## 3 Results
### PdeA Is the Sole c-di-AMP Phosphodiesterase in C. glutamic  
A genome analysis for putative c-di-AMP phosphodiesterase genes revealed a single gene, _cg2174_, containing the DHH/DHHA1 domains found in c-di-AMP phosphodiesteraseerases [67]. It is encoded as the second gene in an operon of five genes [65]. Its 332 amino acid sequence shares 36 and 20% identity with the homologous proteins from _M. tuberculosis_ and _B. burgdorferi_, respectively, and belongs to the group of small, soluble, so-called DhhP-type c-di-AMP PDEs. For initial characterization, a C-terminal Strep-tagged version of the protein was heterologously produced with _E. coli_ and enriched via Strep affinity chromatography. The protein was then used for degradation assays, together with the substrates c-di-AMP and its linear degradation intermediate pApA, as well as a combination of both. As shown in Figure 2a-c, PdeA degraded the substrates to about double the amount of the end product AMP. Noticeably, pApA was degraded faster than c-di-AMP, even when both substrates were provided simultaneously. This also remained the case when pApA was provided in a 3:1 surplus over c-di-AMP (Figure S1), suggesting that the intermediate molecule might be immediately further cleaved to AMP and thus might not play a physiological role in _C. glutamicum_. These results show that _cg2174_ encodes a functional c-di-AMP/pApA phosphodiesteraseerase, and the gene was thus designated as _pdeA_.  
Figure 1: Cyclic diadenosine monophosphate (c-di-AMP) is produced by DisA of _C. glutamicum_ ATCC 13032: (**a**) Chromatograms of HILIC-HPLC analysis of 10 uM c-di-AMP cell-free extract of the c-di-AMP-negative organism _E. coli_ harboring the plasmid pBAD33 and cell-free extract of _E. coli_ harboring the plasmid pBAD3-_disA-strep_. Cells were grown in 2 xTY medium containing 1% arabinose; (**b**) IPC-HPLC analysis showing c-di-AMP synthesis by recombinant DisA-Strep from ATP. DisA-Strep was produced in _E. coli_, enriched via affinity chromatography and incubated with 35 uM ATP at 30 \({}^{\circ}\)C for 15 min.  
For further characterization, a deletion mutant of _C. glutamicum_ lacking the _pdeA_ gene was constructed using a CRISPR-Cpf1 method based on [PERSON] et al. (1999). The strains _C. glutamicum_ wild type and the \(\Delta\)_pdeA_ mutant were then grown over night in 50 mL 2 xTY medium, and cell-free extracts were prepared and analyzed via HPLC. As shown in the close-up box in Figure 2d, no peak at the retention time of c-di-AMP (dashed vertical line) could be detected in the cell-free extract of the wild type, whereas a small peak was observed for the \(\Delta\)_pdeA_ strain. Cell-free extracts of both strains were then mixed with reaction buffer and spiked with 10 uM c-di-AMP to check whether other PDEs involved in c-di-AMP degradation are existent in _C. glutamicum_. Samples were taken after 0, 1 and 2000 steps, respectively. The \(\Delta\  
17 h of incubation and analyzed via HPLC. As shown in Figure 2e,f, spiked c-di-AMP was detected in both assays at the reaction start. After 1 h of incubation, c-di-AMP was completely degraded in cell-free extract of the wild type (Figure 2e), whereas it was still fully present in the cell-free extracts of the deletion mutant, even after 17 h (Figure 2f). These results show that only PdeA is responsible for c-di-AMP degradation in _C. glutamicum_ under the tested conditions.