNA as a probe(24) .

RNA Probes

For riboprobe synthesis, the construct of pGEM 3Zf(+) with fragment A insert was linearized with BamHI, and a 254-bp nucleotide probe was synthesized by using SP6 RNA polymerase (25) in the presence of [alpha-P]UTP (3000 Ci/mmol, Amersham Corp.).

DNA Sequence Analysis

PCR-amplified fragments and their subfragments generated with restriction enzymes were cloned into pGEM 3Zf(+) and sequenced with Sequenase version 2.0 (United States Biochemical Corp.) by the dideoxynucleotide termination method(26) . Reactions were primed with the universal primer, the reverse primer, and oligonucleotides complementary to insert sequences, respectively, as described in Fig. 1.

Primer Extension Mapping of the Transcription Start Site of the gor Gene

RNA was isolated from Anabaena PCC 7120 late exponential phase cultures grown under various nitrogen conditions (NO(3), NH(4), N(2)) as described above. In each reaction, the 5` end-labeled oligonucleotide primer P (+22 to +39, cf.Table 1) was mixed with 30 µg of total RNA. The mixture was hybridized and extended (24) . Extension products were loaded onto a 6% (w/v) polyacrylamide sequencing gel along with a sequencing ladder generated with the same primer.

Expression of GR in E. coli

Using PCR and appropriate primer pairs (primers P and P, cf.Table 1), the coding region of GR was subcloned into the expression vector pGEM-Taq: the Taq promoter was inserted in front of the polylinker region of pGEM 3Zf(+). The recombinant protein was obtained by expression of the construct in a GR-deficient E. coli strain SG5 (data not shown).

Western Blot Analysis

A sample (100 µg) of Anabaena PCC 7120 cell lysate was electrophoresed on a 14% (w/v) SDS-polyacrylamide gel and then transferred to 0.45 micron Hybond C Extra supported nitrocellulose (Amersham International, Buckinghamshire, United Kingdom). Nonspecific binding sites were blocked with phosphate-buffered saline, 3% (w/v) bovine serum albumin. An antiserum obtained from rabbits immunized with the recombinant GR protein was used at a 1:200 dilution. Incubation with the primary antibodies was for 4 h at 4 °C in the same solution. Following five 5-min washes in phosphate-buffered saline, 0.05% (w/v) Tween 20, the membrane was incubated for 60 min with a 1:1000 dilution of a blotting grade affinity-purified goat anti-rabbit IgG antibody-alkaline phosphatase conjugates. The membrane was then washed six times, 5 min each, in phosphate-buffered saline, 0.1% Tween 20. Antigen-antibody complexes were detected using a chemiluminescence system(24) .

Materials

All enzymes and chemicals used were of the highest quality available and were obtained from commercial sources. Oligonucleotides were synthesized by Operon Technologies Inc. (Alameda, CA).

RESULTS

Purification and Sequence Analysis of Peptides of GR from Anabaena PCC 7120

Anabaena PCC 7120 cells (30 g wet weight) N(2)-grown, were used for purification of GR. The enzyme was purified 6000-fold to electrophoretic homogeneity by a five-step procedure, involving ammonium sulfate fractionation, chromatography on DEAE-Sepharose CL-6B, Red Sepharose CL-6B, chromatofocusing, and gel filtration(7) . The purified enzyme exhibited a specific activity of 213 units/mg, and its homogeneity was checked by electrophoresis under denaturing conditions. Fig. 2shows a single protein band with an apparent subunit molecular mass of about 50 kDa after the last purification step. Unexpectedly, the enzyme failed to bind to 2`,5`-ADP Sepharose, commonly used for affinity chromatography purification of the enzyme from other organisms, such as human erythocytes(27) , E. coli(18) , and pea(28) .

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.

