Plasmid shuffling manipulation of essential genes in Synechocystis PCC6803: mutational analysis of the plant-like ferredoxin
Identifieur interne : 005F40 ( PascalFrancis/Checkpoint ); précédent : 005F39; suivant : 005F41Plasmid shuffling manipulation of essential genes in Synechocystis PCC6803: mutational analysis of the plant-like ferredoxin
Auteurs : M. Poncelet [France] ; C. Cassier-Chauvat [France] ; X. Leschelle [France] ; H. Bottin [France] ; F. Chauvat [France]Source :
- Bulletin de l'Institut océanographique : (Monaco) [ 0304-5722 ] ; 1999.
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- Pascal (Inist)
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Abstract
The genes encoding (2Fe-2S) plant-like ferredoxins were studied in the widely-used cyanobacterium Synechocystis PCC6803. The fedl gene (ssl0020) coding for the most abundant ferredoxin product was found to be strongly expressed as a light-induced monocistronic transcript, whereas the other fed genes appeared to be silent (slr1828), or moderately expressed as polycistronic transcripts regulated by either light fluence (slr0150), negative control) or glucose availability (sll1382). fedI was found to be critical to Synechocystis PCC6803 viability in spite of slr0150-, sll1382- or flavodoxin-induction, even after the addition of glucose that compensate for the loss of photosynthesis. Nevertheless, fedI could be deleted from all chromosome copies in cells propagating a fedI gene (even of heterologous origin) on a replicating plasmid. This strain was used as the host for the subsequent introduction of fedI mutant alleles propagated on a second vector. Analysis of the fedI mutant strains generated after plasmid exchange, showed that the C18-C85 disulfide bridge is central neither to the tight compaction of ferredoxin I nor to its reduction by photosystem I, and demonstrate that the length of the FedI carboxy-terminus is important for effective PSI/FedI interactions. The plasmid shuffling strategy presently described has general applicability for mutational analysis of essential genes in many organisms since it is based on promiscuous plasmids.
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Chauvat</name><affiliation wicri:level="3"><inist:fA14 i1="01"><s1>ISBGM bat. 142,CEA Saclay</s1><s2>91191 Gif-sur-Yvette</s2><s3>FRA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>France</country><placeName><region type="region" nuts="2">Île-de-France</region><settlement type="city">Gif-sur-Yvette</settlement></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">INIST</idno><idno type="inist">00-0051909</idno><date when="1999">1999</date><idno type="stanalyst">PASCAL 00-0051909 INIST</idno><idno type="RBID">Pascal:00-0051909</idno><idno type="wicri:Area/PascalFrancis/Corpus">006130</idno><idno type="wicri:Area/PascalFrancis/Curation">000040</idno><idno type="wicri:Area/PascalFrancis/Checkpoint">005F40</idno><idno type="wicri:explorRef" wicri:stream="PascalFrancis" wicri:step="Checkpoint">005F40</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a">Plasmid shuffling manipulation of essential genes in Synechocystis PCC6803: mutational analysis of the plant-like ferredoxin</title><author><name sortKey="Poncelet, M" sort="Poncelet, M" uniqKey="Poncelet M" first="M." last="Poncelet">M. Poncelet</name><affiliation wicri:level="3"><inist:fA14 i1="01"><s1>ISBGM bat. 142,CEA Saclay</s1><s2>91191 Gif-sur-Yvette</s2><s3>FRA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>France</country><placeName><region type="region" nuts="2">Île-de-France</region><settlement type="city">Gif-sur-Yvette</settlement></placeName></affiliation></author><author><name sortKey="Cassier Chauvat, C" sort="Cassier Chauvat, C" uniqKey="Cassier Chauvat C" first="C." last="Cassier-Chauvat">C. Cassier-Chauvat</name><affiliation wicri:level="3"><inist:fA14 i1="01"><s1>ISBGM bat. 142,CEA Saclay</s1><s2>91191 Gif-sur-Yvette</s2><s3>FRA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>France</country><placeName><region type="region" nuts="2">Île-de-France</region><settlement type="city">Gif-sur-Yvette</settlement></placeName></affiliation><affiliation wicri:level="3"><inist:fA14 i1="02"><s1>URA 2096 CNRS, DBCM/DSV, CEA Saclay</s1><s2>91191 Gif-sur-Yvette</s2><s3>FRA</s3><sZ>2 aut.