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Cyanobacterial sulfide-quinone-reductase (SQR): cloning and heterologous expression
Year:
2000
Source of publication :
Journal of Bacteriology
Authors :
שחק, יוספה
;
.
Volume :
182
Co-Authors:

Bronstein, M., Schütz, M., Hauska, G., Padan, E.

Facilitators :
From page:
3336
To page:
3344
(
Total pages:
9
)
Abstract:

The gene encoding sulfide-quinone reductase (SQR; E.C.1.8.5.′), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium Oscillatoria limnetica, was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the Rhodobacter capsulatus SQR and in an open reading frame found in the genome of Aquifex aeolicus. SQR activity was also detected in the unicellular cyanobacterium Aphanothece halophytica following sulfide induction, with a Vmax of 180 μmol of plastoquinone-1 (PQ-1) reduced/mg of chlorophyll/h and apparentKm values of 20 and 40 μM for sulfide and quinone, respectively. Based on the conserved sequences, the gene encoding A. halophytica SQR was also cloned. The SQR polypeptides deduced from the two cyanobacterial genes consist of 436 amino acids for O. limnetica SQR and 437 amino acids forA. halophytica SQR and show 58% identity and 74% similarity. The calculated molecular mass is about 48 kDa for both proteins; the theoretical isoelectric points are 7.7 and 5.6 and the net charges at a neutral pH are 0 and −14 for O. limneticaSQR and A. halophytica SQR, respectively. A search of databases showed SQR homologs in the genomes of the cyanobacteriumAnabaena PCC7120 as well as the chemolithotrophic bacteriaShewanella putrefaciens and Thiobacillus ferrooxidans. All SQR enzymes contain characteristic flavin adenine dinucleotide binding fingerprints. The cyanobacterial proteins were expressed in Escherichia coli under the control of the T7 promoter. Membranes isolated from E. coli cells expressing A. halophytica SQR performed sulfide-dependent PQ-1 reduction that was sensitive to the quinone analog inhibitor 2n-nonyl-4-hydroxyquinoline-N-oxide. The wide distribution of SQR genes emphasizes the important role of SQR in the sulfur cycle in nature.

Of the many organisms performing plant-type oxygenic photosynthesis, only cyanobacteria can facultatively shift to anoxygenic, bacterium-type photosynthesis with sulfide (H2S) as the electron donor in a photosystem I-dependent reaction (31526). This shift, first discovered in the cyanobacterium Oscillatoria limnetica(11), occurs after 2 h of incubation in the presence of sulfide and light and requires protein synthesis (23). The induced cells perform sulfide-dependent CO2 fixation (10111523), H2 evolution (5), or N2 fixation (4), depending on the growth and physiological conditions.

The discovery of anoxygenic photosynthesis in O. limneticaformed the basis for the understanding of an important and unique trait of cyanobacteria. In marked contrast to other plant-type phototrophs, which are sulfide sensitive, these organisms grow and form mats in anaerobic, sulfide-rich niches characteristic of many natural habitats and polluted water (252638). The unique capacity to shift between the two types of photosynthesis has been suggested to represent a primitive relic in the evolution of photosynthesis (25).

Photooxidation of sulfide coupled to CO2 reduction is not unique to cyanobacteria. Sulfide is the most widely used electron donor among photolithotrophic bacteria (7817). The transfer of electrons from sulfide directly into the quinone pool was proposed and supported by the inhibition exerted by quinone analogs as well as energetic considerations (6842).

Tracking of the inducible factor that enables photosynthetic sulfide oxidation in O. limnetica led to the discovery of sulfide-quinone reductase (SQR; E.C.1.8.5.′), a novel enzyme that transfers electrons from sulfide into the quinone pool (36). The SQR was solubilized from membranes of sulfide-induced O. limnetica cells and purified in an active form (2). The isolated active SQR is a hydrophobic membrane enzyme composed of a single polypeptide with an apparent molecular mass of 57 kDa (as determined by sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis [PAGE]). It was shown to have high affinities for sulfide (Km = 8 μM) and quinone (Km = 31 μM for plastoquinone-1 [PQ-1]), to contain a flavin cofactor, and to be sensitive to quinone analogs and KCN (234). The N-terminal sequence was found to contain the characteristic features of an NAD or flavin adenine dinucleotide (FAD) binding domain (244).

