Barbara E. Funnell

P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities

The partition system of P1 plasmids is composed of two proteins, ParA and ParB, and a cis‐acting site parS. parS is wrapped around ParB and Escherichia coli IHF protein in a higher order nucleoprotein complex called the partition complex. ParA is an ATPase that autoregulates the expression of the par operon and has an essential but unknown function in the partition process. In this study we demonstrate a direct interaction between ParA and the P1 partition complex. The interaction was strictly dependent on ParB and ATP. The consequence of this interaction depended on the ParB concentration. At high ParB levels, ParA was recruited to the partition complex via a ParA–ParB interaction, but at low ParB levels, ParA removed or disassembled ParB from the partition complex. ADP could not support these interactions, but could promote the site‐specific DNA binding activity of ParA to parOP, the operator of the par operon. Conversely, ATP could not support a stable interaction of ParA with parOP in this assay. Our data suggest that ParA‐ADP is the repressor of the par operon, and ParA‐ATP, by interacting with the partition complex, plays a direct role in partition. Therefore, one role of adenine nucleotide binding and hydrolysis by ParA is that of a molecular switch controlling entry into two separate pathways in which ParA plays different roles.

ATP control of dynamic P1 ParA-DNA interactions: a key role for the nucleoid in plasmid partition.

P1 ParA is a member of the Walker‐type family of partition ATPases involved in the segregation of plasmids and bacterial chromosomes. ATPases of this class interact with DNA non‐specifically in vitro and colocalize with the bacterial nucleoid to generate a variety of reported patterns in vivo. Here, we directly visualize ParA binding to DNA using total internal reflection fluorescence microscopy. This activity depends on, and is highly specific for ATP. DNA‐binding activity is not coupled to ATP hydrolysis. Rather, ParA undergoes a slow multi‐step conformational transition upon ATP binding, which licenses ParA to bind non‐specific DNA. The kinetics provide a time‐delay switch to allow slow cycling between the DNA binding and non‐binding forms of ParA. We propose that this time delay, combined with stimulation of ParAs ATPase activity by ParB bound to the plasmid DNA, generates an uneven distribution of the nucleoid‐associated ParA, and provides the motive force for plasmid segregation prior to cell division.

Structures of ParB bound to DNA reveal mechanism of partition complex formation.

The faithful inheritance of genetic information, which is essential for all organisms, requires accurate DNA partition (segregation) at cell division. In prokaryotes, partition is mediated by par systems, for which the P1 plasmid system of Escherichia coli is a prototype comprising a partition site and two proteins, ParA and ParB. To form the partition complex necessary for segregation, P1 ParB must recognize a complicated arrangement of A-box and B-box DNA motifs located on opposite ends of a sharply bent parS partition site of ∼74 bp (refs 3–7). Here we describe structures of ParB bound to partition sites. ParB forms an asymmetric dimer with extended amino-terminal HTH (helix–turn–helix) domains that contact A-boxes. The two HTH domains emanate from a dimerized DNA-binding module composed of a six-stranded β-sheet coiled-coil that binds B-boxes. Strikingly, these individual DNA-binding modules rotate freely about a flexible linker, enabling them to contact several arrangements of A- and B-boxes. Most notably, each DNA-binding element binds to and thus bridges adjacent DNA duplexes. These unique structural features of ParB explain how this protein can bind complex arrays of A- and B-box elements on adjacent DNA arms of the looped partition site.

Surfing biological surfaces: Exploiting the nucleoid for partition and transport in bacteria

The ParA family of ATPases is responsible for transporting bacterial chromosomes, plasmids and large protein machineries. ParAs pattern the nucleoid in vivo, but how patterning functions or is exploited in transport is of considerable debate. Here we discuss the process of self‐organization into patterns on the bacterial nucleoid and explore how it relates to the molecular mechanism of ParA action. We review ParA‐mediated DNA partition as a general mechanism of how ATP‐driven protein gradients on biological surfaces can result in spatial organization on a mesoscale. We also discuss how the nucleoid acts as a formidable diffusion barrier for large bodies in the cell, and make the case that the ParA family evolved to overcome the barrier by exploiting the nucleoid as a matrix for movement.

ParA-mediated plasmid partition driven by protein pattern self-organization

DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low‐copy plasmids, such as the plasmids P1 and F, employ a three‐component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker‐type ATPase, typically called ParA, which also binds non‐specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP‐driven patterning is involved in partition is unknown. We reconstituted and visualized ParA‐mediated plasmid partition inside a DNA‐carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB‐stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion‐ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid.

Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis

The ParA family of proteins is involved in partition of a variety of plasmid and bacterial chromosomes. P1 ParA plays two roles in partition: it acts as a repressor of the par operon and has an undefined yet indispensable role in P1 plasmid localization. We constructed seven mutations in three putative ATP‐binding motifs of ParA. Three classes of phenotypes resulted, each represented by mutations in more than one motif. Three mutations created ‘super‐repressors’, in which repressor activity was much stronger than in wild‐type ParA, while the remainder damaged repressor activity. All mutations eliminated partition activities, but two showed a plasmid stability defect that was worse than that of a null mutation. Four mutant ParAs, two super‐repressors and two weak repressors, were analyzed biochemically, and all exhibited damaged ATPase activity. The super‐repressors bound site‐specifically to the par operator sequence, and this activity was strongly stimulated by ATP and ADP. These results support the proposal that ATP binding is essential but hydrolysis is inhibitory for ParAs repressor activity and suggest that ATP hydrolysis is essential for plasmid localization.

