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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

BACTH analysis of interactions of RepA and RepB proteins.(A) The schematic representation of full length RepA and RepB, which were translationally fused to N- and C-terminus of T18 and T25 functional domains of CyaA protein, as well as constructed 'prey' hybrid plasmid which comprised N-terminally truncated RepA fused to T25 domain. (B) RepA/RepA and (C) RepA/RepB interactions in BACTH analysis which were quantified by measuring of β-galactosidase activity (Miller units) in hybrid cotransformants containing bait and prey plasmids. Each value (with standard deviation—extended bars) is the average of at least three independent measurements. Clones of each 'bait'/'prey' plasmid cotransformation were also spotted on LB agar with appropriate antibiotics and X-gal, IPTG, along with positive and negative controls. The colour formation indicates positive clones in which interaction of studied proteins was observed, while the negative clones remain white.
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pone.0131907.g009: BACTH analysis of interactions of RepA and RepB proteins.(A) The schematic representation of full length RepA and RepB, which were translationally fused to N- and C-terminus of T18 and T25 functional domains of CyaA protein, as well as constructed 'prey' hybrid plasmid which comprised N-terminally truncated RepA fused to T25 domain. (B) RepA/RepA and (C) RepA/RepB interactions in BACTH analysis which were quantified by measuring of β-galactosidase activity (Miller units) in hybrid cotransformants containing bait and prey plasmids. Each value (with standard deviation—extended bars) is the average of at least three independent measurements. Clones of each 'bait'/'prey' plasmid cotransformation were also spotted on LB agar with appropriate antibiotics and X-gal, IPTG, along with positive and negative controls. The colour formation indicates positive clones in which interaction of studied proteins was observed, while the negative clones remain white.

Mentions: The results presented above demonstrated the self-association ability of RepA as well as the possibility of reciprocal RepA and RepB interaction in the regulation of repABC operons. To validate these results, a bacterial two-hybrid system (BACTH) [42] was employed for examination of interplays between RepA and RepB. Since numerous recombinant plasmid constructs are usually required in two-hybrid based studies, only proteins originating from the pRleTA1b plasmid were subjected to the analysis. Both the respective repA and repB genes were cloned into pUT18, pUT18C, pKT25, and pKNT25 vectors to construct T18- or T25-gene fusions in various combinations (Fig 9A). Then, the E. coli DHM1 cya reporter strain was sequentially transformed with all plasmids expressing fusion proteins. Positive clones with blue colouring and reporter activity substantially higher than in the control, representing the interacting RepA-RepA proteins, were obtained for one of the tested combinations of fusion plasmids RepA-T18/T25-RepA, demonstrating the ability of RepA to form homooligomeric forms in vivo (Fig 9B). To map the putative oligomerization domain in the N-terminal part of the RepA protein, a truncated version of RepA comprising 79–401 aa was prepared as a 'prey' and cotransformed with a plasmid expressing RepA as a 'bait' (Fig 9A). No positive blue clones were obtained for this combination of fusion plasmids, in contrast to the high β-galactosidase activity observed for intact RepA-RepA proteins (Fig 9B). Altogether, these results suggested the location of the oligomerization domain in the N-terminus of the RepA protein. In the case of the RepA-RepB interaction, a high level of β-galactosidase activity was obtained in one combination of fusion plasmids (RepB-T18/T25-RepA) (Fig 9C). For several other clones, the reporter activity was higher than in the negative control but substantially lower than in positive clones in which RepA-RepA interaction was observed (Fig 9B and 9C). The results obtained confirm the capability of RepA and RepB proteins of interactions in vivo, and the low reporter activities may indicate that such interaction was weak under the experimental conditions applied.


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)

BACTH analysis of interactions of RepA and RepB proteins.(A) The schematic representation of full length RepA and RepB, which were translationally fused to N- and C-terminus of T18 and T25 functional domains of CyaA protein, as well as constructed 'prey' hybrid plasmid which comprised N-terminally truncated RepA fused to T25 domain. (B) RepA/RepA and (C) RepA/RepB interactions in BACTH analysis which were quantified by measuring of β-galactosidase activity (Miller units) in hybrid cotransformants containing bait and prey plasmids. Each value (with standard deviation—extended bars) is the average of at least three independent measurements. Clones of each 'bait'/'prey' plasmid cotransformation were also spotted on LB agar with appropriate antibiotics and X-gal, IPTG, along with positive and negative controls. The colour formation indicates positive clones in which interaction of studied proteins was observed, while the negative clones remain white.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131907.g009: BACTH analysis of interactions of RepA and RepB proteins.(A) The schematic representation of full length RepA and RepB, which were translationally fused to N- and C-terminus of T18 and T25 functional domains of CyaA protein, as well as constructed 'prey' hybrid plasmid which comprised N-terminally truncated RepA fused to T25 domain. (B) RepA/RepA and (C) RepA/RepB interactions in BACTH analysis which were quantified by measuring of β-galactosidase activity (Miller units) in hybrid cotransformants containing bait and prey plasmids. Each value (with standard deviation—extended bars) is the average of at least three independent measurements. Clones of each 'bait'/'prey' plasmid cotransformation were also spotted on LB agar with appropriate antibiotics and X-gal, IPTG, along with positive and negative controls. The colour formation indicates positive clones in which interaction of studied proteins was observed, while the negative clones remain white.
Mentions: The results presented above demonstrated the self-association ability of RepA as well as the possibility of reciprocal RepA and RepB interaction in the regulation of repABC operons. To validate these results, a bacterial two-hybrid system (BACTH) [42] was employed for examination of interplays between RepA and RepB. Since numerous recombinant plasmid constructs are usually required in two-hybrid based studies, only proteins originating from the pRleTA1b plasmid were subjected to the analysis. Both the respective repA and repB genes were cloned into pUT18, pUT18C, pKT25, and pKNT25 vectors to construct T18- or T25-gene fusions in various combinations (Fig 9A). Then, the E. coli DHM1 cya reporter strain was sequentially transformed with all plasmids expressing fusion proteins. Positive clones with blue colouring and reporter activity substantially higher than in the control, representing the interacting RepA-RepA proteins, were obtained for one of the tested combinations of fusion plasmids RepA-T18/T25-RepA, demonstrating the ability of RepA to form homooligomeric forms in vivo (Fig 9B). To map the putative oligomerization domain in the N-terminal part of the RepA protein, a truncated version of RepA comprising 79–401 aa was prepared as a 'prey' and cotransformed with a plasmid expressing RepA as a 'bait' (Fig 9A). No positive blue clones were obtained for this combination of fusion plasmids, in contrast to the high β-galactosidase activity observed for intact RepA-RepA proteins (Fig 9B). Altogether, these results suggested the location of the oligomerization domain in the N-terminus of the RepA protein. In the case of the RepA-RepB interaction, a high level of β-galactosidase activity was obtained in one combination of fusion plasmids (RepB-T18/T25-RepA) (Fig 9C). For several other clones, the reporter activity was higher than in the negative control but substantially lower than in positive clones in which RepA-RepA interaction was observed (Fig 9B and 9C). The results obtained confirm the capability of RepA and RepB proteins of interactions in vivo, and the low reporter activities may indicate that such interaction was weak under the experimental conditions applied.

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