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

Larissa Krüger, Christina Herzberg, View ORCID ProfileHermann Rath, Tiago Pedreira, Anja Poehlein, Jan Gundlach, View ORCID ProfileRolf Daniel, View ORCID ProfileUwe Völker, View ORCID ProfileUlrike Mäder, View ORCID ProfileJörg Stülke
doi: https://doi.org/10.1101/2020.09.03.280651
Larissa Krüger
aDepartment of General Microbiology, GZMB, Georg-August-University Göttingen, Germany
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Christina Herzberg
aDepartment of General Microbiology, GZMB, Georg-August-University Göttingen, Germany
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Hermann Rath
bInterfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany
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Tiago Pedreira
aDepartment of General Microbiology, GZMB, Georg-August-University Göttingen, Germany
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Anja Poehlein
cDepartment of Genomic and Applied Microbiology, GZMB, Georg-August-University Göttingen, Germany
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Jan Gundlach
aDepartment of General Microbiology, GZMB, Georg-August-University Göttingen, Germany
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Rolf Daniel
cDepartment of Genomic and Applied Microbiology, GZMB, Georg-August-University Göttingen, Germany
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Uwe Völker
bInterfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany
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Ulrike Mäder
bInterfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany
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Jörg Stülke
aDepartment of General Microbiology, GZMB, Georg-August-University Göttingen, Germany
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  • ORCID record for Jörg Stülke
  • For correspondence: jstuelk@gwdg.de
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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) 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.

AUTHOR SUMMARY Bacteria are exposed to constantly changing environmental conditions. In order to respond to these changes, they use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. 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 Gram-positive 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. These toxic conditions were the starting point for an extensive suppressor analysis, which led to the identification of a novel glutamate transporter (AimA). If the B. subtilis strain lacking c-di-AMP was cultivated on complex medium, the isolation of suppressor mutants was only possible under anaerobic conditions and if either of the two c-di-AMP-binding signal transduction proteins was absent. This suggests that these proteins are a major burden for the cell on complex medium in their c-di-AMP free state. Our result underline the complexity of c-di-AMP signaling and propose new directions for research.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted September 03, 2020.
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Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis
Larissa Krüger, Christina Herzberg, Hermann Rath, Tiago Pedreira, Anja Poehlein, Jan Gundlach, Rolf Daniel, Uwe Völker, Ulrike Mäder, Jörg Stülke
bioRxiv 2020.09.03.280651; doi: https://doi.org/10.1101/2020.09.03.280651
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Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis
Larissa Krüger, Christina Herzberg, Hermann Rath, Tiago Pedreira, Anja Poehlein, Jan Gundlach, Rolf Daniel, Uwe Völker, Ulrike Mäder, Jörg Stülke
bioRxiv 2020.09.03.280651; doi: https://doi.org/10.1101/2020.09.03.280651

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