Three ParA Dimers Cooperatively Assemble on Type Ia Partition Promoters

Accurate DNA segregation is essential for faithful inheritance of genetic material. In bacteria, this process is mainly ensured by partition systems composed of two proteins, ParA and ParB, and a centromere site. Auto-regulation of Par operon expression is important for efficient partitioning and is primarily mediated by ParA for type Ia plasmid partition systems. For the F-plasmid, four ParAF monomers were proposed to bind to four repeated sequences in the promoter region. By contrast, using quantitative surface-plasmon-resonance, we showed that three ParAF dimers bind to this region. We uncovered that one perfect inverted repeat (IR) motif, consisting of two hexamer sequences spaced by 28-bp, constitutes the primary ParAF DNA binding site. A similar but degenerated motif overlaps the former. ParAF binding to these motifs is well supported by biochemical and modeling analyses. Molecular dynamics simulations predict that the winged-HTH domain displays high flexibility, which may favor the cooperative ParA binding to the promoter. We propose that three ParAF dimers bind cooperatively to overlapping motifs, thus covering the promoter region. A similar organization is found on closely related and distant plasmid partition systems, suggesting that such promoter organization for auto-regulated Par operons is widespread and may have evolved from a common ancestor.

CTP and parS coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning

ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process through which ParBF activates ParAF-ATPase has not been defined. We studied CTP- and parSF-modulated ParAF-ParBF complex assembly, in which DNA-bound ParAF-ATP dimers are activated for ATP hydrolysis by interacting with two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without significantly accelerating ParAF-ParBF complex assembly. Together, parSF and CTP accelerate ParAF-ParBF assembly without further significant increase in ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF-ParBF and ParAF-ParBF interactions promoting efficient partitioning.

Three ParA dimers cooperatively assemble on type Ia partition promoters

Accurate DNA segregation is essential for faithful inheritance of genetic material. In bacteria, this process is mainly ensured by a partition system (Par) composed of two proteins, ParA and ParB, and a centromere site. The auto-regulation of Par operon expression is important for efficient partitioning, and is primarily mediated by ParA for type Ia plasmid partition systems. For the plasmid F, four ParAF monomers were proposed to bind to four repeated sequences in the promoter region. By contrast, using quantitative surface plasmon resonance, we showed that three ParAF dimers bind to this region. We uncovered that one perfect inverted repeat (IR) motif, consisting of two hexamer sequences spaced by 28-bp, constitutes the primary ParAF DNA binding site. A similar but degenerated motif overlaps the former. ParAF binding to these motifs is well supported by biochemical and modeling analyses. In addition, molecular dynamics simulations predict that the winged-HTH domain displays high flexibility, which may favor the cooperative ParA binding to the promoter region. We propose that three ParAF dimers bind cooperatively to overlapping motifs thus covering the promoter region. A similar organization is found on both closely related and distant plasmid partition systems, suggesting that such promoter organization for auto-regulated Par operons is widespread and may have evolved from a common ancestor.

Kinetic pathway of ATP-induced DNA interactions of ParA2, a protein essential for segregation of Vibrio cholerae chromosome 2

Vibrio cholerae chromosome 2 (Chr2) requires its own ParABS system for segregation. Without it, V. cholerae becomes nonviable and loses pathogenicity. ParA2 of Chr2 is a Walker-type ATPase that is the main driver of Chr2 segregation. Most of our understanding of ParA function comes from studying plasmid partition systems. How ParA provides the motive force in segregation of chromosomes, which are much larger than plasmids, is less understood and different models have been proposed. Here we analyzed in vivo behavior and kinetic properties of ParA2 using cell imaging, biochemical and biophysical approaches. ParA2 formed an asymmetric gradient in the cell that localized dynamically in the cell cycle. We found that ParA2 dimers bind ATP and undergo a slow conformational change to an active DNA-binding state, similar to P1 ParA. The presence of DNA catalyzes ParA2 conformational change to allow cooperative binding of active ParA2 dimers to form higher-order oligomers on DNA. Nucleotide exchange rates were also slow, thus providing a control of ParA2 recruitment and dynamic localizations. Although highly conserved in biochemical properties, ParA2 showed faster overall ATP cycling and DNA-rebinding rates than plasmid ParAs, suggesting that this could be shared kinetic features among chromosomal ParAs to regulate the transport of a much larger DNA cargo.

CTP and parS control ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning

ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process of ParBF activation of ParAF-ATPase has not been defined. We studied CTP- and parSF-stimulated ParAF—ParBF complex assembly leading to ParAF-ATPase activation. Activation of DNA-bound ParAF-ATP dimers for ATP hydrolysis requires binding of two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without increasing ParAF—ParBF assembly kinetics. Together, parSF and CTP accelerate ParAF—ParBF assembly without further increasing the ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF—ParBF and ParAF—ParBF interactions promoting efficient partitioning.

ParC, a New Partitioning Protein, Is Necessary for the Active Form of ParA From Myxococcus pMF1 Plasmid

The ParABS partitioning system, a main driver of DNA segregation in bacteria, employs two proteins, ParA and ParB, for plasmid partition. The pMF1 plasmid from Myxococcus fulvus 124B02 has a par operon encoding a small acidic protein, ParC, in addition to type I ParA and ParB homologs. Here, we show that expression of parC upstream of parA (as in the natural case), but not ectopic expression, is essential for the plasmid inheritance in Myxococcus cells. Co-expression of parC upstream of parA was determined to form a soluble ParC–ParA heterodimer at a 1:1 ratio, while individual expression of parA or co-expression of parA with ectopic parC formed insoluble ParA proteins. Purified ParA proteins alone had no ATPase activity and was easily dimerized, while mixing ParA with ParC formed the ParC–ParA heterodimer with the ATPase and polymerization activities. Fusing ParC and ParA also produced soluble proteins and some chimeras restored the ATPase activity and plasmid inheritance. The results highlight that proximal location of parC before parA is critical to realize the functions of ParA in the partition of Myxococcus plasmid pMF1 and shed light on a new mechanism to realize a protein function by two separate proteins.

Nonspecific DNA binding by P1 ParA determines the distribution of plasmid partition and repressor activities

The faithful segregation, or “partition,” of many low-copy number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent nonspecific DNA (nsDNA)-binding activity, which is essential for partition. ParA also has a site-specific DNA-binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid partition and repressor activities, using a ParA with an alanine substitution at Arg351, a residue previously predicted to participate in site-specific DNA binding. In vivo, the parAR351A allele is compromised for partition, but its repressor activity is dramatically improved so that it behaves as a “super-repressor.” In vitro, ParAR351A binds and hydrolyzes ATP, and undergoes a specific conformational change required for nsDNA binding, but its nsDNA-binding activity is significantly damaged. This defect in turn significantly reduces the assembly and stability of partition complexes formed by the interaction of ParA with ParB, the centromere-binding protein, and DNA. In contrast, the R351A change shows only a mild defect in site-specific DNA binding. We conclude that the partition defect is due to altered nsDNA binding kinetics and affinity for the bacterial chromosome. Furthermore, the super-repressor phenotype is explained by an increased pool of non-nucleoid bound ParA that is competent to bind parOP and repress transcription.