register interest

Dr Jeanne Salje

Research Area: Cell and Molecular Biology
Technology Exchange: Microscopy (Confocal), Microscopy (EM), Protein interaction and Transcript profiling
Scientific Themes: Tropical Medicine & Global Health and Immunology & Infectious Disease
The intracellular life cycle of Orientia tsutsugamushi

The intracellular life cycle of Orientia tsutsugamushi

Orientia tsutsugamushi bacteria, labelled with a fluorescent antibody against a major surface protein (the 56kDa protein)

Orientia tsutsugamushi bacteria, labelled with a fluorescent antibody against a major surface ...

Orientia tsutsugamushi bacteria (green) inside a mouse fibroblast cell (red) 50 minutes after infection.

Orientia tsutsugamushi bacteria (green) inside a mouse fibroblast cell (red) 50 minutes after ...

My research group studies the host-pathogen biology of the obligate intracellular bacterium Orientia tsutsugamushi. This vector-borne pathogen causes the life-threatening human disease scrub typhus, that is endemic in large parts of Asia but that is severely under-reported due to difficulties in diagnostics and surveillance. We use a combination of cell biology, biochemistry and systems biology approaches to dissect fundamental questions about the host-pathogen biology of this clinically important bacterium.

Current research is primarily focussed on the following areas: 

  • Developing improved experimental tools for O. tsutsugamushi research. These include methods for bacterial propagation and isolation, developing fluorescent labelling methods for light microscopy imaging and establishing methods for genetic manipulation of Orientia.
  • Studying the structure of the cell wall and cell surface of O. tsutsugamushi, and understanding the implications for bacterial growth and division.
  • Studying the intracellular lifecycle of O. tsutsugamushi. This includes determining molecular mechanisms of bacterial attachment and entry into host cells, escape from the endo-lysosomal pathway, and exit from infected host cells.
  • Using next generation sequencing approaches to study genome organisation and dynamics. We are particularly interested in following genome changes over time and under different selective pressures.

Name Department Institution Country
Professor Nicholas PJ Day FMedSci FRCP Tropical Medicine Oxford University, Bangkok Thailand
Professor Daniel H Paris Tropical Medicine Oxford University, Bangkok Thailand
Dr Rory Bowden Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Atwal S, Giengkam S, VanNieuwenhze M, Salje J. 2016. Live imaging of the genetically intractable obligate intracellular bacteria Orientia tsutsugamushi using a panel of fluorescent dyes. J Microbiol Methods, 130 pp. 169-176. | Show Abstract | Read more

Our understanding of the molecular mechanisms of bacterial infection and pathogenesis are disproportionally derived from a small number of well-characterised species and strains. One reason for this is the enormous time and resources required to develop a new organism into experimental system that can be interrogated at the molecular level, in particular with regards to the development of genetic tools. Live cell imaging by fluorescence microscopy is a powerful technique to study biological processes such as bacterial motility, host cell invasion, and bacterial growth and division. In the absence of genetic tools that enable exogenous expression of fluorescent proteins, fluorescent chemical probes can be used to label and track living cells. A large number of fluorescent chemical probes are commercially available, but these have overwhelmingly been applied to the study of eukaryotic cell systems. Here, we present a methodical analysis of four different classes of probes, which can be used to delineate the cytoplasm, nucleic acids, cell membrane or peptidoglycan of living bacterial cells. We have tested these in the context of the important but neglected human pathogen Orientia tsutsugamushi but expect that the methodology would be broadly applicable to other bacterial species.

Ngamdee W, Tandhavanant S, Wikraiphat C, Reamtong O, Wuthiekanun V, Salje J, Low DA, Peacock SJ, Chantratita N. 2015. Competition between Burkholderia pseudomallei and B. thailandensis. BMC Microbiol, 15 (1), pp. 395. | Show Abstract | Read more