Modulation of the P1 Plasmid Partition Protein ParA by ATP, ADP, and P1 ParB

ParA is an essential P1 plasmid partition protein. It represses transcription of the par genes (parA and parB) and is also required for a second, as yet undefined step in partition. ParA is a ParB-stimulated ATPase that binds to a specific DNA site in the parpromoter region. ATP binding and hydrolysis by ParA affect ParA activities in vitro. ATP and ADP binding stimulate ParA DNA binding and dimerization; however, ATP hydrolysis has a negative effect on DNA binding. Our current experiments reveal that ATP binding and hydrolysis affect ParA conformation and ParA sensitivity to ParB. Nucleotide binding assays show that ParA binds ATP better than ADP (K d values of 33 and 50 μm, respectively). Interaction with these nucleotides as well as ATP hydrolysis by ParA alter ParA conformation as established by CD and ParA sensitivity to heat denaturation. Finally, we show that ParB stimulates ParA DNA binding. This stimulation requires ATP hydrolysisin vitro, suggesting that one role for ATP hydrolysisin vivo is to make ParA repressor sensitive to ParB. Our observations lead to the suggestion that ATP binding and hydrolysis have separable roles in ParA repressor function and perhaps in ParA partition functions as well.

The DNA Binding Domains of P1 ParB and the Architecture of the P1 Plasmid Partition Complex

Stable maintenance of P1 plasmids inEscherichia coli is mediated by a high affinity nucleoprotein complex called the partition complex, which consists of ParB and the E. coli integration host factor (IHF) bound specifically to the P1 parS site. IHF strongly stimulates ParB binding to parS, and the minimal partition complex contains a single dimer of ParB. To examine the architecture of the partition complex, we have investigated the DNA binding activity of various ParB fragments. Gel mobility shift and DNase I protection assays showed that the first 141 residues of ParB are dispensable for the formation of the minimal, high affinity partition complex. A fragment missing only the last 16 amino acids of ParB bound specifically to parS, but binding was weak and was no longer stimulated by IHF. The ability of IHF to stimulate ParB binding to parS correlated with the ability of ParB to dimerize via its C terminus. Using full and partial parS sites, we show that two regions of ParB, one in the center and the other near the C terminus of the protein, interact with distinct sequences withinparS. Based on these data, we have proposed a model of how the ParB dimer binds parS to form the minimal partition complex.

Structural basis for ADP‐mediated transcriptional regulation by P1 and P7 ParA

The accurate segregation of DNA is essential for the faithful inheritance of genetic information. Segregation of the prototypical P1 plasmid par system requires two proteins, ParA and ParB, and a centromere. When bound to ATP, ParA mediates segregation by interacting with centromere‐bound ParB, but when bound to ADP, ParA fulfils a different function: DNA‐binding transcription autoregulation. The structure of ParA is unknown as is how distinct nucleotides arbitrate its different functions. To address these questions, we carried out structural and biochemical studies. Crystal structures show that ParA consists of an elongated N‐terminal α‐helix, which unexpectedly mediates dimerization, a winged‐HTH and a Walker‐box containing C‐domain. Biochemical data confirm that apoParA forms dimers at physiological concentrations. Comparisons of four apoParA structures reveal a strikingly flexible dimer interface that allows ParA to adopt multiple conformations. The ParA–ADP structure shows that ADP‐binding activates DNA binding using a bipartite mechanism. First, it locks in one specific dimer conformation, and second, it induces the folding of two DNA‐binding basic motifs that we show are critical for operator binding.

Plasmid Partition Mechanisms

The stable maintenance of low-copy-number plasmids in bacteria is actively driven by partition mechanisms that are responsible for the positioning of plasmids inside the cell. Partition systems are ubiquitous in the microbial world and are encoded by many bacterial chromosomes as well as plasmids. These systems, although different in sequence and mechanism, typically consist of two proteins and a DNA partition site, or prokaryotic centromere, on the plasmid or chromosome. One protein binds site-specifically to the centromere to form a partition complex, and the other protein uses the energy of nucleotide binding and hydrolysis to transport the plasmid, via interactions with this partition complex inside the cell. For plasmids, this minimal cassette is sufficient to direct proper segregation in bacterial cells. There has been significant progress in the last several years in our understanding of partition mechanisms. Two general areas that have developed are (i) the structural biology of partition proteins and their interactions with DNA and (ii) the action and dynamics of the partition ATPases that drive the process. In addition, systems that use tubulin-like GTPases to partition plasmids have recently been identified. In this chapter, we concentrate on these recent developments and the molecular details of plasmid partition mechanisms.