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  current news   Press   selected story    
     
  11th March 2010  
 

Three tales of bacterial cytomotive polymers

 
 




David Popp

IMCB congratulates David Popp, a Research Scientist in Robert Robinson’s lab, on his three recent publications in The Journal of Biological Chemistry and The Journal of Molecular Biology. His most recent paper – “Structure and filament dynamics of the pSK41 actin-like ParM protein: implications for plasmid DNA segregation” – has been selected by the Faculty of 1000. The Faculty of 1000 highlights the most important articles in biology and medicine, which are selected by a panel of more than 5000 leading researchers. This free online service is continuously updated, and article recommendations by faculty members are based on the merit of the article, not the journal in which it appears. For more information on the Faculty of 1000, please click here: http://facultyof1000.com/ .


Title: Structure and filament dynamics of the pSK41 actin-like ParM protein: implications for plasmid DNA segregation.

Author: Popp D, Xu W, Narita A, Brzoska AJ, Skurray RA, Firth N, Ghoshdastider U, Maeda Y, Robinson RC, Schumacher M.

ERATO "Actin Filament Dynamics" Project, Japan Science and Technology Corporation, c/o RIKEN Harima Institute at Spring 8, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan; Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos 138673, Singapore.

Abstract
Type II plasmid partition systems utilize ParM NTPases in coordination with a centromere-binding protein called ParR to mediate accurate DNA segregation, a process critical for plasmid retention. The Staphylococcus aureus pSK41 plasmid is a medically important plasmid that confers resistance to multiple antibiotics, disinfectants and antiseptics. In the first step of partition, the pSK41 ParR binds its DNA centromere to form a superhelical partition complex that recruits ParM, which then mediates plasmid separation. pSK41 ParM is homologous to R1 ParM, a known actin homologue, suggesting that it may also form filaments to drive partition. To gain insight into the partition function of ParM, we examined its ability to form filaments and determined the crystal structure of apoParM to 1.95 Angstrom. The structure shows that pSK41 ParM belongs to the actin/Hsp70 superfamily. Unexpectedly, however, pSK41 ParM shows the strongest structural homology to the archaeal actin-like protein Thermoplasma acidophilum Ta0583, rather than its functional homologue, R1 ParM. Consistent with this divergence, we find that regions shown to be involved in R1 ParM filament formation are not important in formation of pSK41 ParM polymers. These data are also consonant with our finding that pSK41 ParM forms 1-start 10/4 helices very different from the 37/17 symmetry of R1 ParM. The polymerization kinetics of pSK41 ParM also differed from that of R1 ParM. These results indicate that type II NTPases utilize different polymeric structures to drive plasmid segregation.

 
 


 
 

Figure Legend: psK41ParM: Electrostatic surface potential of pSK41 partition proteins.
In the figure, red surfaces correspond to electronegative regions and blue are electropositive regions. The ParR molecules in the partition complex are shown bound to the centromere as surfaces, the DNA as sticks.

Published in J Biol Chem. (2010) Mar 1 [Epub ahead of print].

 

 
 

 

Title: Polymeric Structures and Dynamic Properties of the Bacterial Actin AlfA.

Authors: Popp D, Narita A, Ghoshdastider U, Maeda K, Maéda Y, Oda T, Fujisawa T, Onishi H, Ito K, Robinson RC.

ERATO "Actin Filament Dynamics" Project, Japan Science and Technology Corporation, c/o RIKEN Harima Institute at Spring 8, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan; Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos 138673, Singapore.

Abstract
Abstract: AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 A, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed. Here AlfA adopted a 16/7 helical symmetry, with a repeat of about 387 A. Thin polymers consisting of several intertwining filaments also formed. Observed helical symmetries of AlfA filaments differed from those of other members of the actin family: F-actin, ParM, or MreB. Both ATP and guanosine 5'-triphosphate are able to promote rapid AlfA filament formation with almost equal efficiencies. The helical structure is only preserved under physiological salt concentrations and at a pH between 6.4 and 7.4, the physiological range of the cytoplasm of B. subtilis. Polymerization kinetics are extremely rapid and compatible with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation, making AlfA one of the most efficient polymerizing motors within the actin family. Phosphate release lags behind polymerization, and time-lapse total internal reflection fluorescence images of AlfA bundles are consistent with treadmilling rather than dynamic microtubule-like instability. High-pressure SAX experiments reveal that the stability of AlfA filaments is intermediate between the stability of ParM and the stability of F-actin. These results emphasize that actin-like polymerizing machineries have diverged to produce a variety of filament geometries with diverse properties that are tailored for specific biological processes.


 
 


 

 
 

Figure Legend: AlfA: Filtered electron micrograph of helical AlfA filaments.

Published in J Mol Biol. (2010) Feb 12. [Epub ahead of print].

 
 

Title: Suprastructures and dynamic properties of mycobacterium tuberculosis FtsZ.

Author: Popp D, Iwasa M, Erickson HP, Narita A, Maeda Y, Robinson RC.

ERATO "Actin Filament Dynamics" Project, Japan Science and Technology Corporation, c/o RIKEN Harima Institute at Spring 8, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan; Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos 138673, Singapore.

Abstract
Tuberculosis causes the most death in humans by any bacterium. Drug targeting of bacterial cytoskeletal proteins requires detailed knowledge of the various filamentous suprastructures and dynamic properties. Here we have investigated by high-resolution electron microscopy the assembly of cell division protein and microtubule homolog FtsZ from Mycobacterium tuberculosis (MtbFtsZ) in vitro in the presence of various monovalent salts, crowding agents and polycations. Supramolecular structures including 2-D rings, 3-D toroids and multistranded helices formed in the presence of molecular crowding were similar to those observed by fluorescence microscopy in bacteria in vivo. Dynamic properties of MtbFtsZ filaments were visualized by light scattering and real time TIRF microscopy. Interestingly MtBFtsZ revealed a form of dynamic instability at steady state. Cation induced condensation phenomena of bacterial cytomotive polymers have not been investigated in any detail, although it is known that many bacteria can contain high amounts of polycations, which may modulate the prokaryotic cytoskeleton. We find that above a threshold concentration of polycations which varied with the valence of the cation, ionic strength and pH, MtbFtsZ mainly formed sheets. The general features of these cation induced condensation phenomena could be explained in the framework of the Manning condensation theory. Chirality and packing defects limited the dimensions of sheets and toroids at steady state as predicted by theoretical models. In first approximation simple physical principles seem to govern the formation of MtbFtsZ suprastructures.

 
 


 
 

Figure Legend: MtBFtsZ: Electron micrograph of a MtbFtsZ toroid. The arrow points to packing defects, scale bar, 200 nm.

Published in J Biol Chem. (2010) Feb 5. [Epub ahead of print]

For more information on Robert Robinson's Lab, please click here.