SQR recently has been proven to be widely spread among anoxygenic phototrophs. SQR activity has been detected in purple “nonsulfur” bacteria (Rhodobacter capsulatus) (35), purple sulfur bacteria (Allochromatium vinosum) (34), green sulfur bacteria (Chlorobium) (37), and green gliding (“nonsulfur”) bacteria (Chloroflexus aurantiacus) (34); in the nonphotosynthetic chemoautotrophs Paracoccus denitrificans (31),Wolinella succinogenes, and Aquifex aeolicus; and in the mitochondria of the sulfide-tolerating marine wormArenicola marina (34).

The SQR of the purple bacterium R. capsulatus was isolated and purified, its gene was cloned, sequenced, and functionally expressed in Escherichia coli (33) and, recently, it was shown that the enzyme is essential for the sulfide-dependent growth of R. capsulatus (32). The R. capsulatus SQR polypeptide consists of 427 amino acids and has a molecular mass of 47 kDa and a net charge of +1. The amino acid sequence of its N terminus shows high similarity (48% identity and 72% similarity) to the amino acid sequence of the O. limnetica N terminus (2), including the FAD binding fingerprint. The complete protein sequence contains two additional FAD binding motifs (33). The Rhodobacter and the cyanobacterial enzymes are rather divergent. For example, they are expected to differ in their quinone binding sites. The natural quinone acceptor for SQR is most probably ubiquinone in R. capsulatus but plastoquinone in the cyanobacterial system (34).

Recently, a gene encoding a mitochondrial polypeptide that exhibits SQR activity was cloned from the fission yeast Schizosaccharomyces pombe. This enzyme, HMT2 (heavy metal tolerance), was proposed to function in the detoxification of endogenous sulfide (43). HMT2 shares high similarity with sequences of unknown functions from the genomes of nematodes, fruit flies, mice, rats, and humans and only low similarity (∼20%) with R. capsulatus SQR (43). In addition to demonstrating further the distribution of SQR in eukaryotes, this recent publication suggests two possible roles for SQR—utilization and detoxification of sulfide—raising the question of whether one type of enzyme or more types are involved.

We have previously shown three functions in which SQR can be involved in cyanobacteria: (i) anoxygenic photosynthetic growth in O. limnetica (23); (ii) anaerobic respiration in O. limnetica (24); and (iii) detoxification of sulfide inAphanothece halophytica, which survives but does not grow in the presence of sulfide (15). In contrast to O. limnetica, in which the SQR has been purified and biochemically characterized, in A. halophytica the sulfide-interacting enzyme has not yet been identified. In the present work, we describe some biochemical properties of A. halophytica SQR as well as the cloning and expression of the sqr genes of both O. limnetica and A. halophytica.

Note:

Registered genes:

Oscillatoria limnetica sulfide quinone reductase gene, complete cds. Accession:

AF242368. GI “8118248” [GenBank].

https://www.ncbi.nlm.nih.gov/nuccore/8118248

 

Aphanothece halophytica sulfide quinone reductase gene, complete cds.

Accession: AF242368. GI “8118248” [GenBank].

https://www.ncbi.nlm.nih.gov/nuccore/AF242369.1

 

Oscillatoria limnetica unknown gene. Accession: AF242370. GI “8118253”

[GenBank].

https://www.ncbi.nlm.nih.gov/nuccore/8118253

 

Aphanothece halophytica unknown gene. Accession: AF242371. GI “8118255”

[GenBank].

https://www.ncbi.nlm.nih.gov/nuccore/AF242371.1

 

 

 

 

 

Related Files :
bacteria
Cyanobacteria
Genetics
photosynthesis
sulfide-quinone reductase (SQR)
עוד תגיות
תוכן קשור
More details
DOI :
10.1128/JB.182.12.3336-3344.2000
Article number:
0
Affiliations:
Database:
Publication Type:
מאמר
;
.
Language:
אנגלית
Editors' remarks:
ID:
52458
Last updated date:
02/03/2022 17:27
Creation date:
20/12/2020 10:52
Scientific Publication
Cyanobacterial sulfide-quinone-reductase (SQR): cloning and heterologous expression
182

Bronstein, M., Schütz, M., Hauska, G., Padan, E.