BACKGROUND: Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, an often fatal disease in tropical countries. Burkholderia thailandensis is a non-virulent but closely related species. Both species are soil saprophytes but are almost never isolated together. RESULTS: We identified two mechanisms by which B. pseudomallei affects the growth of B. thailandensis. First, we found that six different isolates of B. pseudomallei inhibited the growth of B. thailandensis on LB agar plates. Second, our results indicated that 55% of isolated strains of B. pseudomallei produced a secreted compound that inhibited the motility but not the viability of B. thailandensis. Analysis showed that the active compound was a pH-sensitive and heat-labile compound, likely a protein, which may affect flagella processing or facilitate their degradation. Analysis of bacterial sequence types (STs) demonstrated an association between this and motility inhibition. The active compound was produced from B. pseudomallei during the stationary growth phase. CONCLUSION: Taken together, our results indicate that B. pseudomallei inhibits both the growth and motility of its close relative B. thailandensis. The latter phenomenon appears to occur via a previously unreported mechanism involving flagellar processing or degradation.

Giengkam S, Blakes A, Utsahajit P, Chaemchuen S, Atwal S, Blacksell SD, Paris DH, Day NP, Salje J. 2015. Improved Quantification, Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen Orientia tsutsugamushi. PLoS Negl Trop Dis, 9 (8), pp. e0004009. | Show Abstract | Read more

BACKGROUND: Scrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, Orientia tsutsugamushi, is an obligate intracellular bacterium that is transmitted to humans by the bite of a Leptotrombidium mite. Research into the basic mechanisms of cell biology and pathogenicity of O. tsutsugamushi has lagged behind that of other important human pathogens. One reason for this is that O. tsutsugamushi is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on O. tsutsugamushi and also other obligate intracellular pathogens such as those in the Rickettsiales and Chlamydiales families. METHODOLOGY: A clinical isolate of O. tsutsugamushi was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an O. tsutsugamushi-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells. KEY RESULTS: Development of a standardised growth assay to quantify bacterial replication and viability in vitro. Quantitative comparison of different DNA extraction methods. Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement. Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds. Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including different freezing buffers and different rates of freezing. CONCLUSIONS: Here we present a standardised method for comparing the viability of O. tsutsugamushi after purification, treatment and propagation under various conditions. Taken together, we present a body of data to support improved techniques for propagation, purification and storage of this organism. This data will be useful both for improving clinical isolation rates as well as performing in vitro cell biology experiments.

Ngamdee W, Tandhavanant S, Wikraiphat C, Reamtong O, Wuthiekanun V, Salje J, Low DA, Peacock SJ, Chantratita N. 2015. Competition between Burkholderia pseudomallei and B. thailandensis. BMC Microbiol, 15 pp. 56. | Show Abstract | Read more

BACKGROUND: Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, an often fatal disease in tropical countries. Burkholderia thailandensis is a non-virulent but closely related species. Both species are soil saprophytes but are almost never isolated together. RESULTS: We identified two mechanisms by which B. pseudomallei affects the growth of B. thailandensis. First, we found that six different isolates of B. pseudomallei inhibited the growth of B. thailandensis on LB agar plates. Second, our results indicated that 55% of isolated strains of B. pseudomallei produced a secreted compound that inhibited the motility but not the viability of B. thailandensis. Analysis showed that the active compound was a pH-sensitive and heat-labile compound, likely a protein, which may affect flagella processing or facilitate their degradation. Analysis of bacterial sequence types (STs) demonstrated an association between this and motility inhibition. The active compound was produced from B. pseudomallei during the stationary growth phase. CONCLUSION: Taken together, our results indicate that B. pseudomallei inhibits both the growth and motility of its close relative B. thailandensis. The latter phenomenon appears to occur via a previously unreported mechanism involving flagellar processing or degradation.

Salje J. 2014. A single-cell imaging screen reveals multiple effects of secreted small molecules on bacteria. Microbiologyopen, 3 (4), pp. 426-436. | Show Abstract | Read more

Bacteria cells exist in close proximity to other cells of both the same and different species. Bacteria secrete a large number of different chemical species, and the local concentrations of these compounds at the surfaces of nearby cells may reach very high levels. It is fascinating to imagine how individual cells might sense and respond to the complex mix of signals at their surface. However, it is difficult to measure exactly what the local environmental composition looks like, or what the effects of individual compounds on nearby cells are. Here, an electron microscopy imaging screen was designed that would detect morphological changes induced by secreted small molecules. This differs from conventional approaches by detecting structural changes in individual cells rather than gene expression or growth rate changes at the population level. For example, one of the changes detected here was an increase in outer membrane vesicle production, which does not necessarily correspond to a change in gene expression. This initial study focussed on Pseudomonas aeruginosa, Escherichia coli, and Burkholderia dolosa, and revealed an intriguing range of effects of secreted small molecules on cells both within and between species.

