Stable transformation of the Xylella fastidiosa citrus variegated chlorosis strain with oriC plasmids.
Identifieur interne : 000976 ( Main/Corpus ); précédent : 000975; suivant : 000977Stable transformation of the Xylella fastidiosa citrus variegated chlorosis strain with oriC plasmids.
Auteurs : P B Monteiro ; D C Teixeira ; R R Palma ; M. Garnier ; J M Bové ; J. RenaudinSource :
- Applied and environmental microbiology [ 0099-2240 ] ; 2001.
English descriptors
- KwdEn :
- Base Sequence (MeSH), Blotting, Southern (MeSH), Citrus (microbiology), Coffee (microbiology), DNA-Binding Proteins (genetics), Gammaproteobacteria (genetics), Gammaproteobacteria (metabolism), Molecular Sequence Data (MeSH), Origin Recognition Complex (MeSH), Plant Diseases (microbiology), Plasmids (genetics), Recombination, Genetic (MeSH), Restriction Mapping (MeSH), Transformation, Bacterial (MeSH), Viral Proteins (genetics).
- MESH :
- chemical , genetics : DNA-Binding Proteins, Viral Proteins.
- chemical , microbiology : Coffee.
- genetics : Gammaproteobacteria, Plasmids.
- metabolism : Gammaproteobacteria.
- microbiology : Citrus, Plant Diseases.
- Base Sequence, Blotting, Southern, Molecular Sequence Data, Origin Recognition Complex, Recombination, Genetic, Restriction Mapping, Transformation, Bacterial.
Abstract
Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan(r)). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kan(r) gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.
DOI: 10.1128/AEM.67.5.2263-2269.2001
PubMed: 11319110
PubMed Central: PMC92865
Links to Exploration step
pubmed:11319110Le document en format XML
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Garnier</name></author><author><name sortKey="Bove, J M" sort="Bove, J M" uniqKey="Bove J" first="J M" last="Bové">J M Bové</name></author><author><name sortKey="Renaudin, J" sort="Renaudin, J" uniqKey="Renaudin J" first="J" last="Renaudin">J. Renaudin</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2001">2001</date><idno type="RBID">pubmed:11319110</idno><idno type="pmid">11319110</idno><idno type="doi">10.1128/AEM.67.5.2263-2269.2001</idno><idno type="pmc">PMC92865</idno><idno type="wicri:Area/Main/Corpus">000976</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">000976</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Stable transformation of the Xylella fastidiosa citrus variegated chlorosis strain with oriC plasmids.</title><author><name sortKey="Monteiro, P B" sort="Monteiro, P B" uniqKey="Monteiro P" first="P B" last="Monteiro">P B Monteiro</name><affiliation><nlm:affiliation>Fundo de Defesa da Citricultura (Fundecitrus), 14807-040, VI. Melhado-C.P. 391, Araraquara, São Paulo, Brazil. pbmonteiro@fundecitrus.com.br</nlm:affiliation></affiliation></author><author><name sortKey="Teixeira, D C" sort="Teixeira, D C" uniqKey="Teixeira D" first="D C" last="Teixeira">D C Teixeira</name></author><author><name sortKey="Palma, R R" sort="Palma, R R" uniqKey="Palma R" first="R R" last="Palma">R R Palma</name></author><author><name sortKey="Garnier, M" sort="Garnier, M" uniqKey="Garnier M" first="M" last="Garnier">M. Garnier</name></author><author><name sortKey="Bove, J M" sort="Bove, J M" uniqKey="Bove J" first="J M" last="Bové">J M Bové</name></author><author><name sortKey="Renaudin, J" sort="Renaudin, J" uniqKey="Renaudin J" first="J" last="Renaudin">J. Renaudin</name></author></analytic><series><title level="j">Applied and environmental microbiology</title><idno type="ISSN">0099-2240</idno><imprint><date when="2001" type="published">2001</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Base Sequence (MeSH)</term><term>Blotting, Southern (MeSH)</term><term>Citrus (microbiology)</term><term>Coffee (microbiology)</term><term>DNA-Binding Proteins (genetics)</term><term>Gammaproteobacteria (genetics)</term><term>Gammaproteobacteria (metabolism)</term><term>Molecular Sequence Data (MeSH)</term><term>Origin Recognition Complex (MeSH)</term><term>Plant Diseases (microbiology)</term><term>Plasmids (genetics)</term><term>Recombination, Genetic (MeSH)</term><term>Restriction Mapping (MeSH)</term><term>Transformation, Bacterial (MeSH)</term><term>Viral Proteins (genetics)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA-Binding Proteins</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="microbiology" xml:lang="en"><term>Coffee</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Gammaproteobacteria</term><term>Plasmids</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Gammaproteobacteria</term></keywords><keywords scheme="MESH" qualifier="microbiology" xml:lang="en"><term>Citrus</term><term>Plant Diseases</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Base Sequence</term><term>Blotting, Southern</term><term>Molecular Sequence Data</term><term>Origin Recognition Complex</term><term>Recombination, Genetic</term><term>Restriction Mapping</term><term>Transformation, Bacterial</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan(r)). