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Plasmid-Encoded RepA Proteins Specifically Autorepress Individual repABC Operons in the Multipartite Rhizobium leguminosarum bv. trifolii Genome.

Żebracki K, Koper P, Marczak M, Skorupska A, Mazur A - PLoS ONE (2015)

Bottom Line: In the repABC replicons, partitioning and replication functions are transcriptionally linked resulting in complex regulation of rep gene expression.The RepA proteins were able to dimerize/oligomerize: in general dimers formed independently of ATP or ADP, although ATP diminished the concentration of oligomers that were produced.By the comprehensive approach focusing on a set of plasmids instead of individual replicons, the work highlighted subtle differences between the organization and regulation of particular rep operons as well as the structures and specificity of RepA proteins, which contribute to the fine-tuned coexistence of several replicons with similar repABC cassettes in the complex bacterial genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, Institute of Microbiology and Biotechnology, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Lublin, Poland.

ABSTRACT
Rhizobia commonly have very complex genomes with a chromosome and several large plasmids that possess genes belonging to the repABC family. RepA and RepB are members of the ParA and ParB families of partitioning proteins, respectively, whereas RepC is crucial for plasmid replication. In the repABC replicons, partitioning and replication functions are transcriptionally linked resulting in complex regulation of rep gene expression. The genome of R. leguminosarum bv. trifolii TA1 (RtTA1) consists of a chromosome and four plasmids (pRleTA1a-d), equipped with functional repABC genes. In this work, the regulation of transcription of the individual repABC cassettes of the four RtTA1 plasmids was studied. The involvement of the RepA and RepB as well as parS-like centromere sites in this process was depicted, demonstrating some dissimilarity in expression of respective rep regions. RtTA1 repABC genes of individual plasmids formed operons, which were negatively regulated by RepA and RepB. Individual RepA were able to bind to DNA without added nucleotides, but in the presence of ADP, bound specifically to their own operator sequences containing imperfect palindromes, and caused operon autorepression, whereas the addition of ATP stimulated non-specific binding of RepA to DNA. The RepA proteins were able to dimerize/oligomerize: in general dimers formed independently of ATP or ADP, although ATP diminished the concentration of oligomers that were produced. By the comprehensive approach focusing on a set of plasmids instead of individual replicons, the work highlighted subtle differences between the organization and regulation of particular rep operons as well as the structures and specificity of RepA proteins, which contribute to the fine-tuned coexistence of several replicons with similar repABC cassettes in the complex bacterial genome.

No MeSH data available.