Salje J, van den Ent F, de Boer P, Löwe J. 2011. Direct membrane binding by bacterial actin MreB. Mol Cell, 43 (3), pp. 478-487. | Show Abstract | Read more

Bacterial actin MreB is one of the key components of the bacterial cytoskeleton. It assembles into short filaments that lie just underneath the membrane and organize the cell wall synthesis machinery. Here we show that MreB from both T. maritima and E. coli binds directly to cell membranes. This function is essential for cell shape determination in E. coli and is proposed to be a general property of many, if not all, MreBs. We demonstrate that membrane binding is mediated by a membrane insertion loop in TmMreB and by an N-terminal amphipathic helix in EcMreB and show that purified TmMreB assembles into double filaments on a membrane surface that can induce curvature. This, the first example of a membrane-binding actin filament, prompts a fundamental rethink of the structure and dynamics of MreB filaments within cells.

Salje J, Gayathri P, Löwe J. 2010. The ParMRC system: molecular mechanisms of plasmid segregation by actin-like filaments. Nat Rev Microbiol, 8 (10), pp. 683-692. | Show Abstract | Read more

The ParMRC plasmid partitioning apparatus is one of the best characterized systems for bacterial DNA segregation. Bundles of actin-like filaments are used to push plasmids to opposite poles of the cell, whereupon they are stably inherited on cell division. This plasmid-encoded system comprises just three components: an actin-like protein, ParM, a DNA-binding adaptor protein, ParR, and a centromere-like region, parC. The properties and interactions of these components have been finely tuned to enable ParM filaments to search the cell space for plasmids and then move ParR-parC-bound DNA molecules apart. In this Review, we look at some of the most exciting questions in the field concerning the exact molecular mechanisms by which the components of this self-contained system modulate one another's activity to achieve bipolar DNA segregation.

Salje J. 2010. Plasmid segregation: how to survive as an extra piece of DNA. Crit Rev Biochem Mol Biol, 45 (4), pp. 296-317. | Show Abstract | Read more

Non-essential extra-chromosomal DNA elements such as plasmids are responsible for their own propagation in dividing host cells, and one means to ensure this is to carry a miniature active segregation system reminiscent of the mitotic spindle. Plasmids that are maintained at low numbers in prokaryotic cells have developed a range of such active partitioning systems, which are characterized by an impressive simplicity and efficiency and which are united by the use of dynamic, nucleotide-driven filaments to separate and position DNA molecules. A comparison of different plasmid segregation systems reveals (i) how unrelated filament-forming and DNA-binding proteins have been adopted and modified to create a range of simple DNA segregating complexes and (ii) how subtle changes in the few components of these DNA segregation machines has led to a remarkable diversity in the molecular mechanisms of closely related segregation systems. Here, our current understanding of plasmid segregation systems is reviewed and compared with other DNA segregation systems, and this is extended by a discussion of basic principles of plasmid segregation systems, evolutionary implications and the relationship between an autonomous DNA element and its host cell.

Salje J, Zuber B, Löwe J. 2009. Electron cryomicroscopy of E. coli reveals filament bundles involved in plasmid DNA segregation. Science, 323 (5913), pp. 509-512. | Show Abstract | Read more

Bipolar elongation of filaments of the bacterial actin homolog ParM drives movement of newly replicated plasmid DNA to opposite poles of a bacterial cell. We used a combination of vitreous sectioning and electron cryotomography to study this DNA partitioning system directly in native, frozen cells. The diffraction patterns from overexpressed ParM bundles in electron cryotomographic reconstructions were used to unambiguously identify ParM filaments in Escherichia coli cells. Using a low-copy number plasmid encoding components required for partitioning, we observed small bundles of three to five intracellular ParM filaments that were situated close to the edge of the nucleoid. We propose that this may indicate the capture of plasmid DNA within the periphery of this loosely defined, chromosome-containing region.

