Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein

Abstract

Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rodshaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments.

Introduction

Recent advances in prokaryotic cell biology have challenged the long-held notion that bacterial cells exist merely as casings that contain diffusible chemicals and enzymes. Improved bacterial fluorescent imaging techniques, coupled with the abundance of publically available bacterial genome data, has enabled the spatio-temporal localization of novel proteins to be determined in vivo. Strikingly, bacteria contain an array of proteins which not only adopt specific localizations within cells, but form an integral part of a bacterial subcellular cytoskeleton [1]. For example, the bacterial cytoskeletal protein FtsZ, a distant homologue of eukaryotic tubulin, forms a distinct ring-shape at mid-cell (the ‘Z-ring’) that defines the prokaryotic divisional plane and recruits further proteins involved in bacterial cytokinesis, whereas the actin-like protein MreB that is found in rod-shaped cells forms a discontinuous helical structure involved in controlling the width of a bacterium during cellular growth [2]. While only a few examples of prokaryotic tubulin homologues have been found to date [1], genes encoding actin-like proteins (Alps) are prevalent in the chromosomes and mobile genetic elements (such as plasmids) of many diverse bacterial species [3,4]. Phylogenetic analyses have revealed that chromosomally-encoded Alps, such as MreB, are closely related to each other, whereas Alps present on bacterial mobile genetic elements show vast inter-species sequence divergence [3]. Despite the genetic diversity exhibited by bacterial Alps, crystal structures from distantly related Alps have revealed that they share the basic ‘actin-fold’–the cleft present within all homologues of eukaryotic actin that is required for ATP/GTP binding and hydrolysis–and the ability of the Alp monomer to polymerize into filamentous ultrastructures [5].

The 46 kb Staphylococcus aureus plasmid pSK41 harbors a genetic locus, parMRC (Fig 1A), that encodes an actin-like protein, ParM [6,7]. pSK41 is the prototype of a family of medically important conjugative staphylococcal multiresistance plasmids [8] that have most recently been implicated in the development of vanA-mediated vancomycin resistance in S. aureus [9]. We have previously shown that pSK41 parMRC significantly enhances the segregationa stability of an unstable staphylococcal mini-plasmid, and site directed mutagenesis indicated that the NTPase motif of ParM is required for this stability phenotype [6]. The parMRC locus also encodes a DNA binding protein, ParR, which recognizes a series of 10 bp direct repeats, parC, located directly upstream from the parM and parR structural genes. Crystallographic data of ParR bound parC DNA shows that ParR binds as a dimer-of-dimers to the parC repeats, producing an extended macromolecular structure known as the ‘segrosome’ [6]. In the related ParMRC partitioning system of the E. coli multiresistance plasmid R1, ParM interacts with the segrosome to segregate replicated plasmids in a bidirectional fashion. In vitro data indicate that pSK41 ParM adopts a polymeric conformation which is very different to that of actin, MreB and R1 ParM [10]. Whereas pSK41 ParM forms a helical single stranded filament, both R1 ParM and actin adopt a two-start helical conformation, while MreB forms linear protofilaments [10]. Remarkably, database searching using pSK41 ParM crystal structure co-ordinates revealed that this protein is most structurally related to the chromosomally encoded Alp Ta0583 from the archaea Thermoplasma acidophilum, and not the R1 plasmid partitioning protein ParM, underscoring the structural diversity within microbial Alps. Biophysical analyses have suggested that pSK41 ParM filaments undergo a treadmilling-like mechanism of motion in vitro similar to that of F-actin [10]; contrastingly, R1 ParM exhibits a form of dynamic instability similar to that of eukaryotic tubulin [11]. In vivo studies in the native staphylococcal host, using a ParM C-terminal fusion to red fluorescent protein (ParM-RFP), also suggested that pSK41 ParM filaments are not dynamically unstable [12], in agreement with the in vitro observations [10]. Interestingly, the plasmid-partitioning Alp protein Alp7A, from the 55 kb Bacillus subtilis plasmid pLS20, exhibits both treadmilling and dynamic instability [3]. These observations highlight significant diversity in the dynamic properties exhibited by Alps.

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