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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

Oligomerization pattern of His6-RepA/a-d in the presence of ADP/ATP, specific and nonspecific DNA.Western blots are showing the products of cross-linking of individual His6-tagged RepA proteins (0.1–0.2 nM), following 1 h incubation at 28°C with 10 mM DMP and additional compounds. '+' means the presence of: 'C'—nonspecific DNA competitor (10–50 ng); 'Op'—specific operator DNA (10–50 ng); ADP (2 mM) or ATP (2 mM). 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.g008: Oligomerization pattern of His6-RepA/a-d in the presence of ADP/ATP, specific and nonspecific DNA.Western blots are showing the products of cross-linking of individual His6-tagged RepA proteins (0.1–0.2 nM), following 1 h incubation at 28°C with 10 mM DMP and additional compounds. '+' means the presence of: 'C'—nonspecific DNA competitor (10–50 ng); 'Op'—specific operator DNA (10–50 ng); ADP (2 mM) or ATP (2 mM). 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: To gain further insight into the RepA protein oligomerization pattern, additional components such as ATP, ADP, and specific and non-specific DNA in various combinations were included in the cross-linking reactions (Fig 8). In general, the oligomerization pattern of the His6-RepA proteins seemed not to be influenced in vitro by the specific or non-specific DNA. On the other hand, when ATP was present in a cross-linking reaction, the bands corresponding to putative multimers were absent or weakly visible for most of the tested RepA and this phenomenon was independent of the kind of tested DNA (Fig 8A, 8B and 8D). In the case of the RepA/c protein, the presence of ATP in the cross-linking reaction did not affect the multimer formation (Fig 8C). However, the addition of ATP slightly diminished the intensity of bands corresponding to high molecular weight multimers (Fig 8C).


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)

Oligomerization pattern of His6-RepA/a-d in the presence of ADP/ATP, specific and nonspecific DNA.Western blots are showing the products of cross-linking of individual His6-tagged RepA proteins (0.1–0.2 nM), following 1 h incubation at 28°C with 10 mM DMP and additional compounds. '+' means the presence of: 'C'—nonspecific DNA competitor (10–50 ng); 'Op'—specific operator DNA (10–50 ng); ADP (2 mM) or ATP (2 mM). 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.g008: Oligomerization pattern of His6-RepA/a-d in the presence of ADP/ATP, specific and nonspecific DNA.Western blots are showing the products of cross-linking of individual His6-tagged RepA proteins (0.1–0.2 nM), following 1 h incubation at 28°C with 10 mM DMP and additional compounds. '+' means the presence of: 'C'—nonspecific DNA competitor (10–50 ng); 'Op'—specific operator DNA (10–50 ng); ADP (2 mM) or ATP (2 mM). 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: To gain further insight into the RepA protein oligomerization pattern, additional components such as ATP, ADP, and specific and non-specific DNA in various combinations were included in the cross-linking reactions (Fig 8). In general, the oligomerization pattern of the His6-RepA proteins seemed not to be influenced in vitro by the specific or non-specific DNA. On the other hand, when ATP was present in a cross-linking reaction, the bands corresponding to putative multimers were absent or weakly visible for most of the tested RepA and this phenomenon was independent of the kind of tested DNA (Fig 8A, 8B and 8D). In the case of the RepA/c protein, the presence of ATP in the cross-linking reaction did not affect the multimer formation (Fig 8C). However, the addition of ATP slightly diminished the intensity of bands corresponding to high molecular weight multimers (Fig 8C).

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