Cyanobacterial sulfide-quinone-reductase (SQR): cloning and heterologous expression .

The gene encoding sulfide-quinone reductase (SQR; E.C.1.8.5.′), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium Oscillatoria limnetica, was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the Rhodobacter capsulatus SQR and in an open reading frame found in the genome of Aquifex aeolicus. SQR activity was also detected in the unicellular cyanobacterium Aphanothece halophytica following sulfide induction, with a Vmax of 180 μmol of plastoquinone-1 (PQ-1) reduced/mg of chlorophyll/h and apparentKm values of 20 and 40 μM for sulfide and quinone, respectively. Based on the conserved sequences, the gene encoding A. halophytica SQR was also cloned. The SQR polypeptides deduced from the two cyanobacterial genes consist of 436 amino acids for O. limnetica SQR and 437 amino acids forA. halophytica SQR and show 58% identity and 74% similarity. The calculated molecular mass is about 48 kDa for both proteins; the theoretical isoelectric points are 7.7 and 5.6 and the net charges at a neutral pH are 0 and −14 for O. limneticaSQR and A. halophytica SQR, respectively. A search of databases showed SQR homologs in the genomes of the cyanobacteriumAnabaena PCC7120 as well as the chemolithotrophic bacteriaShewanella putrefaciens and Thiobacillus ferrooxidans. All SQR enzymes contain characteristic flavin adenine dinucleotide binding fingerprints. The cyanobacterial proteins were expressed in Escherichia coli under the control of the T7 promoter. Membranes isolated from E. coli cells expressing A. halophytica SQR performed sulfide-dependent PQ-1 reduction that was sensitive to the quinone analog inhibitor 2n-nonyl-4-hydroxyquinoline-N-oxide. The wide distribution of SQR genes emphasizes the important role of SQR in the sulfur cycle in nature.

Of the many organisms performing plant-type oxygenic photosynthesis, only cyanobacteria can facultatively shift to anoxygenic, bacterium-type photosynthesis with sulfide (H2S) as the electron donor in a photosystem I-dependent reaction (31526). This shift, first discovered in the cyanobacterium Oscillatoria limnetica(11), occurs after 2 h of incubation in the presence of sulfide and light and requires protein synthesis (23). The induced cells perform sulfide-dependent CO2 fixation (10111523), H2 evolution (5), or N2 fixation (4), depending on the growth and physiological conditions.

The discovery of anoxygenic photosynthesis in O. limneticaformed the basis for the understanding of an important and unique trait of cyanobacteria. In marked contrast to other plant-type phototrophs, which are sulfide sensitive, these organisms grow and form mats in anaerobic, sulfide-rich niches characteristic of many natural habitats and polluted water (252638). The unique capacity to shift between the two types of photosynthesis has been suggested to represent a primitive relic in the evolution of photosynthesis (25).

Photooxidation of sulfide coupled to CO2 reduction is not unique to cyanobacteria. Sulfide is the most widely used electron donor among photolithotrophic bacteria (7817). The transfer of electrons from sulfide directly into the quinone pool was proposed and supported by the inhibition exerted by quinone analogs as well as energetic considerations (6842).

