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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

Mapping of operator sequences of repABC operons of RtTA1 plasmids: (A) pRleTA1a, (B) pRleTA1b, (C) pRleTA1c, and (D) pRleTA1d.Left panels show schematic depictions of the region upstream of repA of repABC operons. The respective promoters (grey rectangle), operators—imperfect palindromes (black inverted triangles), parS elements (black dots) and repA genes (white broken arrow) were shown. The sequence of each palindrome is highlighted in yellow. Length and relative position of DNA fragments used in operator (Op) mapping were shown with respect to repA start codon. Right panels represent EMSA results with recombinant His6-RepA and respective DNA fragments (5–25 ng). Black triangles indicate the increasing concentration of particular His6-RepA protein (10 and 100 pmol) present in DNA binding reaction. Black arrows indicate the position of the retarded DNA bands.
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pone.0131907.g002: Mapping of operator sequences of repABC operons of RtTA1 plasmids: (A) pRleTA1a, (B) pRleTA1b, (C) pRleTA1c, and (D) pRleTA1d.Left panels show schematic depictions of the region upstream of repA of repABC operons. The respective promoters (grey rectangle), operators—imperfect palindromes (black inverted triangles), parS elements (black dots) and repA genes (white broken arrow) were shown. The sequence of each palindrome is highlighted in yellow. Length and relative position of DNA fragments used in operator (Op) mapping were shown with respect to repA start codon. Right panels represent EMSA results with recombinant His6-RepA and respective DNA fragments (5–25 ng). Black triangles indicate the increasing concentration of particular His6-RepA protein (10 and 100 pmol) present in DNA binding reaction. Black arrows indicate the position of the retarded DNA bands.

Mentions: As demonstrated above, individual RepA can, at least partially, repress its own transcription. In each of the four RtTA1 plasmids, a putative operator sequence (Op) was found upstream of the repA gene initial codon. The operons contained imperfect palindromes, which in the case of the repABC/b, repABC/c, and repABC/d cassettes partially overlapped the identified promoters (Fig 2A–2D).


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)

Mapping of operator sequences of repABC operons of RtTA1 plasmids: (A) pRleTA1a, (B) pRleTA1b, (C) pRleTA1c, and (D) pRleTA1d.Left panels show schematic depictions of the region upstream of repA of repABC operons. The respective promoters (grey rectangle), operators—imperfect palindromes (black inverted triangles), parS elements (black dots) and repA genes (white broken arrow) were shown. The sequence of each palindrome is highlighted in yellow. Length and relative position of DNA fragments used in operator (Op) mapping were shown with respect to repA start codon. Right panels represent EMSA results with recombinant His6-RepA and respective DNA fragments (5–25 ng). Black triangles indicate the increasing concentration of particular His6-RepA protein (10 and 100 pmol) present in DNA binding reaction. Black arrows indicate the position of the retarded DNA bands.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492784&req=5

pone.0131907.g002: Mapping of operator sequences of repABC operons of RtTA1 plasmids: (A) pRleTA1a, (B) pRleTA1b, (C) pRleTA1c, and (D) pRleTA1d.Left panels show schematic depictions of the region upstream of repA of repABC operons. The respective promoters (grey rectangle), operators—imperfect palindromes (black inverted triangles), parS elements (black dots) and repA genes (white broken arrow) were shown. The sequence of each palindrome is highlighted in yellow. Length and relative position of DNA fragments used in operator (Op) mapping were shown with respect to repA start codon. Right panels represent EMSA results with recombinant His6-RepA and respective DNA fragments (5–25 ng). Black triangles indicate the increasing concentration of particular His6-RepA protein (10 and 100 pmol) present in DNA binding reaction. Black arrows indicate the position of the retarded DNA bands.
Mentions: As demonstrated above, individual RepA can, at least partially, repress its own transcription. In each of the four RtTA1 plasmids, a putative operator sequence (Op) was found upstream of the repA gene initial codon. The operons contained imperfect palindromes, which in the case of the repABC/b, repABC/c, and repABC/d cassettes partially overlapped the identified promoters (Fig 2A–2D).

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