Related in: MedlinePlus

Transcriptional activity of repABC operons of Rhizobium leguminosarum bv. trifolii TA1 plasmids: (A) pRleTA1a, (B) pRleTA1b, (C) pRleTA1c, and (D) pRleTA1d.Left panels show schematic genetic organization of the repABC cassettes. White arrows represent repA, repB and repC genes, and white broken arrows depict repA genes. Black dots show the position of parS-sites. The identified promoters of repABC operons were marked as grey rectangular boxes. The respective DNA fragments necessary for particular promoter identification, operon structure assignment, as well as their sequential deletions used for operon regulation studies, which were cloned into pMPK reporter and pBBR1MCS-5 vectors, were shown as black lines with positions indicated relative to repA start codon. The following system was applied for pMPK- and pBBR-1MCS5-based constructs nomenclature: e.g. in pMa/A2 recombinant plasmid the first two letters (pM) mean the shortcut of vector name (pMPK in this case), the next small letter 'a' means that it is derivative of pRleTA1a repABC cassette, the capital letter A followed by slash means that the cloned fragment comprise entire repA gene (in the case of pMa-d/ABC2 constructs the cloned fragments encompass entire repA and repB genes and fragment of repC fused with lacZ). Respectively, pBa/A2 means the same fragment recloned to the pBBR-1MCS5. Right panels represent β-galactosidase activities of respective lacZ transcriptional fusions measured in A. tumefaciens and expressed in Miller units. Each value (with standard deviation—extended bars) is the average of at least three independent measurements.
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pone.0131907.g001: Transcriptional activity of repABC operons of Rhizobium leguminosarum bv. trifolii TA1 plasmids: (A) pRleTA1a, (B) pRleTA1b, (C) pRleTA1c, and (D) pRleTA1d.Left panels show schematic genetic organization of the repABC cassettes. White arrows represent repA, repB and repC genes, and white broken arrows depict repA genes. Black dots show the position of parS-sites. The identified promoters of repABC operons were marked as grey rectangular boxes. The respective DNA fragments necessary for particular promoter identification, operon structure assignment, as well as their sequential deletions used for operon regulation studies, which were cloned into pMPK reporter and pBBR1MCS-5 vectors, were shown as black lines with positions indicated relative to repA start codon. The following system was applied for pMPK- and pBBR-1MCS5-based constructs nomenclature: e.g. in pMa/A2 recombinant plasmid the first two letters (pM) mean the shortcut of vector name (pMPK in this case), the next small letter 'a' means that it is derivative of pRleTA1a repABC cassette, the capital letter A followed by slash means that the cloned fragment comprise entire repA gene (in the case of pMa-d/ABC2 constructs the cloned fragments encompass entire repA and repB genes and fragment of repC fused with lacZ). Respectively, pBa/A2 means the same fragment recloned to the pBBR-1MCS5. Right panels represent β-galactosidase activities of respective lacZ transcriptional fusions measured in A. tumefaciens and expressed in Miller units. Each value (with standard deviation—extended bars) is the average of at least three independent measurements.

Mentions: In silico promoter prediction suggested operon organization of all RtTA1 repABC regions, with the putative promoter located upstream of the repA genes [31]. Moreover, in the putative promoter regions of all RtTA1 rep cassettes, operator-like palindromes were identified suggesting complex regulation of repABC expression. To study the transcription of the repABC genes of individual RtTA1 plasmids and regulation of the putative repABC operons with special attention to the contribution of RepA in this process, series of rep-lacZ transcriptional fusions were constructed and analysed (Fig 1).


Plasmid-Encoded RepA Proteins Specifically Autorepress Individual repABC Operons in the Multipartite Rhizobium leguminosarum bv. trifolii Genome.

Żebracki K, Koper P, Marczak M, Skorupska A, Mazur A - PLoS ONE (2015)

Transcriptional activity of repABC operons of Rhizobium leguminosarum bv. trifolii TA1 plasmids: (A) pRleTA1a, (B) pRleTA1b, (C) pRleTA1c, and (D) pRleTA1d.Left panels show schematic genetic organization of the repABC cassettes. White arrows represent repA, repB and repC genes, and white broken arrows depict repA genes. Black dots show the position of parS-sites. The identified promoters of repABC operons were marked as grey rectangular boxes. The respective DNA fragments necessary for particular promoter identification, operon structure assignment, as well as their sequential deletions used for operon regulation studies, which were cloned into pMPK reporter and pBBR1MCS-5 vectors, were shown as black lines with positions indicated relative to repA start codon. The following system was applied for pMPK- and pBBR-1MCS5-based constructs nomenclature: e.g. in pMa/A2 recombinant plasmid the first two letters (pM) mean the shortcut of vector name (pMPK in this case), the next small letter 'a' means that it is derivative of pRleTA1a repABC cassette, the capital letter A followed by slash means that the cloned fragment comprise entire repA gene (in the case of pMa-d/ABC2 constructs the cloned fragments encompass entire repA and repB genes and fragment of repC fused with lacZ). Respectively, pBa/A2 means the same fragment recloned to the pBBR-1MCS5. Right panels represent β-galactosidase activities of respective lacZ transcriptional fusions measured in A. tumefaciens and expressed in Miller units. Each value (with standard deviation—extended bars) is the average of at least three independent measurements.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492784&req=5

