Abstract
The broadly conserved bacterial signalling molecule cyclic-di-adenosine monophosphate (c-di-AMP) controls osmoresistance via its regulation of potassium (K+) and compatible solute uptake. High levels of c-di-AMP resulting from inactivation of c-di-AMP phosphodiesterase activity leads to poor growth of bacteria under high osmotic conditions. To better understand how bacteria can adjust in response to excessive c-di-AMP levels and to identify signals that feed into the c-di-AMP network, we characterised genes identified in a screen for osmoresistant suppressor mutants of the high c-di-AMP Lactococcus ΔgdpP strain. Mutations were identified which increased the uptake of osmoprotectants, including gain-of-function mutations in a Kup family K+ importer (KupB) and inactivation of the glycine betaine transporter transcriptional repressor BusR. The KupB mutations increased the intracellular K+ level while BusR inactivation increased the glycine betaine level. In addition, BusR was found to directly bind c-di-AMP and repress expression of the glycine betaine transporter in response to elevated c-di-AMP. Interestingly, overactive KupB activity or loss of BusR triggered c-di-AMP accumulation, suggesting turgor pressure changes act as a signal for this second messenger. In another group of suppressors, overexpression of an operon encoding an EmrB family multidrug resistance protein allowed cells to lower their intracellular level of c-di-AMP through active export. Lastly evidence is provided that c-di-AMP levels in several bacteria are rapidly responsive to environmental osmolarity changes. Taken together, this work provides evidence for a model in which high c-di-AMP containing cells are dehydrated due to lower K+ and compatible solute levels and that this osmoregulation system is able to sense and respond to cellular water stress.
Highlights
In order to survive and grow, bacteria must be able to sense and respond to a multitude of environmental conditions
We show that a Lactococcus lactis GdpP phosphodiesterase mutant with a high intracellular pool of c-di-AMP is able to grow under hyperosmotic conditions after acquiring mutations which increase osmolyte uptake or by actively exporting c-diAMP
In addition we show that c-di-AMP accumulates in response to elevated K+ and glycine betaine uptake, and in several bacteria the c-di-AMP level is rapidly responsive to environmental osmolarity changes, allowing it to sense and respond to water stress
Summary
In order to survive and grow, bacteria must be able to sense and respond to a multitude of environmental conditions. In response to an osmotic upshift (hyperosmotic stress), bacteria import potassium ions (K+) which is followed by a secondary response involving uptake or synthesis of compatible solutes such as glycine betaine, carnitine and proline [2] This allows the cell to limit the loss of water and maintain turgor. The speed at which cells need to detect and respond to the external osmolarity change is critical and the early responses in many cases involves posttranslational modulation of existing transporter activity, since the synthesis of new proteins can take too long [3] These transporters include membrane stretch-activated mechanosensitive channels that activate during an osmotic downshift and intracellular ionic strength or K+ activated compatible solute uptake systems that function during an osmotic upshift [4]
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