c-di-AMP, a likely master regulator of bacterial K ⁺ homeostasis machinery, activates a K ⁺ exporter

Abstract

bis-(3′,5′)-cyclic diadenosine monophosphate (c-di-AMP) is a second messenger with roles in virulence, cell wall and biofilm formation, and surveillance of DNA integrity in many bacterial species, including pathogens. Strikingly, it has also been proposed to coordinate the activity of the components of K+ homeostasis machinery, inhibiting K+ import, and activating K+ export. However, there is a lack of quantitative evidence supporting the direct functional impact of c-di-AMP on K+ transporters. To gain a detailed understanding of the role of c-di-AMP on the activity of a component of the K+ homeostasis machinery in B. subtilis, we have characterized the impact of c-di-AMP on the functional, biochemical, and physiological properties of KhtTU, a K+/H+ antiporter composed of the membrane protein KhtU and the cytosolic protein KhtT. We have confirmed c-di-AMP binding to KhtT and determined the crystal structure of this complex. We have characterized in vitro the functional properties of KhtTU and KhtU alone and quantified the impact of c-di-AMP and of pH on their activity, demonstrating that c-di-AMP activates KhtTU and that pH increases its sensitivity to this nucleotide. Based on our functional and structural data, we were able to propose a mechanism for the activation of KhtTU by c-di-AMP. In addition, we have analyzed the impact of KhtTU in its native bacterium, providing a physiological context for the regulatory function of c-di-AMP and pH. Overall, we provide unique information that supports the proposal that c-di-AMP is a master regulator of K+ homeostasis machinery.

A Central Role for Magnesium Homeostasis during Adaptation to Osmotic Stress

“…Cyclic di-AMP has been implicated in growth under osmotic stress due to its central role in coordinating K + homeostasis ( 26 , 35 , 36 ). However, c-di-AMP also binds MgtE ( 37 ).…”

“…Recently, the dinucleotide second messenger c-di-AMP has been implicated in the control of K + and compatible solute transport, suggesting that it acts as a central regulator of osmoadaptation ( 24 ). Increased c-di-AMP levels inhibit both transcription and activity of the osmotically induced K + transporters KimA and KtrAB and activate the K + exporters CpaA and KhtU ( 36 , 37 , 65 ). Furthermore, increased c-di-AMP inhibits compatible solute uptake by the Opu-family proteins ( 35 , 46 ).…”

Homeostatic control of c-di-AMP synthase (MsDisA) and hydrolase (MsPDE) fromMycobacterium smegmatis

“…These processes recruit different antiporter systems. A well-known example is the potassium/proton antiport system in B. subtilis , regulated by c-di-AMP during osmotic imbalance (34). An interesting observation was made when we, further measured the intracellular and extracellular pH of wild type M. smegmatis mc 2 155 and M. smegmatis transformed with msdisA overexpression gene (MsDisAOE strain) during their growth in the alkaline media.…”

Adaptation ofListeria monocytogenesto perturbation ofc‐di‐AMPmetabolism underpins its role in osmoadaptation and identifies a fosfomycin uptake system

“…c‐di‐AMP is essential for viability of many bacteria and archaea under standard growth conditions, suggesting that the second messenger fulfils important functions in the cell (Woodward et al ., 2010; Luo and Helmann, 2012; Barker et al ., 2013; Blötz et al ., 2017; Braun et al ., 2019). Indeed, c‐di‐AMP controls potassium homeostasis and transport of other osmolytes (Nelson et al ., 2013; Bai et al ., 2014; Chin et al ., 2015; Moscoso et al ., 2015; Huynh et al ., 2016; Schuster et al ., 2016; Devaux et al ., 2018a; Devaux et al ., 2018b; Rubin et al ., 2018; Quintana et al ., 2019; Wang et al ., 2019; Sikkema et al ., 2020; Zeden et al ., 2018, 2020; Cereija et al ., 2021). Therefore, c‐di‐AMP is a key component in the regulation of the cellular turgor, a physical variable that needs to be tightly regulated (Commichau et al ., 2018).…”