Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

Abstract

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

Cyclic di-AMP Oversight of Counter-Ion Osmolyte Pools Impacts Intrinsic Cefuroxime Resistance in Lactococcus lactis

“…Recent work in S. aureus and B. subtilis identified suppressor mutations in Gln and Glu transporters, respectively, which rescued the growth of mutants devoid of c-di-AMP (9,38). These results align well with those found in L. lactis and suggest that cells defective in c-di-AMP production are unable to regulate intracellular levels of major osmolyte amino acids.…”

Bacterial transcription during growth arrest

“…Intracellular potassium levels are actively modulated by regulatory mechanisms that are sensitive to the metabolic state of the cell. In many Gram-positive bacteria, this regulation involves the small signaling molecule cyclic di-AMP [ 56 ], while in E. coli the nitrogen-sensing branch of the phosphotransferase signal transduction system (PTS Ntr ), which transfers phosphate from phosphoenolpyruvate to the effector protein PtsN in a multistep relay, plays an important role [ 57 , 58 ]. Increased intracellular potassium was shown to increase σ 38 (a.k.a.…”

Sustained Control of Pyruvate Carboxylase by the Essential Second Messenger Cyclic di-AMP in Bacillus subtilis

“…In many bacteria, c-di-AMP is essential for growth on complex media ( 13 , 22 ). In B. subtilis , c-di-AMP is even essential on minimal media in the presence of potassium or glutamate ( 23 , 24 ). While the toxicity of potassium or glutamate can easily be overcome by the acquisition of suppressor mutations, this is not possible on complex media.…”

“…While the toxicity of potassium or glutamate can easily be overcome by the acquisition of suppressor mutations, this is not possible on complex media. However, the deletion of either darA or darB in a B. subtilis strain lacking c-di-AMP (Δ dac ) allows the emergence of suppressor mutations even on complex medium ( 24 ). This observation suggests that the DarA and DarB proteins are involved in harmful interactions in their c-di-AMP free apo-form.…”