Figure 5. TOXGREEN analysis of bacterial divisome proteins.

a) Schematic representation of the essential membrane proteins of the bacterial divisome of E. coli. The six bitopic proteins are highlighted in blue. ZipA contributes to tethering to the membrane the polymeric FtsZ, which forms the scaffold of the divisome. FtsB and FtsL form a hetero-tetramer mediated by their TM helices and the periplasmic coiled coiled domains. They are recruited to the divisome by FtsQ, which forms with them a ternary complex. FtsI is a Pennicilline Binding Protein important for the synthesis of septal cell wall. FtsN plays an important roled in the regulation of cell divison. It contains a SPOR domain that recognizes the septal peptidoglycan. b) Evolutionary tree of the 11 bacterial species selected for the analysis. These include the gram negative alpha- (C. crescentus, R. prowazekii), beta- (N. meningitidis) and gamma-proteobacteria species (Y. pestis, E. coli, H. influenzae, L. pneumophila, V. cholera), as well as gram positive bacilli (B. subtilis) and cocci (S. pneumonia, S. pyogenes) species. c–h) TOXGREEN analysis of FtsB, FtsL, FtsQ, ZipA/EzrA, FtsI and FtsN sequences from the 11 species. Not all proteins are present in all the species. In particular, ZipA (f) is present only some classes of proteobacteria; for the gram positive species we analyzed EzrA, which is an FtsZ modulator topologically similar to ZipA. TOXGREEN chimera expression levels were verified by Western blot using anti-MBP antibodies (bands displayed under the histograms). It is notable how the chimeras expression levels vary among the samples. In general, it is not possible to draw a precise relationship between the physical strength of association and reporter gene expression in TOXCAT/TOXGREEN, even when the chimera’s expression levels are considered. Empirically, 40% GpA can be taken as a reasonable limit under which the confidence in discriminating specific association from background expression is low.