pone.0131907.g001: Transcriptional activity of repABC operons of Rhizobium leguminosarum bv. trifolii TA1 plasmids: (A) pRleTA1a, (B) pRleTA1b, (C) pRleTA1c, and (D) pRleTA1d.Left panels show schematic genetic organization of the repABC cassettes. White arrows represent repA, repB and repC genes, and white broken arrows depict repA genes. Black dots show the position of parS-sites. The identified promoters of repABC operons were marked as grey rectangular boxes. The respective DNA fragments necessary for particular promoter identification, operon structure assignment, as well as their sequential deletions used for operon regulation studies, which were cloned into pMPK reporter and pBBR1MCS-5 vectors, were shown as black lines with positions indicated relative to repA start codon. The following system was applied for pMPK- and pBBR-1MCS5-based constructs nomenclature: e.g. in pMa/A2 recombinant plasmid the first two letters (pM) mean the shortcut of vector name (pMPK in this case), the next small letter 'a' means that it is derivative of pRleTA1a repABC cassette, the capital letter A followed by slash means that the cloned fragment comprise entire repA gene (in the case of pMa-d/ABC2 constructs the cloned fragments encompass entire repA and repB genes and fragment of repC fused with lacZ). Respectively, pBa/A2 means the same fragment recloned to the pBBR-1MCS5. Right panels represent β-galactosidase activities of respective lacZ transcriptional fusions measured in A. tumefaciens and expressed in Miller units. Each value (with standard deviation—extended bars) is the average of at least three independent measurements.
Mentions: In silico promoter prediction suggested operon organization of all RtTA1 repABC regions, with the putative promoter located upstream of the repA genes [31]. Moreover, in the putative promoter regions of all RtTA1 rep cassettes, operator-like palindromes were identified suggesting complex regulation of repABC expression. To study the transcription of the repABC genes of individual RtTA1 plasmids and regulation of the putative repABC operons with special attention to the contribution of RepA in this process, series of rep-lacZ transcriptional fusions were constructed and analysed (Fig 1).

Bottom Line: In the repABC replicons, partitioning and replication functions are transcriptionally linked resulting in complex regulation of rep gene expression.The RepA proteins were able to dimerize/oligomerize: in general dimers formed independently of ATP or ADP, although ATP diminished the concentration of oligomers that were produced.By the comprehensive approach focusing on a set of plasmids instead of individual replicons, the work highlighted subtle differences between the organization and regulation of particular rep operons as well as the structures and specificity of RepA proteins, which contribute to the fine-tuned coexistence of several replicons with similar repABC cassettes in the complex bacterial genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, Institute of Microbiology and Biotechnology, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Lublin, Poland.

ABSTRACT
Rhizobia commonly have very complex genomes with a chromosome and several large plasmids that possess genes belonging to the repABC family. RepA and RepB are members of the ParA and ParB families of partitioning proteins, respectively, whereas RepC is crucial for plasmid replication. In the repABC replicons, partitioning and replication functions are transcriptionally linked resulting in complex regulation of rep gene expression. The genome of R. leguminosarum bv. trifolii TA1 (RtTA1) consists of a chromosome and four plasmids (pRleTA1a-d), equipped with functional repABC genes. In this work, the regulation of transcription of the individual repABC cassettes of the four RtTA1 plasmids was studied. The involvement of the RepA and RepB as well as parS-like centromere sites in this process was depicted, demonstrating some dissimilarity in expression of respective rep regions. RtTA1 repABC genes of individual plasmids formed operons, which were negatively regulated by RepA and RepB. Individual RepA were able to bind to DNA without added nucleotides, but in the presence of ADP, bound specifically to their own operator sequences containing imperfect palindromes, and caused operon autorepression, whereas the addition of ATP stimulated non-specific binding of RepA to DNA. The RepA proteins were able to dimerize/oligomerize: in general dimers formed independently of ATP or ADP, although ATP diminished the concentration of oligomers that were produced. By the comprehensive approach focusing on a set of plasmids instead of individual replicons, the work highlighted subtle differences between the organization and regulation of particular rep operons as well as the structures and specificity of RepA proteins, which contribute to the fine-tuned coexistence of several replicons with similar repABC cassettes in the complex bacterial genome.

No MeSH data available.


Related in: MedlinePlus