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

Abstract

The antibiotic-resistant pathogen Acinetobacter baumannii has diverged from model {psi}-proteobacteria in fundamental ways, complicating the development of new antimicrobial strategies. A major area of divergence is cell division. A. baumannii lacks several widely conserved division enzymes, such as FtsEX, and instead possesses a suite of atypical gene products with no similarity to well-characterized proteins. Key among these is AdvA, which we previously identified by Tn-seq 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, and most advA transposon insertions were lethal unless they occurred within the linker between these regions. The roles of AdvA in cell division and the basis for these positional transposon effects were unclear. Here, we combine mutagenesis with fluorescence localization, two-hybrid, and structural analyses to define how AdvA domains function in assembling and activating the A. baumannii divisome. AdvA depletion profoundly disrupts divisome construction at Z-rings. This dependence reflects numerous interactions with divisome proteins, with AdvAs N-terminal region binding multiple components and 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 activation as well as recruitment. Finally, we determined the structure of the cytoplasmic domain, revealing a novel adenylyl/guanylyl cyclase-like fold that lacks canonical catalytic and dimerization sites and instead features a positively charged tip key to fluoroquinolone resistance and a C-terminal helix essential to division. The critical C-terminal structure helps explain the positional transposon effects and facilitated identification of a distant homolog in Pseudomonas aeruginosa. These results reveal a new control protein governing bacterial division that could be exploited to combat nosocomial infections. ImportanceThe multidrug-resistant sepsis pathogen Acinetobacter baumannii poses an urgent threat to public health. Fundamental features of its cell cycle, such as how it controls cell division, are not well understood, but this information could lead to improved antimicrobial strategies. We demonstrate that a protein (AdvA) bearing a previously unrecognized structure has a critical role in cell division and fluoroquinolone antibiotic resistance in the pathogen. The structure resembles the nucleotide cyclase class of enzymes, but it has lost the typical catalytic properties and instead uses novel sites to enable assembly and activation of the cell division machine. The novel fold is found in other pathogens such as Pseudomonas aeruginosa, in which it is also connected to drug resistance and cell division. This work opens new avenues to understand and interrupt cell division in multidrug-resistant hospital-acquired bacteria.