Bacteria often use cyclic dinucleotides as second messengers for signal transduction. While the classical molecule c-di-GMP is involved in lifestyle selection, the functions of the more recently discovered signaling nucleotide cyclic di-AMP are less defined. For many Gram-positive bacteria, c-di-AMP is essential for growth suggesting its involvement in a key cellular function. We have analyzed c-di-AMP signaling in the genome-reduced pathogenic bacterium Mycoplasma pneumoniae. Our results demonstrate that these bacteria produce c-di-AMP, and we could identify the diadenylate cyclase CdaM (MPN244). This enzyme is the founding member of a novel family of diadenylate cyclases. Of two potential c-di-AMP degrading phosphodiesterases, only PdeM (MPN549) is active in c-di-AMP degradation, whereas NrnA (MPN140) was reported to degrade short oligoribonucleotides. As observed in other bacteria, both the c-di-AMP synthesizing and the degrading enzymes are essential for M. pneumoniae suggesting control of a major homeostatic process. To obtain more insights into the nature of this process, we have identified a c-di-AMP-binding protein from M. pneumoniae, KtrC. KtrC is the cytoplasmic regulatory subunit of the low affinity potassium transporter KtrCD. It is established that binding of c-di-AMP inhibits the KtrCD activity resulting in a limitation of potassium uptake. Our results suggest that the control of potassium homeostasis is the essential function of c-di-AMP in M. pneumoniae.

The following Supporting Information is available for this article: Fig. S1 Principal component analysis of the normalized transcriptome data obtained from RNAseq analysis.Fig. S2 Salicylic acid (SA) treatment does not increase TGA-dependent activation of the DLO1 promoter in mesophyll protoplasts.Fig. S3 TGA1 with mutated cysteines does not lead to increased basal SARD1 transcript levels.Fig. S4 Clade-II TGAs are not important for ICS1 expression after infection with Pseudomonas syringae pv. maculicola ES4356 (Psm). Table S1Primers used for qRT-PCR. Table S2Expression Data of 2090 salicylic acid-inducible genes. Table S3Fold change in selected transcripts as identified by RNAseq analysis of fourweek old Arabidopsis sid2 and sid2 tga1 tga4 treated with water (mock) or 1 mM salicylic acid (SA) for 8 hours.Methods S1 Detailed description of methods.Notes S1 Maps and sequences of plasmids used in this work.

Glutaredoxins (GRXs) are small proteins that interact with the atypical tripeptide glutathione (GSH), a redox active metabolite that forms a disulfide (GSSG) upon oxidation. GRXs encoding variants of a CPYC motif at a conserved active site act as GSH-dependent thiol-disulfide oxidoreductases (class I). GRXs with a CGFS site (class II) bind GSH in a way that is non-permissive for thiol-disulfide oxidoreductase reactions and favours transient iron-sulfur cluster binding. The biochemical functions of CCxC/S-type (class III) GRXs, which are found only in land plants, have remained largely unexplored. In this study, we characterized the in vitro properties of one of the Arabidopsis thaliana class III GRXs, namely ROXY9. In contrast to class I GRX AtGRXC2, ROXY9 was inactive as a reductase on the small substrate bis(2-hydroxyethyl)disulfide (HEDS) and as an oxidase on the redox-sensitive fluorescent protein roGFP2. Redox titrations with different GSH/GSSG ratios revealed formation of a disulfide bond (S2) between the first and the last cysteine of the CCLC motif at -220 to -230 mV. Glutathionylation (SG) at the first reactive cysteine was not observed. In contrast, AtGRXC2 and another class I GRX, HsGRX1, were oxidized to roughly equal amounts of GRX-SG and GRX-S2 over a wide range of GSH/GSSG ratios. The different reactivity of ROXY9 towards GSSG as compared to class I GRXs is most likely due to subtle differences in the GSH binding mode and explains why ROXY9 does not function as a GSH-dependent oxidoreductase on standard substrates. This feature might have evolved to avoid overlapping functions with class I GRXs in planta.

SummarySalicylic acid (SA) is an important signaling molecule of the plant immune system.SA biosynthesis is indirectly modulated by the closely related transcription factors TGA1 (TGACG-BINDING FACTOR 1) and TGA4. They activate expression of SARD1 (SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1), the gene product of which regulates the key SA biosynthesis gene ICS1 (ISOCHORISMATE SYNTHASE 1).Since TGA1 interacts with the SA receptor NPR1 (NON EXPRESSOR OF PATHOGENESIS-RELATED GENES 1) in a redox-dependent manner and since the redox state of TGA1 is altered in SA-treated plants, TGA1 was assumed to play a role in the NPR1-dependent signaling cascade. Here we identified 193 out of 2090 SA-induced genes that require TGA1/TGA4 for maximal expression after SA treatment. One robustly TGA1/TGA4-dependent gene encodes for the SA hydroxylase DLO1 (DOWNY MILDEW RESISTANT 6-LIKE OXYGENASE 1) suggesting an additional regulatory role of TGA1/TGA4 in SA catabolism.Expression of TGA1/TGA4-dependent genes in mock/SA-treated or Pseudomonas-infected plants was rescued in the tga1 tga4 double mutant after introduction of a mutant genomic TGA1 fragment encoding a TGA1 protein without any cysteines. Thus, the functional significance of the observed redox modification of TGA1 in SA-treated tissues has remained enigmatic.SIGNIFICANCE STATEMENTPrevious findings demonstrating a redox-dependent interaction between transcription factor TGA1 and NPR1 attracted considerable attention. Here we show that TGA1 can act in the NPR1- and SA-dependent signaling cascade, but that its SA-regulated redox-active cysteines do not affect its function in this process.

To identify cytosolic proteins that bind to cyclic di-AMP, a biotinylated analog of the nucleotide is used for protein pull-down experiments. In this approach, biotinylated c-di-AMP is coupled to Streptactin-covered beads. After protein separation using standard SDS-PAGE, the protein(s) of interest are identified by mass spectrometric analyses.