Critical Role for the Unique N-Terminus of Chlamydial MreB in Directing Its Membrane Association and Interaction with Elements of the Divisome

Abstract

9Chlamydiae lack the conserved central coordinator protein of cell division FtsZ, a 10 tubulin-like homolog. Current evidence indicates Chlamydia uses the actin-like homolog, 11 MreB, to substitute for the role of FtsZ. Interestingly, we observed MreB as a ring at the 12 septum in dividing cells of Chlamydia. We hypothesize that MreB, to substitute for FtsZ in 13 Chlamydia, must possess unique properties compared to canonical MreB orthologs. 14 Sequence differences between chlamydial MreB and orthologs in other bacteria revealed that 15 chlamydial MreB possesses an extended N-terminal region and the conserved amphipathic 16 helix found in other bacterial MreBs. The extended N-terminal region was sufficient to 17 restore the localization of a truncated E. coli MreB mutant lacking its amphipathic helix to 18 the membrane and was crucial for interactions with cell division components RodZ and FtsK, 19 though the region was not required for homotypic interactions. Importantly, the N-terminal 20 region was sufficient to direct GFP to the membrane when expressed in Chlamydia. A mutant 21 N-terminal region with reduced amphipathicity was unable to perform these functions. From 22 these data, the extended N-terminal region of chlamydial MreB is critical for localization and 23 interactions of this protein. Our data provide mechanistic support for chlamydial MreB to 24 serve as a substitute for FtsZ. 25 Word Count: 200/200 26 27 28 29 30 31 Importance 32Chlamydia trachomatis is an obligate intracellular pathogen, causing sexual 33 transmitted diseases and trachoma. Studying chlamydial physiology, especially its cell 34 division mechanism, is important for developing novel therapeutic strategies for the 35 treatment of these diseases. Since chlamydial cell division has unique features, including a 36 polarized cell division process independent of FtsZ, a canonical cell division coordinator, 37 studying the subject is helpful for understanding undefined aspects of chlamydial growth. In 38 this study, we characterized MreB, a substitute for FtsZ, as a cell division coordinator. It 39 forms a filamentous ring at the septum, like FtsZ in E. coli. We show that the localization of 40 MreB is dependent upon the amphipathic nature of its extended N-terminus. Furthermore, 41 this region is crucial for its interaction with other proteins involved in cell division. Given 42 these results, chlamydial MreB may function as a scaffold for cell divisome proteins at the 43 septum and regulate cell division in this organism. 44 Word count: 150/150 45 48 two morphologically and functionally distinct cellular forms during their developmental 49 cycle: the elementary body (EB) and the reticulate body (RB)(1). The EB is the infectious but 50 non-dividing form whereas the RB is the dividing but non-infectious form. After entering the 51 host cell, Chlamydia remains within a membrane-bound parasitic organelle, termed an 52 inclusion(2). RBs undergo multiple rounds of cell division until they engage a secondary 53 differentiation program and...