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kan(r) gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">11319110</PMID><DateCompleted><Year>2001</Year><Month>09</Month><Day>06</Day></DateCompleted><DateRevised><Year>2018</Year><Month>11</Month><Day>13</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0099-2240</ISSN><JournalIssue CitedMedium="Print"><Volume>67</Volume><Issue>5</Issue><PubDate><Year>2001</Year><Month>May</Month></PubDate></JournalIssue><Title>Applied and environmental microbiology</Title><ISOAbbreviation>Appl Environ Microbiol</ISOAbbreviation></Journal><ArticleTitle>Stable transformation of the Xylella fastidiosa citrus variegated chlorosis strain with oriC plasmids.</ArticleTitle><Pagination><MedlinePgn>2263-9</MedlinePgn></Pagination><Abstract><AbstractText>Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan(r)). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kan(r) gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Monteiro</LastName><ForeName>P B</ForeName><Initials>PB</Initials><AffiliationInfo><Affiliation>Fundo de Defesa da Citricultura (Fundecitrus), 14807-040, VI. Melhado-C.P. 391, Araraquara, São Paulo, Brazil. pbmonteiro@fundecitrus.com.br</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Teixeira</LastName><ForeName>D C</ForeName><Initials>DC</Initials></Author><Author ValidYN="Y"><LastName>Palma</LastName><ForeName>R R</ForeName><Initials>RR</Initials></Author><Author ValidYN="Y"><LastName>Garnier</LastName><ForeName>M</ForeName><Initials>M</Initials></Author><Author ValidYN="Y"><LastName>Bové</LastName><ForeName>J M</ForeName><Initials>JM</Initials></Author><Author ValidYN="Y"><LastName>Renaudin</LastName><ForeName>J</ForeName><Initials>J</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Appl Environ Microbiol</MedlineTA><NlmUniqueID>7605801</NlmUniqueID><ISSNLinking>0099-2240</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D003069">Coffee</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D004268">DNA-Binding Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="C114650">OriC chromosomal replication origin</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D051738">Origin Recognition Complex</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D014764">Viral Proteins</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D001483" MajorTopicYN="N">Base Sequence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D015139" MajorTopicYN="N">Blotting, 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Complex</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010935" MajorTopicYN="N">Plant Diseases</DescriptorName><QualifierName UI="Q000382" MajorTopicYN="N">microbiology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010957" MajorTopicYN="N">Plasmids</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011995" MajorTopicYN="N">Recombination, Genetic</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D015183" MajorTopicYN="N">Restriction Mapping</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D014169" MajorTopicYN="Y">Transformation, Bacterial</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D014764" MajorTopicYN="N">Viral Proteins</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2001</Year><Month>4</Month><Day>25</Day><Hour>10</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2001</Year><Month>9</Month><Day>8</Day><Hour>10</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2001</Year><Month>4</Month><Day>25</Day><Hour>10</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">11319110</ArticleId><ArticleId IdType="doi">10.1128/AEM.67.5.2263-2269.2001</ArticleId><ArticleId IdType="pmc">PMC92865</ArticleId></ArticleIdList><ReferenceList><Reference><Citation>Plasmid. 1998;39(3):205-14</Citation><ArticleIdList><ArticleId IdType="pubmed">9571137</ArticleId></ArticleIdList></Reference><Reference><Citation>Plant Cell. 1994 Jul;6(7):927-33</Citation><ArticleIdList><ArticleId IdType="pubmed">8069104</ArticleId></ArticleIdList></Reference><Reference><Citation>J Bacteriol. 1999 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