Cyclic-di-AMP is a ubiquitous bacterial second messenger playing a vital role in growth, cell wall homeostasis, antibiotic, heat- and salt-resistance. In Lactococcus, spontaneous mutation in the c-di-AMP phosphodiesterase gene (gdpP) caused a dual heat-resistant and salt-sensitive phenotype. Plating of a gdpP mutant on high salt agar resulted in the growth of a few colonies at low frequency. Genome sequencing of 2 selected salt resistant mutants revealed mutations in the gene encoding the diadenylate cyclase (CdaA) enzyme involved in c-di-AMP synthesis. Analysis of a further 100 salt resistant suppressor mutants led to the identification of >40 independent CdaA mutant variants. Those without mutations (n=4) were found to have revertant changes in the gdpP gene which likely restored phosphodiesterase activity. To confirm that these mutations affect c-di-AMP levels, we established an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to quantify cellular c-di-AMP levels. The results showed that intracellular c-di-AMP levels are undetectable in wild type L. lactis MG1363 and ~3-5x higher in a gdpP mutant compared to the cdaA/gdpP double mutants, demonstrating the c-di-AMP in Lactococcus is modulated by these enzymes. The inability to detect c-di-AMP in strain MG1363 could be due to this strain containing a natural frameshift mutation in the CdaR homolog encoding gene immediately downstream of cdaA. CdaR has been shown to strongly upregulate CdaA mediated c-di-AMP synthesis. We therefore analysed four cheese making Lactococcus strains and found that they all contain a complete unaltered cdaR gene homolog. However the level of c-di-AMP in one of these wild type strains (ASCC892185) was undetectable, while derivatives from it with gdpP and cdaA/gdpP mutations had high and low c-di-AMP levels, respectively. These results confirm that GdpP and CdaA control c-di-AMP levels in different strains of Lactococcus.