Cytoskeletal Proteins of Bacteria

Our goal is to identify and characterize the major families of dynamic cytoskeletal proteins that exist in bacteria, to determine the various functions that they perform, and to understand how these polymers are regulated spatially and temporally by other factors within the cell. We specialize in using genetics and cell biology to study the function and in vivo assembly dynamics of cytoskeletal polymers in many different species of prokaryotes, including E.coli, Bacillus subtillis, B.megaterium, B.thuringiensis, Mycobacterium smegmatis, and Pseudomonas species.  To complete some of our long term goals, we collaborate with biochemists, structural biologists and physicists to study polymer assembly in vitro and in silico to generate an integrated and detailed mechanistic understanding of cytoskeletal dynamics in prokaryotes.  Studies of the bacterial cytoskeleton will lead to an understanding of how prokaryotic cells generate and maintain their subcellular organization and will provide insight into how the eukaryotic cytoskeleton evolved. 

Identification of a diverse superfamily of cytoskeletal Actin-like proteins (Alps) in bacteria


Bacteria have more than 40 divergent actin-like proteins or Alps (Becker, et. al. 2006; Derman et. al. 2009), most of which are encoded by mobile genetic elements such as plasmids or phage. The expansive actin protein family demonstrates that actins are ancient proteins that likely arose several billion
years ago in a prokaryote.  One of these, Alp7A, is a key component of the segregation system of Bacillus subtilis plasmid pLS20 (Derman et. al. 2009) that works by pushing plasmids apart. Remarkably, Alp7A displays both dynamic instability and treadmilling behavior.  Alp7A assembly in vivo depends upon the Alp7R DNA binding protein and its plasmid binding sites (alp7C). This suggests that Alp7A polymerization is regulated by Alp7R, but exactly how is a question we are now trying to answer.

Alp7A-GFP filaments (green) pushing plasmids (blue) apart over time (seconds, white) (Mol Microbiol. 2009Aug;73(4):534-52)

Work with bacterial actins show that they are remarkably diverse, both in their amino acid sequences and in their biochemical properties, therefore representing an ideal system for understanding the biochemical basis for the distinct polymerization dynamics of different cytoskeletal proteins. 
 

A movie showing a 3D rotation of Alp7A-GFP filaments (green) and plasmids (blue) in B. subtilis. (Mol Microbiol. 2009Aug;73(4):534-52). Scale bar is 5 microns.

Time-lapse movie showing Alp7A-GFP filaments (green) pushing plasmids (blue) apart over the course of 24 seconds in B. subtilis (Mol Microbiol. 2009Aug;73(4):534-52).  Scale bar is 5 microns.

Phylogenetic tree of actin-like proteins (Derman et. al. 2009)




We are characterizing divergent tubulin-related proteins in bacteria.  We first identified TubZ from pBtoxis, and demonstrated that it forms dynamic treadmilling filaments involved in DNA segregation (Larsen et al. 2007). We have recently characterized PhuZ, a divergent tubulin relative encoded by bacteriophage (201phi2-1) that infects Pseudomonas species (Kraemer et al. 2012). To understand the function of PhuZ, we developed a single cell assay to study phage reproduction in vivo. We discovered that phage 201phi2-1 forms a DNA replication factory that is positioned in the center of the cell.
The PhuZ protein forms a cytoskeletal element that acts to position the phage DNA at midcell. After replication, the DNA is packaged into viral capsids on the surface of the replication factory. Our collaborators James Kraemer and David Agard at UCSF determined the crystal structure of PhuZ, and showed that it has a unique C-terminal extension which plays a key role in polymerization.  Our results describe a new paradigm for how phage replicate in bacteria. Surprisingly, the in vivo assembly pathway of this phage is similar to that of  certain eukaryotic herpes viruses, which also assemble viral particles around a DNA replication factory. Herpes viruses are thought to be distantly related to double stranded bacteriophage and share mechanisms involved in capsid assembly and DNA packaging.  Our work establishes for the first time a potential connection between the in vivo assembly pathway between these highly divergent viruses.
 
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A model for replication of large bacteriophage.  After 2012-1 infects a cell, the host chromosome is degraded and short PhuZ filaments appear that eventually extend from the poles of the cell to the phage nucleoid in the center. The PhuZ spindle positions the phage DNA in the center of the cell to allow 2012-1 genomes to be efficiently replicated and/or packaged into the capsids. After the completion of phage assembly, the cell lyses, expelling mature phage into the environment (Kraemer et al. 2012)

A phylogenetic tree showing PhuZ forms a divergent family of tubulins encoded by Pseudomonas phage genomes (PhuZ, green); Clostridial (blue) chromosomes (Cb,Ck,Cl,CA), plasmids (pCL2) and phage (Cst, Cbc); Bacillus plasmids (TubZ, red), representative bacterial FtsZ (black) and eukaryotic α/β tubulina (cyan).  Bootstrap values are given for selected branches. (Kraemer et al. 2012)

PhuZ-GFP forms filaments (green) that position phage DNA (blue) in the center of the cell. (Kraemer et al. 2012)

Characterization of the prokaryotic tubulin family