Construction of a Nucleotide Probe for the gor Gene and Southern Blot Analysis

Three degenerate oligonucleotide primers synthesized on the basis of the amino acid sequences of three internal peptides are shown in Fig. 1and Table 1. Several different DNA fragments were produced by PCR amplification using primers P(1) and P(2). Therefore, primer P(3) was used internally for reamplification by nested PCR. By using primers P(1) and P(3), a major PCR fragment of 254 bp (fragment A) was isolated. The deduced amino acid sequence was highly similar to other known GR sequences. Hence, an RNA probe generated from fragment A was used to analyze Anabaena PCC 7120 genomic DNA. The probe hybridized to a single band in all restriction digests (Fig. 3), indicating that the gor gene exists as a single copy gene in the Anabaena PCC 7120 genome. A similar result was obtained from low-stringency hybridization. The weak band (6 kb) in the high M(r) region seen using a DraI+HindIII digestion (Fig. 3) may be due to the incomplete digestion by DraI. The nucleotide sequence of fragment A also provided the information needed for synthesis of the specific primers used to isolate the entire 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(r) standards are indicated on the left. The sizes of fragments hybridizing with the gor probe are given on the right.


Isolation and Sequence Analysis of the gor Gene

Southern blot analysis of Anabaena PCC 7120 genomic DNA showed that the gor gene was contained within a 3-kb HindIII fragment. HindIII adapters were added to size-fractionated 3-kb HindIII fragments. Hence, fragments B and C were isolated following the PCR amplification procedures described under ``Experimental Procedures.'' The PCR fragments and subfragments generated from the restriction enzyme digestion were cloned and sequenced. pUC/M13 forward primer (5`-CGCCAGGGTTTTCCCAGTCACGAC-3`) and reverse primer (5`-AACAGCTATGAC-3`) as well as additional synthesized oligonucleotide primers (Table 1) were used for sequencing. A detailed restriction endonuclease site map and the sequencing strategy used are depicted in Fig. 1. Sequencing of DNA in this region revealed an open reading frame of 458 codons with a putative ribosome-binding site 4 bp upstream of the ATG start codon (Fig. 4). The codon usage is typical of Anabaena PCC 7120 coding sequences(29) , e.g. codons which are low in G+C content in the third position are favored (Table 2). In addition, 360 bp of the 5`- and 430 bp of the 3`-flanking sequences were determined.

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^1) 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) .


Origins of gor Transcription

The existence of three putative E. coli-type promoters and one putative BifA/NtcA-binding site upstream of the coding sequence indicated a potential differential regulatory mechanism at the transcriptional level. In order to determine whether gor transcription was initiated from more than one promoter, the 5` initiation sites of the gor transcripts were mapped by primer extension of RNA obtained from Anabaena PCC 7120 grown on different nitrogen sources. Two sizes of RNA were observed after primer extension by use of the synthetic oligonucleotide primer P, covering positions +22 to +39. A shorter transcript (RNA(I)) started at -23 nucleotides upstream of the translation initiation site and apparently derived from the proximal promoter. This transcript was detected under all three culture conditions used. A longer transcript (RNA) originated from the middle promoter at -36. However, this latter transcript was observed only in ammonium grown cultures (Fig. 7). The extension product of RNA was about 4-fold less abundant than that of RNA(I), indicating that the promoter for RNA is used to a smaller extent.

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(2)-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.


GR Expression under Various Nitrogen Regimes

The GR expression construct was transfected into the GR-deficient E. coli strain SG5, and clones with high specific GR activity were selected. The recombinant protein synthesized in E. coli was purified and used for immunization of rabbits. Western blot analysis (Fig. 9) showed that the rabbit-anti GR antibody specifically recognized a polypeptide of about 50 kDa in crude extracts of Anabaena PCC 7120 and that the GR protein level was higher in ammonium-grown cultures than in cultures grown on the other nitrogen sources (NO(3), N(2)). This result is consistent with our observation that cellular GR activity in-creases when Anabaena PCC 7120 is transferred from nitrate to ammonium medium (data not shown).

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(2)), or in media containing nitrate (NO(3)) or ammonium (NH(4)), 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.


DISCUSSION

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(3) and N(2), 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.

FOOTNOTES

*
This work was supported by a grant from the Swedish Council for Forestry and Agriculture Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X89712[GenBank].

