Table 1.
Mutations identified in osmoresistant suppressor mutants of ΔgdpP.
Fig 1.
Mutations in kupB restore osmoresistance to Lc. lactis ΔgdpP independently of c-di-AMP.
(A) Comparison of growth of Lc. lactis MG1363 background strains on GM17 agar or GM17 agar + 0.2M NaCl following spotting of serial dilutions. (B) Levels of c-di-AMP (mean ± SEM) in Lc. lactis strains from three independent biological replications. P < 0.001 (***), P < 0.01 (**) and P < 0.05 (*) indicate significant differences compared to ΔgdpP (one-way ANOVA followed by Student’s t test). NS = not significant. (C) Location of suppressor mutations in KupB. (D) Amino acid identities of the two Kup domains in other organisms compared to Lc. lactis KupB.
Fig 2.
Constitutive KupB activity results in higher salt resistance and c-di-AMP levels in bacteria.
(A) Overexpression of kupB and kupBA618V in Lc. lactis ΔgdpP enhances osmoresistance. Spots (5μl) are from log-phase cells diluted from 10−1 (top) to 10−5 (bottom). (B) K+ levels in WT Lc. lactis, ΔgdpP, ΔgdpPkupBA618V and ΔgdpP overexpressing kupBA618V. (C) C-di-AMP levels in ΔgdpP and ΔgdpPkupBA618V and ΔgdpP overexpressing kupB and kupBA618. (D) C-di-AMP levels in WT Lc. lactis and WT Lb. reuteri overexpressing kupB and kupBA618V. Data are the mean ± SEM from three independent biological replications. Significant results P < 0.001 (***), P < 0.01 (**) and P < 0.05 (*) are indicated (one-way ANOVA followed by Student’s t test).
Fig 3.
BusR binds c-di-AMP and exacerbates the osmosensitive phenotype of ΔgdpP.
(A) Domains in BusR. (B) DRaCALA of cell lysates containing full length BusR, the BusR TrkA_C domain or MBP with 32P-c-di-AMP. (C) DRaCALA and binding affinity of the purified BusR TrkA_C domain with 32P-c-di-AMP. (D) Expression of busAA promoter as a lacZ fusion in different Lc. lactis strains on GM17 agar + 0.1M NaCl. (E) Intracellular glycine betaine levels. Strains in red bars were grown in GM17 + 0.1M NaCl due to the osmoresistant suppressor ΔgdpPcdaAD123Y growing poorly in GM17 alone. Data are the mean ± SEM from three independent biological replications. P < 0.001 (***) indicate significant differences compared to ΔgdpP (one-way ANOVA followed by Tukey’s test). (F) Comparison of growth of strains on GM17 agar or GM17 agar + 0.2M NaCl following spotting of serial dilutions. (G) C-di-AMP levels in WT Lc. lactis and ΔbusR. Data are the mean ± SEM from three independent biological replications. Significant results P < 0.001 (***) are indicated (Student’s t test).
Fig 4.
Active export of c-di-AMP lowers intracellular c-di-AMP and restores osmoresistance in Lc. lactis ΔgdpP.
(A) Location of two independent intergenic deletions between rplL and rmaX. Arrows and lollipops indicate predicted transcription start sites and terminators, respectively. (B) Expression of genes in ΔgdpP and the two suppressor mutants containing intergenic deletions (ΔgdpP rplLtermΔ209 and ΔgdpP rplLtermΔ85) using qRT-PCR. (C) Levels of intracellular and extracellular c-di-AMP in ΔgdpP and osmoresistant suppressor derivatives. Data are the mean ± SEM from three independent biological replications. (D) Comparison of growth of strains on GM17 agar or GM17 agar + 0.2M NaCl following spotting of serial dilutions. Includes ΔgdpP strains overexpressing different genes in pGh9 plasmid using the promoter from rplJ. (E) Levels of intracellular and extracellular c-di-AMP in ΔgdpP strains overexpressing different genes from the pGh9 plasmid using the promoter from rplJ. Data are the mean ± SEM from three independent biological replications.
Fig 5.
C-di-AMP levels in non-growing energised cells respond rapidly to environmental osmolarity changes.
(A) Schematic diagram showing the steps in the non-growing cell suspension assay. Cells are suspended in a low osmolarity buffer causing water influx. Glucose is added to energise the cells at time 0 to provide ATP for c-di-AMP synthesis. After 10 mins, either water or NaCl/KCl is added causing further cellular hydration or cellular dehydration, respectively. (B) C-di-AMP levels in Lc. lactis WT suspended in different solvents/solutions: ACN-MeOH (acetonitrile:methanol) or buffer (1/10 KPM) with and without glucose and deoxyglucose. (C) C-di-AMP levels in Lc. lactis WT cells in 1/10 KPM buffer + glucose. After the 10 min (arrow), cells were treated with water, 0.1M NaCl, 0.3M NaCl, 0.1 KCl and 0.3M KCl and c-di-AMP levels were measured at 20 mins. The same experiment was carried out for cells of Lb. plantarum (D), L. monocytogenes (E), S. aureus (F), and Lc. lactis ΔgdpP/cdaAT273fs (H) with the key for treatments the same as that shown in (C). Non-ionic treatments (sucrose and sorbitol) were also performed using L. monocytogenes (G). Mean ± SEM levels of c-di-AMP were measured from three independent cell suspensions. P < 0.001 (***), P < 0.01 (**) and P < 0.05 (*) indicate significant differences at the 20 minute time point relative to the water addition sample (one-way ANOVA followed by Student’s t test).