</sZ><sZ>4 aut.</sZ></inist:fA14><country>France</country><placeName><region type="region" nuts="2">Île-de-France</region><settlement type="city">Gif-sur-Yvette</settlement></placeName></affiliation></author><author><name sortKey="Leschelle, X" sort="Leschelle, X" uniqKey="Leschelle X" first="X." last="Leschelle">X. Leschelle</name><affiliation wicri:level="3"><inist:fA14 i1="01"><s1>ISBGM bat. 142,CEA Saclay</s1><s2>91191 Gif-sur-Yvette</s2><s3>FRA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>France</country><placeName><region type="region" nuts="2">Île-de-France</region><settlement type="city">Gif-sur-Yvette</settlement></placeName></affiliation></author><author><name sortKey="Bottin, H" sort="Bottin, H" uniqKey="Bottin H" first="H." last="Bottin">H. Bottin</name><affiliation wicri:level="3"><inist:fA14 i1="01"><s1>ISBGM bat. 142,CEA Saclay</s1><s2>91191 Gif-sur-Yvette</s2><s3>FRA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>France</country><placeName><region type="region" nuts="2">Île-de-France</region><settlement type="city">Gif-sur-Yvette</settlement></placeName></affiliation><affiliation wicri:level="3"><inist:fA14 i1="02"><s1>URA 2096 CNRS, DBCM/DSV, CEA Saclay</s1><s2>91191 Gif-sur-Yvette</s2><s3>FRA</s3><sZ>2 aut.</sZ><sZ>4 aut.</sZ></inist:fA14><country>France</country><placeName><region type="region" nuts="2">Île-de-France</region><settlement type="city">Gif-sur-Yvette</settlement></placeName></affiliation></author><author><name sortKey="Chauvat, F" sort="Chauvat, F" uniqKey="Chauvat F" first="F." last="Chauvat">F. Chauvat</name><affiliation wicri:level="3"><inist:fA14 i1="01"><s1>ISBGM bat. 142,CEA Saclay</s1><s2>91191 Gif-sur-Yvette</s2><s3>FRA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>France</country><placeName><region type="region" nuts="2">Île-de-France</region><settlement type="city">Gif-sur-Yvette</settlement></placeName></affiliation></author></analytic><series><title level="j" type="main">Bulletin de l'Institut océanographique : (Monaco)</title><title level="j" type="abbreviated">Bull. Inst. océanogr. : (Monaco)</title><idno type="ISSN">0304-5722</idno><imprint><date when="1999">1999</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Bulletin de l'Institut océanographique : (Monaco)</title><title level="j" type="abbreviated">Bull. Inst. océanogr. : (Monaco)</title><idno type="ISSN">0304-5722</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Ferredoxin</term><term>Gene expression</term><term>Photosynthesis</term><term>Plasmid</term><term>Regulation(control)</term><term>Site directed mutagenesis</term><term>Synechocystis</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Synechocystis</term><term>Photosynthèse</term><term>Ferrédoxine</term><term>Expression génique</term><term>Régulation</term><term>Mutagenèse dirigée</term><term>Plasmide</term><term>Gène fedI</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The genes encoding (2Fe-2S) plant-like ferredoxins were studied in the widely-used cyanobacterium Synechocystis PCC6803. The fedl gene (ssl0020) coding for the most abundant ferredoxin product was found to be strongly expressed as a light-induced monocistronic transcript, whereas the other fed genes appeared to be silent (slr1828), or moderately expressed as