§
To whom correspondence should be addressed. Tel.: 46-18-174539; Fax: 46-18-558431.

(^1)
The abbreviations used are: GR, glutathione reductase; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s).

ACKNOWLEDGEMENTS

We thank Professor Richard N. Perham, Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom for generously providing E. coli strain SG5.

REFERENCES

  1. Williams, C. H., Jr. (1976) in The Enzymes (Boyer, P. D., ed) 3rd Ed., Vol. XIII, pp. 89-173, Academic Press, New York
  2. Meister, A., and Anderson, M. E. (1983) Annu. Rev. Biochem. 52,711-760 [CrossRef][Medline] [Order article via Infotrieve]
  3. Alscher, R. G. (1989) Physiol. Plant. 77,457-464 [CrossRef]
  4. Foyer, C. H., and Halliwell, B. (1976) Planta 133,21-25 [CrossRef]
  5. Bielawski, W., and Joy, K. W. (1986) Photochem. 25,2261-2265 [CrossRef]
  6. Dalton, D. A., Langeberg, L., and Treneman, N. C. (1993) Physiol. Plant. 87,365-370 [CrossRef]
  7. Serrano, A., Rivas, J., and Losada, M. (1984) J. Bacteriol. 158,317-324 [Abstract/Free Full Text]
  8. Rendón, J. L., Calcagno, M., Mendoza-Hernández, G., and Ondarza, R. N. (1986) Arch. Biochem. Biophys. 248,215-223 [CrossRef][Medline] [Order article via Infotrieve]
  9. Kalt-Torres, W., Burke, J. J., and Anderson, J. M. (1984) Physiol. Plant. 61,271-278 [CrossRef]
  10. Dilek-Tözüm, S. R., and Gallon, J. R. (1979) J. Gen. Microbiol. 111,313-326
  11. Schirmer, R. H., Krauth-Siegel, R. L., and Schulz, G. E. (1989) in Coenzymes and Cofactors (Dolphin, D., Poulson, R., and Avramovic, O. eds) Vol. III A, pp. 553-596, Wiley, New York
  12. Karplus, P. A., and Schulz, G. E. (1987) J. Mol. Biol. 195,701-729 [CrossRef][Medline] [Order article via Infotrieve]
  13. Mittl, P. R. E., and Schulz, G. E. (1994) Protein Sci. 3,799-809 [Medline] [Order article via Infotrieve]
  14. Greer, S., and Perham, R. N. (1986) Biochemistry 25,2736-2742 [CrossRef][Medline] [Order article via Infotrieve]
  15. Perry, A. C. F., Ni Bhriain, N., Brown, N. L., and Rouch, D. A. (1991) Mol. Microbiol. 5,163-171 [CrossRef][Medline] [Order article via Infotrieve]
  16. Creissen, G., Edwards, E. A., Enard, C., Wellburn, A., and Mullineaux, P. (1991) Plant J. 2,129-131 [CrossRef]
  17. Kubo, A., Sano, T., Saji, H., Tanaka, K., Kondo, N., and Tanaka, K. (1993) Plant Cell Physiol. 34,1259-1266 [Abstract/Free Full Text]
  18. Tutic, M., Lu, X., Schirmer, R. H., and Werner, D. (1990) Eur. J. Biochem. 188,523-528 [Medline] [Order article via Infotrieve]
  19. Christman, M. F., Storz, G., and Ames, B. N. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,3484-3488 [Abstract/Free Full Text]
  20. Storz, G., Tartaglia, L. A., and Ames, B. N. (1990) Science 248,189-194 [Abstract/Free Full Text]
  21. Golden, J. W., Whorff, L. L., and Wiest, D. R. (1991) J. Bacteriol. 173,7098-7105 [Abstract/Free Full Text]
  22. Carlberg, I., and Mannervik, B. (1975) J. Biol. Chem. 250,5475-5480 [Abstract/Free Full Text]
  23. Dzelzkalns, V. A., Szekeres, M., and Mulligan, B. J. (1988) in Plant Molecular Biology, A Practical Approach (Shawn, C. H., ed) pp. 277-299, IRL Press, Oxford
  24. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  25. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Nucleic Acids Res. 12,7035-7056 [Abstract/Free Full Text]
