The KupA and KupB Proteins of Lactococcus lactis IL1403 Are Novel c-di-AMP Receptor Proteins Responsible for Potassium Uptake

Abstract

Cyclic di-AMP (c-di-AMP) is a second messenger involved in diverse metabolic processes, including osmolyte uptake, cell wall homeostasis, and antibiotic and heat resistance. In Lactococcus lactis, a lactic acid bacterium which is used in the dairy industry and as a cell factory in biotechnological processes, the only reported interaction partners of c-di-AMP are the pyruvate carboxylase and BusR, the transcription regulator of the busAB operon for glycine betaine uptake. However, recent studies uncovered a major role of c-di-AMP in the control of potassium homeostasis, and potassium is the signal that triggers c-di-AMP synthesis. In this study, we have identified KupA and KupB, which belong to the Kup/HAK/KT family, as novel c-di-AMP binding proteins. Both proteins are high-affinity potassium transporters, and their transport activities are inhibited by binding of c-di-AMP. Thus, in addition to the well-studied Ktr/Trk potassium channels, KupA and KupB represent a second class of potassium transporters that are subject to inhibition by c-di-AMP. IMPORTANCE Potassium is an essential ion in every living cell. Even though potassium is the most abundant cation in cells, its accumulation can be toxic. Therefore, the level of potassium has to be tightly controlled. In many Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in the control of potassium homeostasis by binding to potassium transporters and regulatory proteins and RNA molecules. In the lactic acid bacterium Lactococcus lactis, none of these conserved c-di-AMP-responsive molecules are present. In this study, we demonstrate that the KupA and KupB proteins of L. lactis IL1403 are high-affinity potassium transporters and that their transport activity is inhibited by the second messenger c-di-AMP.

c-di-AMP hydrolysis by the phosphodiesterase AtaC promotes differentiation of multicellular bacteria

“…Changes in external osmolarity trigger water fluxes across the membrane, which can lead to cell dehydration or swelling and, finally, collapse or burst when osmobalance mechanisms fail to respond properly (5). As a key component of these mechanisms, c-di-AMP directly targets transport systems for osmoactive and osmoprotective substances such as potassium ions and low-molecular-weight compatible solutes in many bacteria (6)(7)(8)(9)(10).…”

Environmental potassium regulates bacterial flotation, antibiotic production and turgor pressure in Serratia through the TrkH transporter

“…Interestingly, experiments in Gram‐positive bacteria showed that the sensor kinase, KdpD is inhibited after cyclic‐di‐AMP (c‐di‐AMP) binding (Bai et al, ; Moscoso et al, ). Multiple recent reports lately have shown the impact of c‐di‐AMP in osmoregulation and its interaction with a TrkH effector protein (Zarrella et al, ; Pham and Turner, ; Quintana et al, ). However, c‐di‐AMP is not synthesized in Gammaproteobacteria (Commichau et al, ), such as S39006.…”

Identification of the main glutamine and glutamate transporters inStaphylococcus aureusand their impact on c-di-AMP production

“…Over the last decade, considerable evidence has emerged that c-di-AMP plays a major role in osmotic regulation, primarily by positively regulating potassium export or negatively regulating potassium and osmolyte uptake (Rocha et al ., 2019, Quintana et al ., 2019, Kim et al ., 2015, Corrigan et al ., 2013, Moscoso et al ., 2015, Chin et al ., 2015, Huynh et al ., 2016, Schuster et al ., 2016, Pham & Turner, 2019, Pham et al ., 2018, Devaux et al ., 2018, Zarrella et al ., 2018, Gundlach et al ., 2017b, Gundlach et al ., 2017a, Gundlach et al ., 2017c, Gundlach et al ., 2019). However, individual c-di-AMP target proteins identified thus far are themselves not essential.…”

“…There is now considerable evidence that one such messenger, cyclic di-adenosine monophosphate (c-di-AMP) plays a significant role in osmoregulation in bacteria (Pham et al ., 2018, Pham & Turner, 2019, Quintana et al ., 2019, Zarrella et al ., 2018, Teh et al ., 2019, Fahmi et al ., 2019, Devaux et al ., 2018, Bai et al ., 2014, Zeden et al ., 2018, Corrigan et al ., 2011, Rocha et al ., 2019, Gundlach et al ., 2017b, Gundlach et al ., 2017a, Witte et al ., 2013, Whiteley et al ., 2015, Whiteley et al ., 2017). c-di-AMP binds to and negatively regulates a number of different potassium and osmolyte importers (Rocha et al ., 2019, Quintana et al ., 2019, Kim et al ., 2015, Corrigan et al ., 2013, Moscoso et al ., 2015, Chin et al ., 2015, Huynh et al ., 2016, Schuster et al ., 2016, Pham & Turner, 2019, Pham et al ., 2018, Devaux et al ., 2018, Zarrella et al ., 2018, Gundlach et al ., 2017b, Gundlach et al ., 2017a, Gundlach et al ., 2017c). c-di-AMP is essential for bacterial growth under standard growth conditions but it is also toxic at high levels in many Firmicutes, hence its cellular levels must be tightly regulated (Gundlach et al ., 2015b, Mehne et al ., 2013, Corrigan et al ., 2011, Corrigan et al ., 2015, Woodward et al ., 2010, Witte et al ., 2013).…”

“…Previously, we found that inactivation of the main glycine betaine transporter OpuD as well as the predicted amino acid transporter AlsT (SAUSA300_1252) allows an S. aureus dacA mutant to grow in rich medium in the absence of c-di-AMP (Zeden et al ., 2018). In several other Firmicutes, including B. subtilis, Lactococcus lactis, Streptococcus pneumoniae, Streptococcus pyogenes and L. monocytogenes , inactivating mutations have also been identified in osmolyte and potassium transport systems that allow these bacteria to grow in the absence of c-di-AMP (Pham et al ., 2018, Pham & Turner, 2019, Quintana et al ., 2019, Zarrella et al ., 2018, Teh et al ., 2019, Fahmi et al ., 2019, Devaux et al ., 2018, Bai et al ., 2014, Zeden et al ., 2018, Corrigan et al ., 2011, Rocha et al ., 2019, Gundlach et al ., 2017b, Gundlach et al ., 2017a, Witte et al ., 2013, Whiteley et al ., 2015, Whiteley et al ., 2017). This is consistent with the idea that in the absence of c-di-AMP, potassium and osmolyte transporters are more active, resulting in the accumulation of toxic levels of potassium and osmolytes in the cell.…”