polycistronic transcripts regulated by either light fluence (slr0150), negative control) or glucose availability (sll1382). fedI was found to be critical to Synechocystis PCC6803 viability in spite of slr0150-, sll1382- or flavodoxin-induction, even after the addition of glucose that compensate for the loss of photosynthesis. Nevertheless, fedI could be deleted from all chromosome copies in cells propagating a fedI gene (even of heterologous origin) on a replicating plasmid. This strain was used as the host for the subsequent introduction of fedI mutant alleles propagated on a second vector. Analysis of the fedI mutant strains generated after plasmid exchange, showed that the C18-C85 disulfide bridge is central neither to the tight compaction of ferredoxin I nor to its reduction by photosystem I, and demonstrate that the length of the FedI carboxy-terminus is important for effective PSI/FedI interactions. The plasmid shuffling strategy presently described has general applicability for mutational analysis of essential genes in many organisms since it is based on promiscuous plasmids.</div></front></TEI><inist><standard h6="B"><pA><fA01 i1="01" i2="1"><s0>0304-5722</s0></fA01><fA02 i1="01"><s0>BULCAA</s0></fA02><fA03 i2="1"><s0>Bull. 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All rights reserved.</s1></fA44><fA45><s0>1 p.1/4</s0></fA45><fA47 i1="01" i2="1"><s0>00-0051909</s0></fA47><fA60><s1>P</s1></fA60><fA61><s0>A</s0></fA61><fA64 i1="01" i2="1"><s0>Bulletin de l'Institut océanographique : (Monaco)</s0></fA64><fA66 i1="01"><s0>MCO</s0></fA66><fC01 i1="01" l="ENG"><s0>The genes encoding (2Fe-2S) plant-like ferredoxins were studied in the widely-used cyanobacterium Synechocystis PCC6803. The fedl gene (ssl0020) coding for the most abundant ferredoxin product was found to be strongly expressed as a light-induced monocistronic transcript, whereas the other fed genes appeared to be silent (slr1828), or moderately expressed as polycistronic transcripts regulated by either light fluence (slr0150), negative control) or glucose availability (sll1382). fedI was found to be critical to Synechocystis PCC6803 viability in spite of slr0150-, sll1382- or flavodoxin-induction, even after the addition of glucose that compensate for the loss of photosynthesis. Nevertheless, fedI could be deleted from all chromosome copies in cells propagating a fedI gene (even of heterologous origin) on a replicating plasmid. This strain was used as the host for the subsequent introduction of fedI mutant alleles propagated on a second vector. Analysis of the fedI mutant strains generated after plasmid exchange, showed that the C18-C85 disulfide bridge is central neither to the tight compaction of ferredoxin I nor to its reduction by photosystem I, and demonstrate that the length of the FedI carboxy-terminus is important for effective PSI/FedI interactions. 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Poncelet</name></region><name sortKey="Bottin, H" sort="Bottin, H" uniqKey="Bottin H" first="H." last="Bottin">H. Bottin</name><name sortKey="Bottin, H" sort="Bottin, H" uniqKey="Bottin H" first="H." last="Bottin">H. Bottin</name><name sortKey="Cassier Chauvat, C" sort="Cassier Chauvat, C" uniqKey="Cassier Chauvat C" first="C." last="Cassier-Chauvat">C. Cassier-Chauvat</name><name sortKey="Cassier Chauvat, C" sort="Cassier Chauvat, C" uniqKey="Cassier Chauvat C" first="C." last="Cassier-Chauvat">C. Cassier-Chauvat</name><name sortKey="Chauvat, F" sort="Chauvat, F" uniqKey="Chauvat F" first="F." last="Chauvat">F. Chauvat</name><name sortKey="Leschelle, X" sort="Leschelle, X" uniqKey="Leschelle X" first="X." last="Leschelle">X. Leschelle</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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