Bacillus anthracis Tubulin-Related Protein Ba-TubZ Assembles Force-Generating Polymers Ramanujam Srinivasan, 1 Mithilesh Mishra, 2 Fong Yew Leong, 3 Keng-Hwee Chiam, 1,3 and Mohan Balasubramanian 1,2,4 * 1 Mechanobiology Institute, The National University of Singapore, 5A Engineering Drive 1, Singapore 117411, Singapore 2 Temasek Life Sciences Laboratory, The National University of Singapore, 1 Research Link, Singapore 117604, Singapore 3 A*STAR Institute of High Performance Computing, 1 Fusionopolis Way, #16-16 Connexis, Singapore 138632, Singapore 4 Department of Biological Sciences, The National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore Received 4 January 2011; Revised 8 July 2011; Accepted 13 July 2011 Monitoring Editor: George Bloom Pathogenicity of Bacillus anthracis depends on the faithful inheritance of plasmid pXO1, in a process that requires the plasmid encoded tubulin-related protein Ba-TubZ. Here we show, using heterologous expression in Schizosaccharomyces pombe, that Ba-TubZ assembles into a dynamic polymer in the absence of other B. anthracis proteins and can generate force capable of deforming the fission yeast nuclear envelope. The poly- mer bundles contain 27 6 15 protofilaments/lm assuming that each protofilament spans the entire length. Thinner appearing buckled and thicker appear- ing straight filaments of Ba-TubZ were both capable of inducing nuclear envelope deformation. Unlike the related protein Bt-TubZ from Bacillus thuringiensis, which undergoes treadmilling upon expression in fis- sion yeast, Ba-TubZ polymers did not undergo detecta- ble treadmilling. Instead, in fluorescence recovery after photobleaching experiments, it displayed a different turnover behavior characterized by moderate fluores- cence recovery along the entire length of the polymer. Modeling Ba-TubZ bundles as Euler–Bernoulli beams that buckle under compressive loads when pushed against the nuclear envelope allowed us to estimate that Ba-TubZ generates forces in the order of 1–10 nN. We propose that polymerization based filament elonga- tion and force generation might aid faithful segregation of the virulence plasmid. V C 2011 Wiley-Liss, Inc. Key Words: bacteria, fission yeast, cytoskeleton, plasmid segregation, virulence Introduction A lthough the lack of a detectable cytoskeleton was once considered a hallmark of prokaryotes, this view has changed dramatically in the last two decades. Cur- rently four major families of cytoskeletal proteins have been identified in prokaryotes [Michie and Lowe, 2006; Erickson, 2007; Adams and Errington, 2009; Margolin, 2009]. These include the three major families (actin, tubulin, and intermediate filaments) known in eukarya, as well as polymers composed of Walker A domain contain- ing ATPases. Members of the actin family, such as MreB and ParM, function in cell shape maintenance and/or DNA segregation [Jones et al., 2001; Moller-Jensen et al., 2002; Garner et al., 2004; Gitai et al., 2005; Kruse et al., 2005; Kruse and Gerdes, 2005]. Members of the tubulin- related FtsZ family largely participate in the physical mechanism of cytokinesis, although some members of the tubulin family, such as TubZ participate in DNA segrega- tion [Larsen et al., 2007; Anand et al., 2008; Chen and Erickson, 2008; Akhtar et al., 2009]. Proteins such as crescentin from Caulobacter crescentus assemble into poly- mers resembling intermediate filaments and are known to regulate morphogenesis [Ausmees et al., 2003; Cabeen et al., 2009]. The diversity in the known polymer-forming protein families in prokaryotes and the ability of prokary- otic proteins, such as Walker A ATPases, as yet unknown to form cytoskeletal structures in eukaryotes, presents an unprecedented opportunity to generate new insight into the evolution and function of the cytoskeleton. The bacterial cytoskeleton is also used by pathogens to faithfully segregate plasmids encoding pathogenicity fac- tors. In recent years, at least two members of the DNA segregating tubulin family of cytoskeletal proteins have been discovered in Bacilli [Tinsley and Khan, 2006; Larsen et al., 2007; Margolin, 2007; Anand et al., 2008; Chen and Erickson, 2008; Akhtar et al., 2009; Lowe and Additional Supporting Information may be found in the online version of this article. R. S. and M. M. contributed equally to this work. *Address correspondence to: Mohan Balasubramanian, Temasek Life Sciences Laboratory, The National University of Singapore, Singapore 117604, Singapore. E-mail: mohan@tll.org.sg Published online 20 July 2011 in Wiley Online Library (wileyonlinelibrary.com). RESEARCH ARTICLE Cytoskeleton, September 2011 68:501–511 (doi: 10.1002/cm.20526) V C 2011 Wiley-Liss, Inc. 501 n