Salje J, Löwe J. 2008. Bacterial actin: architecture of the ParMRC plasmid DNA partitioning complex. EMBO J, 27 (16), pp. 2230-2238. | Show Abstract | Read more

The R1 plasmid employs ATP-driven polymerisation of the actin-like protein ParM to move newly replicated DNA to opposite poles of a bacterial cell. This process is essential for ensuring accurate segregation of the low-copy number plasmid and is the best characterised example of DNA partitioning in prokaryotes. In vivo, ParM only forms long filaments when capped at both ends by attachment to a centromere-like region parC, through a small DNA-binding protein ParR. Here, we present biochemical and electron microscopy data leading to a model for the mechanism by which ParR-parC complexes bind and stabilise elongating ParM filaments. We propose that the open ring formed by oligomeric ParR dimers with parC DNA wrapped around acts as a rigid clamp, which holds the end of elongating ParM filaments while allowing entry of new ATP-bound monomers. We propose a processive mechanism by which cycles of ATP hydrolysis in polymerising ParM drives movement of ParR-bound parC DNA. Importantly, our model predicts that each pair of plasmids will be driven apart in the cell by just a single double helical ParM filament.

Salje J, Löwe J. 2008. Bacterial actin: Architecture of the ParMRC plasmid DNA partitioning complex Chemtracts, 21 (6), pp. 231-232. | Show Abstract

During eukaryotic cell division, the DNA molecules are partitioned to opposite poles of the cell, ensuring accurate distribution of chromosomes between the dividing cells. In prokaryotes, the mechanism for accurate segregation is unknown. Previous studies have focused on partitioning of a plasmid, Rl, which encodes a stability operon, par, that is required for proper segregation of this low-copy-number plasmid. The par operon of Rl encodes three components: ParM, an actin-like ATPase, ParR, a DNA-bind- ing protein, and parC, a centromere-like DNA sequence element to which ParR binds. In the presence of ATP, ParM polymerizes and then disassembles. Disassembly is prevented, however, by the binding of ParR to parC, which stabilizes the ParM filaments. In the current model for DNA segregation, the ATP-dependent polymerization of ParM is believed to move newly replicated plasmids to the opposite poles. This study therefore determined how the ParM filaments are formed and stabilized and ascertained the sites involved to effect both mechanisms. Mutational studies, pull-down and gel- shift assays for point and deletion mutants of Rl ParR, and electron microscopy at varying concentrations of labeled parC established that the ParR- parC complex forms the clamp that binds at both the C-terminal ends of ParM, forming ring structures that bind ATP and stabilize the ParM filaments. ParR binds through its N-terminus to parC, forming a rigid scaffold of protein (ParR) wrapped by a DNA helix (parC). This binding does not include the Rl promoter region within the parC domain, because the promoter region extends out as a loop from the ParR binding region. Additionally, studies with mutants of ParM also identified the binding sites of the ParR-parC complex on ParM. A model for ParM polymerization consistent with electron micrograph images was thus proposed whereby ATP binding polymerizes ParM. Binding of the ParR-parC complex clamps the C-terminal ParM-ATP complex stabilizing filament formation. Hydrolysis of ATP to ADP dislocates ParR-parC and translocates ParR to one side to allow binding of a new ParM-ATP monomer. ParR translocates back, rocking the clamp, and the cycle of hydrolysis, translocation, and monomer addition is repeated until elongation is completed (Fig. 1). © 2008 Data Trace Publishing Company.

