Hexameric rings of the scaffolding protein CheW enhance response sensitivity and cooperativity in Escherichia coli chemoreceptor arrays.

The Escherichia coli chemoreceptor array is a supramolecular assembly that enables cells to respond to extracellular cues dynamically and with great precision and sensitivity. In the array, transmembrane receptors organized as trimers of dimers are connected at their cytoplasmic tips by hexameric rings of alternating subunits of the kinase CheA and the scaffolding protein CheW (CheA-CheW rings). Interactions of CheW molecules with the members of receptor trimers not directly bound to CheA-CheW rings may lead to the formation of hexameric CheW rings in the chemoreceptor array. Here, we detected such CheW rings with a cellular cysteine-directed cross-linking assay and explored the requirements for their formation and their participation in array assembly. We found that CheW ring formation varied with cellular CheW abundance, depended on the presence of receptors capable of a trimer-of-dimers arrangement, and did not require CheA. Cross-linking studies of a CheA~CheW fusion protein incapable of forming homomeric CheW oligomers demonstrated that CheW rings were not essential for the assembly of CheA-containing arrays. Förster resonance energy transfer (FRET)-based kinase assays of arrays containing variable amounts of CheW rings revealed that CheW rings enhanced the cooperativity and the sensitivity of the responses to attractants. We propose that six-membered CheW rings provide the additional interconnectivity required for optimal signaling and gradient tracking performance by chemosensory arrays.

A Bacterial Inflammation Sensor Regulates c-di-GMP Signaling, Adhesion, and Biofilm Formation.

Bacteria that colonize animals must overcome, or coexist, with the reactive oxygen species products of inflammation, a front-line defense of innate immunity. Among these is the neutrophilic oxidant bleach, hypochlorous acid (HOCl), a potent antimicrobial that plays a primary role in killing bacteria through nonspecific oxidation of proteins, lipids, and DNA. Here, we report that in response to increasing HOCl levels, Escherichia coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We identify the mechanism of DgcZ sensing of HOCl to be direct oxidation of its regulatory chemoreceptor zinc-binding (CZB) domain. Dissection of CZB signal transduction reveals that oxidation of the conserved zinc-binding cysteine controls CZB Zn2+ occupancy, which in turn regulates the catalysis of c-di-GMP by the associated GGDEF domain. We find DgcZ-dependent biofilm formation and HOCl sensing to be regulated in vivo by the conserved zinc-coordinating cysteine. Additionally, point mutants that mimic oxidized CZB states increase total biofilm. A survey of bacterial genomes reveals that many pathogenic bacteria that manipulate host inflammation as part of their colonization strategy possess CZB-regulated diguanylate cyclases and chemoreceptors. Our findings suggest that CZB domains are zinc-sensitive regulators that allow host-associated bacteria to perceive host inflammation through reactivity with HOCl. IMPORTANCE Immune cells are well equipped to eliminate invading bacteria, and one of their primary tools is the synthesis of bleach, hypochlorous acid (HOCl), the same chemical used as a household disinfectant. In this work, we present findings showing that many host-associated bacteria possess a bleach-sensing protein that allows them to adapt to the presence of this chemical in their environment. We find that the bacterium Escherichia coli responds to bleach by hunkering down and producing a sticky matrix known as biofilm, which helps it aggregate and adhere to surfaces. This behavior may play an important role in pathogenicity for E. coli and other bacteria, as it allows the bacteria to detect and adapt to the weapons of the host immune system.