Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems

Radnedge, Lyndsay; Youngren, Brenda; Davis, Michael A.; Austin, Stuart

doi:10.1093/emboj/17.20.6076

Cited by 83 publications

(93 citation statements)

References 22 publications

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“…The P1 species specificity difference from P7 is determined by the interaction of a short tract of amino acids in ParB with a unique BoxB motif (21). The species specificity of pMT1 also proved to reside in a unique BoxB motif.…”

Section: Discussionmentioning

confidence: 95%

Plasmid Partition System of the P1parFamily from the pWR100 Virulence Plasmid ofShigella flexneri

2005

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P1par family members promote the active segregation of a variety of plasmids and plasmid prophages in gram-negative bacteria. Each has genes for ParA and ParB proteins, followed by a parS partition site. The large virulence plasmid pWR100 of Shigella flexneri contains a new P1par family member: pWR100par. Although typical parA and parB genes are present, the putative pWR100parS site is atypical in sequence and organization. However, pWR100parS promoted accurate plasmid partition in Escherichia coli when the pWR100 Par proteins were supplied. Unique BoxB hexamer motifs within parS define species specificities among previously described family members. Although substantially different from P1parS from the P1 plasmid prophage of E. coli, pWR100parS has the same BoxB sequence. As predicted, the species specificity of the two types proved identical. They also shared partition-mediated incompatibility, consistent with the proposed mechanistic link between incompatibility and species specificity. Among several informative sequence differences between pWR100parS and P1parS is the presence of a 21-bp insert at the center of the pWR100parS site. Deletion of this insert left much of the parS activity intact. Tolerance of central inserts with integral numbers of helical DNA turns reflects the critical topology of these sites, which are bent by binding the host IHF protein.

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Section: Discussionmentioning

confidence: 95%

Plasmid Partition System of the P1parFamily from the pWR100 Virulence Plasmid ofShigella flexneri

2005

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“…Despite the evolutionary proximity of these partition regions, the individual elements of the cassettes are not functionally interchangeable. This specificity is conferred by relatively subtle variations among the P1 and P7 components that are crucial for protein-protein and protein-DNA interactions during segregation (19,20,34,35). The homologous partition regions of plasmids F and N15 similarly exhibit species specificity (36).…”

Section: Discussionmentioning

confidence: 99%

Protein Diversity Confers Specificity in Plasmid Segregation

2005

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The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNAbinding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomermonomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation.Protein machines for DNA transactions, including transcription, replication, recombination. and segregation, involve highly specific protein-protein and protein-nucleic acid contacts. These interactions are crucial for the precise assembly of the requisite multiprotein complexes and for the biological processes that these complexes mediate (1). A broad, but defined, spectrum of protein-protein and protein-DNA interactions have evolved that determine exactly the architecture of nucleoprotein complexes (5, 30, 43 [and other references therein]).The segregation of plasmids in bacteria also involves specific protein-protein and protein-DNA interactions. Plasmid partition most commonly involves a pair of plasmid-encoded proteins, often denoted ParA and ParB, that assemble on a cisacting partition site, sometimes known as parS (39). The ParA protein does not directly contact the partition site DNA but is instead recruited into the partition nucleoprotein complex through interactions with the ParB protein (4, 6). ParA possesses weak intrinsic ATPase activity which is stimulated by the ParB factor. The assembly of the partition nucleoprotein complex is required for the proper movement of plasmid pairs in opposite poleward directions prior to cell division (9,15,26,27). This movement is likely to occur as a result of extensive ATP-mediated polymerization and/or depolymerization of the ParA component (D. Barillà et al., EMBO J., in press).The partition cassette of the multidrug resistance plasmid TP228 specifies a ParA homolog, P...

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“…Strain CC4253 has an additional ϳ170 bp of P1 DNA downstream of the parS site. catp, the promoter for the cat gene; lacZp, the lacUV5 promoter; repAp, the promoter for the P1 repA gene (23). 5xT1 and 4xT1, five and four copies, respectively, of the T1 transcriptional terminator from rrnB.…”

Section: Figmentioning

confidence: 99%

Transcriptional Interference by a Complex Formed at the Centromere-Like Partition Site of Plasmid P1

Sawitzke

Sergueev

et al. 2002

J Bacteriol

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The partition site, parS, promotes accurate segregation of the replicated P1 plasmid to daughter cells when the P1-encoded ParA and ParB proteins are supplied. The parS site was inserted into the Escherichia coli chromosome between the promoter and the structural gene for ␤-galactosidase, lacZ. There was little interference with lacZ expression when ParA and ParB were supplied in trans. However, when a mutant ParA protein, ParAM314I, was supplied along with ParB, expression of lacZ was shut down. ParAM314I, ParB, and parS appear to form a nucleoprotein complex that blocks transcription. Mutations in parA and parB that relieved the parAM314I-dependent block were found. In addition, new mutations which impose the block were selected. Five of the latter mapped to parA and one to parB; all had a propagation-defective phenotype (Par PD ) similar to that of parAM314I. Thus, whereas a null par mutant P1 plasmid segregates its DNA randomly, these mutants prevent even random distribution of the plasmid. We propose that ParA protein normally interacts transiently with the ParB-parS complex for partition to proceed but that the mutations block ParA dissociation. This "permanent" ParA-ParB-parS complex acts as a transcription block. Consistent with this hypothesis, we found that three of the seven blocking mutations lie within regions of ParA and ParB that are known to interact with each other. When the transcription block is imposed, regional silencing of nearby genes occurs. However, the requirement for ParA and a mutant parA or parB allele distinguishes the transcription block from the regional ParB-dependent gene silencing previously described.

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Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems

Cited by 83 publications

References 22 publications

Plasmid Partition System of the P1parFamily from the pWR100 Virulence Plasmid ofShigella flexneri

Plasmid Partition System of the P1parFamily from the pWR100 Virulence Plasmid ofShigella flexneri

Protein Diversity Confers Specificity in Plasmid Segregation

Transcriptional Interference by a Complex Formed at the Centromere-Like Partition Site of Plasmid P1

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