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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

Time course cross-linking analysis of His6-RepA/a-d.His6-tagged RepA proteins (0.1–0.2 nM) were incubated with DMP concentration fixed at 10 mM, at 28°C, from 30 s to 120 min, separated on 10% SDS-PAGE and visualized by Western blot with anti-His6 antibodies. The thin lines indicate the marker bands whose molecular masses are expressed in kDa. Arrows indicate different species formed by RepA proteins corresponding to monomers and dimers, while the position of multimers was marked with brackets.
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pone.0131907.g007: Time course cross-linking analysis of His6-RepA/a-d.His6-tagged RepA proteins (0.1–0.2 nM) were incubated with DMP concentration fixed at 10 mM, at 28°C, from 30 s to 120 min, separated on 10% SDS-PAGE and visualized by Western blot with anti-His6 antibodies. The thin lines indicate the marker bands whose molecular masses are expressed in kDa. Arrows indicate different species formed by RepA proteins corresponding to monomers and dimers, while the position of multimers was marked with brackets.

Mentions: A time course experiment with a fixed concentration of DMP (10 mM) revealed that each of the tested RepA was initially fixed into covalently bound dimers, but yet the multimeric forms were observed very soon (Fig 7). Three of the tested His6-RepA proteins, namely RepA/a, RepA/c, and RepA/d displayed similar kinetics of the respective dimer/multimer formation: they required from 3 to 5 min to form dimers/multimers, while after 15 min the reaction reached a plateau (Fig 7A, 7C and 7D). On the contrary, the RepA/b protein formed dimers rapidly in the solution: after 30 s of incubation with 10 mM of DMP at 28°C; after another 30 s, multimers were visible and after 3 min the reaction reached a plateau (Fig 7B).


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)

Time course cross-linking analysis of His6-RepA/a-d.His6-tagged RepA proteins (0.1–0.2 nM) were incubated with DMP concentration fixed at 10 mM, at 28°C, from 30 s to 120 min, separated on 10% SDS-PAGE and visualized by Western blot with anti-His6 antibodies. The thin lines indicate the marker bands whose molecular masses are expressed in kDa. Arrows indicate different species formed by RepA proteins corresponding to monomers and dimers, while the position of multimers was marked with brackets.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131907.g007: Time course cross-linking analysis of His6-RepA/a-d.His6-tagged RepA proteins (0.1–0.2 nM) were incubated with DMP concentration fixed at 10 mM, at 28°C, from 30 s to 120 min, separated on 10% SDS-PAGE and visualized by Western blot with anti-His6 antibodies. The thin lines indicate the marker bands whose molecular masses are expressed in kDa. Arrows indicate different species formed by RepA proteins corresponding to monomers and dimers, while the position of multimers was marked with brackets.
Mentions: A time course experiment with a fixed concentration of DMP (10 mM) revealed that each of the tested RepA was initially fixed into covalently bound dimers, but yet the multimeric forms were observed very soon (Fig 7). Three of the tested His6-RepA proteins, namely RepA/a, RepA/c, and RepA/d displayed similar kinetics of the respective dimer/multimer formation: they required from 3 to 5 min to form dimers/multimers, while after 15 min the reaction reached a plateau (Fig 7A, 7C and 7D). On the contrary, the RepA/b protein formed dimers rapidly in the solution: after 30 s of incubation with 10 mM of DMP at 28°C; after another 30 s, multimers were visible and after 3 min the reaction reached a plateau (Fig 7B).

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