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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

EMSA based analysis of His6-RepA proteins binding specificity to operator (Op) sequences originating from parental and non-parental plasmids.DNA fragments (15 ng) (identical as those marked in Fig 2) designated a2, b2, c2 and d2 comprised mapped operators. Red '+' means presence of respective His6-RepA protein (100 pmol) in the binding reaction. Black arrow indicates position of the retarded DNA bands.
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pone.0131907.g004: EMSA based analysis of His6-RepA proteins binding specificity to operator (Op) sequences originating from parental and non-parental plasmids.DNA fragments (15 ng) (identical as those marked in Fig 2) designated a2, b2, c2 and d2 comprised mapped operators. Red '+' means presence of respective His6-RepA protein (100 pmol) in the binding reaction. Black arrow indicates position of the retarded DNA bands.

Mentions: In the N-terminal part of the individual RepA proteins, elongated α-helices and helix-turn-helix (HTH) DNA binding motifs were predicted (Fig 3). To study the binding of RepA proteins to putative Op DNA sequences, we undertook EMSA analyses. Particular RepA were overexpressed in E. coli as N-terminally His6-tagged recombinant proteins, purified by affinity chromatography under native conditions, and used in series of non-radioactive EMSA with PCR-amplified DNA fragments of various lengths, comprising putative Op sequences of individual repABC operons (Fig 2A–2D). Using this in vitro approach for all of the His6-RepA RtTA1 proteins, their ability for binding to their own Op element was demonstrated (Fig 2A–2D). Moreover, the length of the operator sequence necessary for respective RepA binding was mapped and ranged from -55 to -6 bp for repABC/a, downstream of -96 bp for repABC/b, downstream of -40 bp for repABC/c, and downstream of -66 for repABC/d relative to the repA ATG codon (Fig 2A–2D). Noteworthy, no cross reactivity between individual RepA proteins and Op elements from non-parental repABC cassettes was demonstrated (Fig 4). The RepA proteins bound to their own repABC operator sites in a very specific manner, which is likely to correlate with operon autorepression. The negative regulation of transcription of RtTA1 rep operons was strongly enhanced by RepB, as demonstrated above. We performed the EMSA with respective recombinant RepB proteins and DNA fragments denoted a5, b2, c3, and d3 (Fig 2), comprising the minimal-length DNA segments upstream of repA necessary for respective His6-RepA binding, but no shifted bands were observed in this assay. These results indicate that individual RepB proteins may exert their corepressor action by binding to corresponding RepA (described later) rather than directly to the Op region.


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)

EMSA based analysis of His6-RepA proteins binding specificity to operator (Op) sequences originating from parental and non-parental plasmids.DNA fragments (15 ng) (identical as those marked in Fig 2) designated a2, b2, c2 and d2 comprised mapped operators. Red '+' means presence of respective His6-RepA protein (100 pmol) in the binding reaction. Black arrow indicates position of the retarded DNA bands.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131907.g004: EMSA based analysis of His6-RepA proteins binding specificity to operator (Op) sequences originating from parental and non-parental plasmids.DNA fragments (15 ng) (identical as those marked in Fig 2) designated a2, b2, c2 and d2 comprised mapped operators. Red '+' means presence of respective His6-RepA protein (100 pmol) in the binding reaction. Black arrow indicates position of the retarded DNA bands.
Mentions: In the N-terminal part of the individual RepA proteins, elongated α-helices and helix-turn-helix (HTH) DNA binding motifs were predicted (Fig 3). To study the binding of RepA proteins to putative Op DNA sequences, we undertook EMSA analyses. Particular RepA were overexpressed in E. coli as N-terminally His6-tagged recombinant proteins, purified by affinity chromatography under native conditions, and used in series of non-radioactive EMSA with PCR-amplified DNA fragments of various lengths, comprising putative Op sequences of individual repABC operons (Fig 2A–2D). Using this in vitro approach for all of the His6-RepA RtTA1 proteins, their ability for binding to their own Op element was demonstrated (Fig 2A–2D). Moreover, the length of the operator sequence necessary for respective RepA binding was mapped and ranged from -55 to -6 bp for repABC/a, downstream of -96 bp for repABC/b, downstream of -40 bp for repABC/c, and downstream of -66 for repABC/d relative to the repA ATG codon (Fig 2A–2D). Noteworthy, no cross reactivity between individual RepA proteins and Op elements from non-parental repABC cassettes was demonstrated (Fig 4). The RepA proteins bound to their own repABC operator sites in a very specific manner, which is likely to correlate with operon autorepression. The negative regulation of transcription of RtTA1 rep operons was strongly enhanced by RepB, as demonstrated above. We performed the EMSA with respective recombinant RepB proteins and DNA fragments denoted a5, b2, c3, and d3 (Fig 2), comprising the minimal-length DNA segments upstream of repA necessary for respective His6-RepA binding, but no shifted bands were observed in this assay. These results indicate that individual RepB proteins may exert their corepressor action by binding to corresponding RepA (described later) rather than directly to the Op region.

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