Tracking of the inducible factor that enables photosynthetic sulfide oxidation in O. limnetica led to the discovery of sulfide-quinone reductase (SQR; E.C.1.8.5.′), a novel enzyme that transfers electrons from sulfide into the quinone pool (36). The SQR was solubilized from membranes of sulfide-induced O. limnetica cells and purified in an active form (2). The isolated active SQR is a hydrophobic membrane enzyme composed of a single polypeptide with an apparent molecular mass of 57 kDa (as determined by sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis [PAGE]). It was shown to have high affinities for sulfide (Km = 8 μM) and quinone (Km = 31 μM for plastoquinone-1 [PQ-1]), to contain a flavin cofactor, and to be sensitive to quinone analogs and KCN (234). The N-terminal sequence was found to contain the characteristic features of an NAD or flavin adenine dinucleotide (FAD) binding domain (244).

SQR recently has been proven to be widely spread among anoxygenic phototrophs. SQR activity has been detected in purple “nonsulfur” bacteria (Rhodobacter capsulatus) (35), purple sulfur bacteria (Allochromatium vinosum) (34), green sulfur bacteria (Chlorobium) (37), and green gliding (“nonsulfur”) bacteria (Chloroflexus aurantiacus) (34); in the nonphotosynthetic chemoautotrophs Paracoccus denitrificans (31),Wolinella succinogenes, and Aquifex aeolicus; and in the mitochondria of the sulfide-tolerating marine wormArenicola marina (34).

The SQR of the purple bacterium R. capsulatus was isolated and purified, its gene was cloned, sequenced, and functionally expressed in Escherichia coli (33) and, recently, it was shown that the enzyme is essential for the sulfide-dependent growth of R. capsulatus (32). The R. capsulatus SQR polypeptide consists of 427 amino acids and has a molecular mass of 47 kDa and a net charge of +1. The amino acid sequence of its N terminus shows high similarity (48% identity and 72% similarity) to the amino acid sequence of the O. limnetica N terminus (2), including the FAD binding fingerprint. The complete protein sequence contains two additional FAD binding motifs (33). The Rhodobacter and the cyanobacterial enzymes are rather divergent. For example, they are expected to differ in their quinone binding sites. The natural quinone acceptor for SQR is most probably ubiquinone in R. capsulatus but plastoquinone in the cyanobacterial system (34).

Recently, a gene encoding a mitochondrial polypeptide that exhibits SQR activity was cloned from the fission yeast Schizosaccharomyces pombe. This enzyme, HMT2 (heavy metal tolerance), was proposed to function in the detoxification of endogenous sulfide (43). HMT2 shares high similarity with sequences of unknown functions from the genomes of nematodes, fruit flies, mice, rats, and humans and only low similarity (∼20%) with R. capsulatus SQR (43). In addition to demonstrating further the distribution of SQR in eukaryotes, this recent publication suggests two possible roles for SQR—utilization and detoxification of sulfide—raising the question of whether one type of enzyme or more types are involved.

We have previously shown three functions in which SQR can be involved in cyanobacteria: (i) anoxygenic photosynthetic growth in O. limnetica (23); (ii) anaerobic respiration in O. limnetica (24); and (iii) detoxification of sulfide inAphanothece halophytica, which survives but does not grow in the presence of sulfide (15). In contrast to O. limnetica, in which the SQR has been purified and biochemically characterized, in A. halophytica the sulfide-interacting enzyme has not yet been identified. In the present work, we describe some biochemical properties of A. halophytica SQR as well as the cloning and expression of the sqr genes of both O. limnetica and A. halophytica.

Registered genes:

Oscillatoria limnetica sulfide quinone reductase gene, complete cds. Accession:

AF242368. GI “8118248” [GenBank].

https://www.ncbi.nlm.nih.gov/nuccore/8118248

 

Aphanothece halophytica sulfide quinone reductase gene, complete cds.

Accession: AF242368. GI “8118248” [GenBank].

https://www.ncbi.nlm.nih.gov/nuccore/AF242369.1

 

Oscillatoria limnetica unknown gene. Accession: AF242370. GI “8118253”

[GenBank].

https://www.ncbi.nlm.nih.gov/nuccore/8118253

 

Aphanothece halophytica unknown gene. Accession: AF242371. GI “8118255”

[GenBank].

https://www.ncbi.nlm.nih.gov/nuccore/AF242371.1

 

 

 

 

 

Scientific Publication
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