How does the ParABC system segregate low copy number plasmids in bacteria?

Martin Howard Dept of Systems BiologyJohn Innes CentreNorwich, UK

Patterning in Bacteria• Traditional view of bacterial organization:

“randomly filled bag”

• But this isn’t true at all!

• Many processes where proteins are precisely localized in space and time

• Several key examples have emerged, including:

Chemotaxis (polar localization of chemotactic receptors)

Min system (how to divide accurately)

• How is precise positioning achieved?• Self-organization: Howard & Kruse, J. Cell Biol. 168 533 (2005)

MinCDE Oscillations

• MinE stimulates coherent pole to pole oscillations of MinCDE

• Centre of cell marked by minimum MinC/MinD concentration

MinD Oscillations

Hale, Meinhardt, de Boer:

EMBO J. 20 1563 (2001)

MinCDE Oscillations

• Formation of oscillating MinE ring structure

Fu, Shih, Zhang, Rothfield: PNAS 98 980 (2001)

• Centre of cell marked by minimum MinC/MinD concentration

MinE Oscillations

Hale, Meinhardt, de Boer:

EMBO J. 20 1563 (2001)

MinD in Filamentous Cells

Raskin & de Boer: PNAS (1999)

• Induce filamentous cells by deleting FtsZ

• Clear evidence for characteristic wavelength

Modelling the Min System

• Many mathematical models of the Min system

Howard et al: Phys. Rev. Lett. (2001) Meinhardt et al: PNAS (2001) Kruse: Biophys J. (2002)

• All have common basis: oscillations result from a dynamical instability resulting from intrinsic interactions of Min proteins

Howard & Kruse: JCB (2005) Kruse, Howard & Margolin: Mol. Microbiol. (2007)

• Models can explain characteristic wavelength of Min patterning important in identifying underlying mechanism

Introduction to Plasmids

• DNA not on main chromosomes that can replicate independently

• Sometimes present at very low copy numbers

• Causes a problem at cell division: how to ensure plasmid are transmitted reliably to both daughter cells?

Importance:

• Encode important functions e.g. antibiotic resistance or virulence

• Method of segregation prior to cell division is a primitive ancestor of mitotic apparatus in eukaryotes

Par Dynamics and Plasmid Segregation

• How are low copy number plasmids segregated in bacteria?

• Often through oscillatory dynamics of ParABC system

• Without oscillations plasmids are not segregated

• Somehow Par oscillations generate force that moves plasmids!

• What is the mechanism behind the oscillations?

ParA dynamics in E. coli plasmid pB171

Ebersbach & Gerdes: Mol. Microbiol. (2004)

The ParABC System

• ParA: ATPase that binds nonspecifically to DNA

polymerizes on nucleoid surface

• ParB: protein that binds to DNA at parC regions

together form “partition complex”

• ParB interacts with ParA functioning as “adaptor” between ParA and parC

• Dynamics of plasmids/Par proteins occurs along nucleoid surface

Par and Min: Similarities

• ParA/MinD both form polymers

• ParA/MinD are both ATPases

• ParA/MinD both undergo spatiotemporal oscillations, though in difference locations (nucleoid vs membrane)

• ParA oscillations require ParB and parC centromere-like site

• MinD oscillations require MinE

• ParB/MinE stimulate ATPase activity of ParA/MinD

• Are Par dynamics also driven by reaction-diffusion dynamic instability? As in Adachi et al: JMB (2006)

• Is there a characteristic wavelength for Par oscillations?

Ebersbach et al: Mol. Microbiol. (2006)

• Position of plasmid foci seem to depend on the number of plasmids

• For Min-like dynamic instability, number of foci would depend on the length of the nucleoid

• Mechanism of oscillation could be fundamentally different in Par vs Min

Par and Min Oscillations Arise From Different Mechanisms

Niki et al: Mol. Microbiol. (2007)

Pushing or Pulling?• Do ParA filaments push (similar to ParM) or pull?

• No evidence that plasmids directly nucleate ParA

• How are plasmids close together separated?

• More likely to be by pulling

Plasmid: red ParA: green

Conclusions• Many similarities between Min and Par

• Nevertheless, new mechanism may be needed for Par dynamics

• Pulling force generated by depolymerisation of ParA filaments by ParB/parC