Control of cell division by an Acinetobacter baumannii protein with a novel nucleotidyl-cyclase-like fold

The multidrug-resistant sepsis pathogen Acinetobacter baumannii has diverged from model {gamma}-proteobacteria in fundamental ways, hindering efforts to develop new lines of attack against the microbe. A major area of divergence is cell division. The pathogen lacks several widely conserved division enzymes, such as FtsEX, and instead possesses a suite of atypical gene products showing no sequence similarity to any well-characterized proteins. Key among these unusual proteins is AdvA. In previous Tn-seq studies, we identified AdvA as essential for A. baumannii division and fluoroquinolone resistance. The protein comprises an N-terminal transmembrane/periplasmic region connected to a C-terminal unannotated cytoplasmic domain. Interestingly, most advA transposon insertions were lethal unless they occurred within the linker between these two regions. The roles of AdvA in cell division and the basis for positional transposon effects were unclear. Here, we combine mutagenesis with fluorescence localization, two-hybrid, and structural analyses to investigate how AdvA domains function in assembling and activating the A. baumannii divisome. We show that AdvA depletion profoundly disrupts divisome construction at Z-rings. This dependence is based on numerous interactions by AdvA with divisome proteins, with its N-terminal region binding multiple components and the cytoplasmic domain binding one (the early protein ZipA). In addition, we identified substitutions in FtsB and FtsW that suppress AdvA essentiality, consistent with a role in divisome protein activation as well as recruitment. Finally, we determined the structure of the AdvA cytoplasmic domain, which revealed a novel 3D-fold resembling adenylyl/guanylyl cyclases with striking deviations. Most notably, AdvA lacks canonical catalytic and dimerization sites and contains unusual features, including a positively charged tip promoting fluoroquinolone resistance and an extra C-terminal helix essential to divisome interactions and cell division. The critical nature of the most C-terminal structure in AdvA helps explain the positional effects of transposon insertions and facilitated identification of a distant structural homolog in the pathogen Pseudomonas aeruginosa. These results illuminate a new type of control protein governing bacterial division that could be exploited for improved antimicrobial strategies targeting nosocomial infections.