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

Larissa Krüger,Rolf Daniel,Ulrike Mäder,Tiago Pedreira,Till Ischebeck,Jörg Stülke,Anja Poehlein,Christina Herzberg,Hermann Rath,Jan Gundlach,Uwe Völker,Jue D Wang

doi:10.1371/journal.pgen.1009092

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.

Highlights

To achieve appropriate responses to changing environmental conditions, all organisms use second messengers which are formed in response to an environmental cue
Among the repertoire of bacterial second messenger nucleotides, cyclic di-AMP (c-di-AMP) stands out as it is the only second messenger that is essential for the bacteria that produce it, including the Grampositive model organism Bacillus subtilis
C-di-AMP plays a major role in the control of potassium homeostasis, and we found that glutamate is toxic to a B. subtilis strain lacking c-di-AMP to the same extent as potassium

Summary

Introduction

To achieve appropriate responses to changing environmental conditions, all organisms use second messengers which are formed in response to an environmental cue. The common nucleotide second messengers cyclic AMP, cyclic di-GMP (c-di-GMP), and guanosine tetra/pentaphosphate ((p)ppGpp) are used to prioritize the utilization of carbon sources and to regulate virulence, to control the choice between sessile and motile lifestyles, and to downregulate essential cellular functions upon amino acid starvation, respectively [2,3,4,5,6,7]. The more recently discovered second messenger cyclic di-AMP is unique in several respects. This nucleotide is essential in many bacteria that produce it, suggesting that this molecule plays a role in central cellular activities. C-di-AMP is recognized by the human immune system It binds the STING protein resulting the induction of an innate immune response [10]

Methods

Results

Discussion

Conclusion