Kelly S, Reed J, Kramer S, Ellis L, Webb H, Sunter J, Salje J, Marinsek N, Gull K, Wickstead B, Carrington M. 2007. Functional genomics in Trypanosoma brucei: a collection of vectors for the expression of tagged proteins from endogenous and ectopic gene loci. Mol Biochem Parasitol, 154 (1), pp. 103-109. | Read more

Salje J, Ludwig B, Richter OM. 2005. Is a third proton-conducting pathway operative in bacterial cytochrome c oxidase? Biochem Soc Trans, 33 (Pt 4), pp. 829-831. | Show Abstract | Read more

Despite the existence of several three-dimensional structures of cytochrome c oxidases, a detailed understanding of pathways involved in proton movements through the complex remains largely elusive. Next to the two well-established pathways (termed D and K), an additional proton-conducting network ('H-channel') has been proposed for the beef heart enzyme. Yet, our recent mutational studies on corresponding residues of the Paracoccus denitrificans cytochrome c oxidase provide no clues that such a pathway operates in the prokaryotic enzyme.

Atwal S, Giengkam S, VanNieuwenhze M, Salje J. 2016. Live imaging of the genetically intractable obligate intracellular bacteria Orientia tsutsugamushi using a panel of fluorescent dyes. J Microbiol Methods, 130 pp. 169-176. | Show Abstract | Read more

Our understanding of the molecular mechanisms of bacterial infection and pathogenesis are disproportionally derived from a small number of well-characterised species and strains. One reason for this is the enormous time and resources required to develop a new organism into experimental system that can be interrogated at the molecular level, in particular with regards to the development of genetic tools. Live cell imaging by fluorescence microscopy is a powerful technique to study biological processes such as bacterial motility, host cell invasion, and bacterial growth and division. In the absence of genetic tools that enable exogenous expression of fluorescent proteins, fluorescent chemical probes can be used to label and track living cells. A large number of fluorescent chemical probes are commercially available, but these have overwhelmingly been applied to the study of eukaryotic cell systems. Here, we present a methodical analysis of four different classes of probes, which can be used to delineate the cytoplasm, nucleic acids, cell membrane or peptidoglycan of living bacterial cells. We have tested these in the context of the important but neglected human pathogen Orientia tsutsugamushi but expect that the methodology would be broadly applicable to other bacterial species.

Giengkam S, Blakes A, Utsahajit P, Chaemchuen S, Atwal S, Blacksell SD, Paris DH, Day NP, Salje J. 2015. Improved Quantification, Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen Orientia tsutsugamushi. PLoS Negl Trop Dis, 9 (8), pp. e0004009. | Show Abstract | Read more

BACKGROUND: Scrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, Orientia tsutsugamushi, is an obligate intracellular bacterium that is transmitted to humans by the bite of a Leptotrombidium mite. Research into the basic mechanisms of cell biology and pathogenicity of O. tsutsugamushi has lagged behind that of other important human pathogens. One reason for this is that O. tsutsugamushi is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on O. tsutsugamushi and also other obligate intracellular pathogens such as those in the Rickettsiales and Chlamydiales families. METHODOLOGY: A clinical isolate of O. tsutsugamushi was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an O. tsutsugamushi-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells. KEY RESULTS: Development of a standardised growth assay to quantify bacterial replication and viability in vitro. Quantitative comparison of different DNA extraction methods. Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement. Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds. Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including different freezing buffers and different rates of freezing. CONCLUSIONS: Here we present a standardised method for comparing the viability of O. tsutsugamushi after purification, treatment and propagation under various conditions. Taken together, we present a body of data to support improved techniques for propagation, purification and storage of this organism. This data will be useful both for improving clinical isolation rates as well as performing in vitro cell biology experiments.

Salje J, van den Ent F, de Boer P, Löwe J. 2011. Direct membrane binding by bacterial actin MreB. Mol Cell, 43 (3), pp. 478-487. | Show Abstract | Read more

Bacterial actin MreB is one of the key components of the bacterial cytoskeleton. It assembles into short filaments that lie just underneath the membrane and organize the cell wall synthesis machinery. Here we show that MreB from both T. maritima and E. coli binds directly to cell membranes. This function is essential for cell shape determination in E. coli and is proposed to be a general property of many, if not all, MreBs. We demonstrate that membrane binding is mediated by a membrane insertion loop in TmMreB and by an N-terminal amphipathic helix in EcMreB and show that purified TmMreB assembles into double filaments on a membrane surface that can induce curvature. This, the first example of a membrane-binding actin filament, prompts a fundamental rethink of the structure and dynamics of MreB filaments within cells.

