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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

Analysis of ADP/ATP role in the DNA binding specificity of RepA proteins: (A) pRleTA1a, (B) pRleTA1b, (C) pRleTA1c, and (D) pRleTA1d.'Op' indicates DNA fragment (15 ng) comprising mapped operator sequences of individual repABC operons, while 'C' means non-specific DNA competitor (15 ng) (148 bp DNA fragment of the gene coding for Kmr of the pBBR1MCS-2). The Op fragments (a2, b2, c2, d2) correspond to the ones shown in Fig 2. Black triangles indicate the increasing concentration of ADT/ATP (0.1, 1, and 2 mM) while '+' means presence of respective His6-RepA (10 pmol) protein in the binding reaction. Black arrows indicate the position of the retarded DNA bands.
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pone.0131907.g005: Analysis of ADP/ATP role in the DNA binding specificity of RepA proteins: (A) pRleTA1a, (B) pRleTA1b, (C) pRleTA1c, and (D) pRleTA1d.'Op' indicates DNA fragment (15 ng) comprising mapped operator sequences of individual repABC operons, while 'C' means non-specific DNA competitor (15 ng) (148 bp DNA fragment of the gene coding for Kmr of the pBBR1MCS-2). The Op fragments (a2, b2, c2, d2) correspond to the ones shown in Fig 2. Black triangles indicate the increasing concentration of ADT/ATP (0.1, 1, and 2 mM) while '+' means presence of respective His6-RepA (10 pmol) protein in the binding reaction. Black arrows indicate the position of the retarded DNA bands.

Mentions: The role of ATP/ADP in the DNA binding specificity of RepA proteins was examined in the EMSA, in which two DNA fragments were used: a specific—including the respective Op sequence—and a non-specific competitor (an internal fragment of the Kmr gene of the pBBR1MCS-2) (Fig 5). For each of the recombinant RtTA1 His6-RepA proteins, the addition of ATP stimulated its non-specific DNA binding, while in the presence of ADP the individual RepA bound specifically to Op sequences (Fig 5A–5D). These results show that, in the presence of ADP, individual RepA bind specifically to their own Op sequence for rep operon repression.


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)

Analysis of ADP/ATP role in the DNA binding specificity of RepA proteins: (A) pRleTA1a, (B) pRleTA1b, (C) pRleTA1c, and (D) pRleTA1d.'Op' indicates DNA fragment (15 ng) comprising mapped operator sequences of individual repABC operons, while 'C' means non-specific DNA competitor (15 ng) (148 bp DNA fragment of the gene coding for Kmr of the pBBR1MCS-2). The Op fragments (a2, b2, c2, d2) correspond to the ones shown in Fig 2. Black triangles indicate the increasing concentration of ADT/ATP (0.1, 1, and 2 mM) while '+' means presence of respective His6-RepA (10 pmol) protein in the binding reaction. Black arrows indicate the position of the retarded DNA bands.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131907.g005: Analysis of ADP/ATP role in the DNA binding specificity of RepA proteins: (A) pRleTA1a, (B) pRleTA1b, (C) pRleTA1c, and (D) pRleTA1d.'Op' indicates DNA fragment (15 ng) comprising mapped operator sequences of individual repABC operons, while 'C' means non-specific DNA competitor (15 ng) (148 bp DNA fragment of the gene coding for Kmr of the pBBR1MCS-2). The Op fragments (a2, b2, c2, d2) correspond to the ones shown in Fig 2. Black triangles indicate the increasing concentration of ADT/ATP (0.1, 1, and 2 mM) while '+' means presence of respective His6-RepA (10 pmol) protein in the binding reaction. Black arrows indicate the position of the retarded DNA bands.
Mentions: The role of ATP/ADP in the DNA binding specificity of RepA proteins was examined in the EMSA, in which two DNA fragments were used: a specific—including the respective Op sequence—and a non-specific competitor (an internal fragment of the Kmr gene of the pBBR1MCS-2) (Fig 5). For each of the recombinant RtTA1 His6-RepA proteins, the addition of ATP stimulated its non-specific DNA binding, while in the presence of ADP the individual RepA bound specifically to Op sequences (Fig 5A–5D). These results show that, in the presence of ADP, individual RepA bind specifically to their own Op sequence for rep operon repression.

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