  26. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5463-5467 [Abstract/Free Full Text]
  27. Mannervik, B., Jacobsson, K., and Boggaram, V. (1976) FEBS Lett. 66,221-224 [CrossRef][Medline] [Order article via Infotrieve]
  28. Connell, J. P., and Mullet, J. E. (1986) Plant Physiol. 82,351-356 [Abstract/Free Full Text]
  29. Herdman, M., Janvier, M., Waterbury, J. B., Rippka, R., Stanier, R. Y., and Mandel, M. (1979) J. Gen. Microbiol. 111,63-71
  30. Ramasubramanian, T. S., Wei, T. F., and Golden, J. W. (1994) J. Bacteriol. 176,1214-1223 [Abstract/Free Full Text]
  31. Wei, T. F., Ramasubramanian, T. S., Pu, F., and Golden, J. W. (1993) J. Bacteriol. 175,4025-4035 [Abstract/Free Full Text]
  32. Vega-Palas, M. A., Flores, E., and Herrero, A. (1992) Mol. Microbiol. 6,1853-1859 [CrossRef][Medline] [Order article via Infotrieve]
  33. de Crombrugghe, B., Busby, S., and Buc, H. (1984) Science 224,831-838 [Abstract/Free Full Text]
  34. Andersson, S. G. E., and Kurland, C. G. (1990) Microbiol. Rev. 54,198-210 [Abstract/Free Full Text]
  35. Douglas, S. E. (1994) in The Molecular Biology of Cyanobacteria ( Bryant, D. A., ed) pp. 91-118, Kluwer Academic Publishers, Dordrecht
  36. Giovannoni, S. J., Turner, S., Olsen, G. J., Barns, S., Lane, D. J., and Pace, N. R. (1988) J. Bacteriol. 170,3584-3592 [Abstract/Free Full Text]
  37. Scrutton, N. S., Berry, A., and Perham, R. N. (1990) Nature 343,38-43 [CrossRef][Medline] [Order article via Infotrieve]
  38. Mittl, P. R. E., Berry, A., Scrutton, N. S., Perham, R. N., and Schulz, G. E. (1993) J. Mol. Biol. 231,191-195 [CrossRef][Medline] [Order article via Infotrieve]
  39. Tang, X. Y., and Webb, M. A. (1994) Plant Physiol. 104,1081-1082 [CrossRef][Medline] [Order article via Infotrieve]
  40. Hausladen, A., and Alscher, R. G. (1994) Plant Physiol. 105,205-213 [Abstract]
  41. Madamanchi, N. R., Anderson, J. V., Alscher, R. G., Cramer, C. L., and Hess, J. L. (1992) Plant Physiol. 100,138-145 [Abstract/Free Full Text]
  42. Boussiba, S., and Gibson, J. (1991) FEMS Microbiol. Rev. 88,1-14 [CrossRef]
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
Z. Su, F. Mao, P. Dam, H. Wu, V. Olman, I. T. Paulsen, B. Palenik, and Y. Xu
Computational inference and experimental validation of the nitrogen assimilation regulatory network in cyanobacterium Synechococcus sp. WH 8102
Nucleic Acids Res., February 10, 2006; 34(3): 1050 - 1065.
[Abstract] [Full Text] [PDF]

Home page
 Plant Cell PhysiolHome page
H. Ashida, Y. Sawa, and H. Shibata
Cloning, Biochemical and Phylogenetic Characterizations of {gamma}-Glutamylcysteine Synthetase from Anabaena sp. PCC 7120
Plant Cell Physiol., April 1, 2005; 46(4): 557 - 562.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
A. Herrero, A. M. Muro-Pastor, and E. Flores
Nitrogen Control in Cyanobacteria
J. Bacteriol., January 15, 2001; 183(2): 411 - 425.
[Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, F.
Right arrow Articles by Mannervik, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, F.
Right arrow Articles by Mannervik, B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement