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

Abstract

Acinetobacter baumannii has diverged substantially from model {gamma}-proteobacteria, complicating our understanding of the antibiotic-resistant pathogen. A major area of divergence is cell division. A. baumannii lacks widely conserved division enzymes such as FtsEX and relies on gene products with no similarity to well-characterized proteins. Key among these is AdvA, which we previously identified by Tn-seq as essential for division. AdvA comprises an N-terminal membrane/periplasmic region linked to an unannotated cytoplasmic domain, and most advA transposon insertions were lethal except within the linker. How AdvA functions and why insertions showed this positional bias were unclear. Here we combine genetic, localization, two-hybrid, and structural analyses to define roles for AdvA domains in cell division. Removing the N-terminal region or partially truncating the cytoplasmic domain caused lethal division defects. The lethal division block in AdvA-deficient bacteria was associated with failure to recruit early and late divisome proteins to Z-rings but was suppressed by mutations in late septal synthesis genes (ftsB and ftsW). The cytoplasmic domain bound the early Z-ring protein ZipA, while the N-terminal region mediated dimerization, and both contributed to Z-ring localization. Finally, the crystal structure of the cytoplasmic domain revealed a highly divergent adenylyl/guanylyl cyclase-like fold lacking canonical catalytic and dimerization sites and possessing unusual features, including a positively charged tip and extra C-terminal helix, critical for ZipA binding and division. The essential C-terminal addition helps explain the transposon and truncation effects and facilitated identification of a distant homolog in Pseudomonas aeruginosa. These results reveal an atypical cyclase-like protein governing bacterial division. Importance: The multidrug-resistant sepsis pathogen Acinetobacter baumannii poses an urgent threat to public health. How A. baumannii accomplishes cell division without key conserved factors is 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 in the pathogen. The structure resembles nucleotide cyclase enzymes, but it has lost their typical catalytic properties and instead uses noncanonical sites and structural additions to enable assembly of the cell division machine. This atypical domain is found in other pathogens such as Pseudomonas aeruginosa, in which it is also connected to cell division. This work opens new avenues to understand division in multidrug-resistant hospital-acquired bacteria.