Essentiality of c-di-AMP inBacillus 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 ( A mino acid im porter 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) suggesting 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.

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“…This is known as bypass of essentiality (BoE), with systematic studies of essential gene bypass having been conducted in yeast (Li et al ., 2019; van Leeuwen et al ., 2020). BoE has been shown for several essential genes in bacteria including zipA , c-di-AMP null mutants, RNase E, bamD , and ftsH (Anupama et al ., 2011; Pichoff et al ., 2015; Krüger et al ., 2021; Wang et al ., 2021; Hart and Silhavy, n.d.). Utilizing a BoE screen we were able to isolate eight double mutants capable of tolerating erfRP deletion (Figure 2A).…”

Molecular mechanisms underlying glyphosate resistance in bacteria

“…Both GltP and GltT belong to the dicarboxylate/amino acid:cation (Na + or H + ) symporter family (Slotboom et al, 1999;Saier et al, 2000b). It is interesting to note that the suppressor screen by Wicke and co-workers (Wicke et al, 2019) did not led to the inactivation of the aimA gene that was recently shown to encode the major glutamate transporter AimA in B. subtilis (Krüger et al, 2021). It is tempting to speculate that AimA has a higher specificity for transporting glutamate as compared to GltT and GltP.…”

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“…Glutamat, sie hemmen die Aufnahme der beiden Ionen oder stimulieren den Abbau von Glutamat. Grüner Stern: aktivierende Mutation; roter Stern: inaktivierende Mutation (modifi ziert aus [11]).…”