Salje J, Gayathri P, Löwe J. 2010. The ParMRC system: molecular mechanisms of plasmid segregation by actin-like filaments. Nat Rev Microbiol, 8 (10), pp. 683-692. | Show Abstract | Read more

The ParMRC plasmid partitioning apparatus is one of the best characterized systems for bacterial DNA segregation. Bundles of actin-like filaments are used to push plasmids to opposite poles of the cell, whereupon they are stably inherited on cell division. This plasmid-encoded system comprises just three components: an actin-like protein, ParM, a DNA-binding adaptor protein, ParR, and a centromere-like region, parC. The properties and interactions of these components have been finely tuned to enable ParM filaments to search the cell space for plasmids and then move ParR-parC-bound DNA molecules apart. In this Review, we look at some of the most exciting questions in the field concerning the exact molecular mechanisms by which the components of this self-contained system modulate one another's activity to achieve bipolar DNA segregation.

Salje J. 2010. Plasmid segregation: how to survive as an extra piece of DNA. Crit Rev Biochem Mol Biol, 45 (4), pp. 296-317. | Show Abstract | Read more

Non-essential extra-chromosomal DNA elements such as plasmids are responsible for their own propagation in dividing host cells, and one means to ensure this is to carry a miniature active segregation system reminiscent of the mitotic spindle. Plasmids that are maintained at low numbers in prokaryotic cells have developed a range of such active partitioning systems, which are characterized by an impressive simplicity and efficiency and which are united by the use of dynamic, nucleotide-driven filaments to separate and position DNA molecules. A comparison of different plasmid segregation systems reveals (i) how unrelated filament-forming and DNA-binding proteins have been adopted and modified to create a range of simple DNA segregating complexes and (ii) how subtle changes in the few components of these DNA segregation machines has led to a remarkable diversity in the molecular mechanisms of closely related segregation systems. Here, our current understanding of plasmid segregation systems is reviewed and compared with other DNA segregation systems, and this is extended by a discussion of basic principles of plasmid segregation systems, evolutionary implications and the relationship between an autonomous DNA element and its host cell.

Salje J, Zuber B, Löwe J. 2009. Electron cryomicroscopy of E. coli reveals filament bundles involved in plasmid DNA segregation. Science, 323 (5913), pp. 509-512. | Show Abstract | Read more

Bipolar elongation of filaments of the bacterial actin homolog ParM drives movement of newly replicated plasmid DNA to opposite poles of a bacterial cell. We used a combination of vitreous sectioning and electron cryotomography to study this DNA partitioning system directly in native, frozen cells. The diffraction patterns from overexpressed ParM bundles in electron cryotomographic reconstructions were used to unambiguously identify ParM filaments in Escherichia coli cells. Using a low-copy number plasmid encoding components required for partitioning, we observed small bundles of three to five intracellular ParM filaments that were situated close to the edge of the nucleoid. We propose that this may indicate the capture of plasmid DNA within the periphery of this loosely defined, chromosome-containing region.

Salje J, Löwe J. 2008. Bacterial actin: architecture of the ParMRC plasmid DNA partitioning complex. EMBO J, 27 (16), pp. 2230-2238. | Show Abstract | Read more

The R1 plasmid employs ATP-driven polymerisation of the actin-like protein ParM to move newly replicated DNA to opposite poles of a bacterial cell. This process is essential for ensuring accurate segregation of the low-copy number plasmid and is the best characterised example of DNA partitioning in prokaryotes. In vivo, ParM only forms long filaments when capped at both ends by attachment to a centromere-like region parC, through a small DNA-binding protein ParR. Here, we present biochemical and electron microscopy data leading to a model for the mechanism by which ParR-parC complexes bind and stabilise elongating ParM filaments. We propose that the open ring formed by oligomeric ParR dimers with parC DNA wrapped around acts as a rigid clamp, which holds the end of elongating ParM filaments while allowing entry of new ATP-bound monomers. We propose a processive mechanism by which cycles of ATP hydrolysis in polymerising ParM drives movement of ParR-bound parC DNA. Importantly, our model predicts that each pair of plasmids will be driven apart in the cell by just a single double helical ParM filament.

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