c-di-AMP accumulation impairs toxin expression of Bacillus anthracis by down-regulating potassium importers

ABSTRACT The Gram-positive bacterium Bacillus anthracis is the causative agent of anthrax and a bioterrorism threat worldwide. As a crucial second messenger in many bacterial species, cyclic di-AMP (c-di-AMP) modulates various key processes for bacterial homeostasis and pathogenesis. Overaccumulation of c-di-AMP alters cellular growth and reduces anthrax toxin expression as well as virulence in Bacillus anthracis by unresolved underlying mechanisms. In this report, we discovered that c-di-AMP binds to a series of receptors involved in potassium uptake in B. anthracis. By analyzing Kdp and Ktr mutants for osmotic stress, gene expression, and anthrax toxin expression, we also showed that c-di-AMP inhibits Kdp operon expression through binding to the KdpD and ydaO riboswitch; up-regulating intracellular potassium promotes anthrax toxin expression in c-di-AMP accumulated B. anthracis. Decreased anthrax toxin expression at high c-di-AMP occurs through the inhibition of potassium uptake. Understanding the molecular basis of how potassium uptake affects anthrax toxin has the potential to provide new insight into the control of B. anthracis. IMPORTANCE The bacterial second messenger cyclic di-AMP (c-di-AMP) is a conserved global regulator of potassium homeostasis. How c-di-AMP regulates bacterial virulence is unknown. With this study, we provide a link between potassium uptake and anthrax toxin expression in Bacillus anthracis. c-di-AMP accumulation might inhibit anthrax toxin expression by suppressing potassium uptake.

from E. coli would also down-regulate the expression of KdpFABC.Therefore, the potassium uptake systems KtrCB/D and KdpFABC from B. anthracis are both inhibited by c-di-AMP.To rule out the possibility of c-di-AMP affecting bacteria growth, the intracellu lar c-di-AMP concentrations were measured in CdaA+KdpFABC and CdaA+KdpFABC/D (Fig. 2C).The fact that there was no change in intracellular c-di-AMP concentration suggested that KdpD from B. anthracis is functional and the KdpFABC is more severely suppressed by c-di-AMP in the presence of KdpD B. anthracis .
Impairment of KtrCB/D and KdpFABC systems by c-di-AMP in E. coli LB2003 prompted us to examine the impact of c-di-AMP on KtrCB/D and KdpFABC expression.Previous research showed that elevated levels of c-di-AMP result in increased susceptibility to osmotic stress.The ΔΔPDE mutants (37) were unable to grow even at a mild salt concentration.A growth assay showed that the expression of KtrC and KdpD partially restored the growth defects of ΔΔPDE in the brain heart infusion (BHI) (2.5% NaCl) medium (Fig. 3A).Quantitative reverse transcription (qRT)-PCR results of the c-di-AMP accumulated strain (ΔΔPDE) revealed that the transcriptional levels of the Kdp system were reduced 5-to 56-fold, including kdpA, kdpB, kdpC, and kdpD (Fig. 3B).The Ktr  system, including ktrC, ktrB, and ktrD, also attenuated 1.9-to 4.7-fold in c-di-AMP accumulated strain (Fig. 3B).After transforming pDG148-kdpD or pDG148-ktrC into ΔΔPDE, we observed that the overexpression of kdpD or ktrC promoted the expression of kdpA/B/C and ktrB/C/D (Fig. 3B).The overexpression of KdpD or KtrC also restored the anthrax toxin deficiency in the c-di-AMP accumulated strain (Fig. 3C).As the c-di-AMP receptor, the overexpression of KdpD indeed decreased the intracellular c-di-AMP whereas the overexpression of KtrC did not affect the c-di-AMP concentration (Fig. 3D).These results suggested that upregulating potassium uptake instead of intracellular c-di-AMP change is critical for anthrax toxin regulation.
Wang et al. (23) found a c-di-AMP riboswitch ydaO sequence upstream of the kdp transcript in B. thuringiensis.The riboswitch encoding region was present in the 5′-untranslated region (UTR) encoding region (147-346 bp upstream of the start codon).c-di-AMP represses kdp operon transcription by enhancing the transcriptional termination of ydaO.A BLAST N search revealed that the same c-di-AMP riboswitch that regulated the kdp operon also existed in B. anthracis (147-346 bp upstream of the start codon).The sequence of the riboswitch was identical to B. thuringiensis.The role of the c-di-AMP riboswitch in regulation was assessed by constructing a ΔUTRΔΔPDE strain where the c-di-AMP riboswitch encoding region from the B. anthracis Sterne genome was deleted.Immunoblotting results demonstrated that anthrax toxin expression was restored in the ΔUTRΔΔPDE strain (Fig. 4A).qRT-PCR results suggested that the transcriptional level of kdpD increased by approximately threefold upon deletion of UTR (Fig. 4B).The transcriptional levels of the Kdp system were increased 1.5-to 4.4-fold, including kdpA, kdpB, and kdpC.The transcriptional levels of ktrB, ktrC, and ktrD were significantly enhanced and rebound to parental strain levels upon the deletion of UTR.For the anthrax toxin regulator, the transcriptional level of atxA increased by ninefold and rebounded to the parental strain levels (Fig. 4B).The transcriptional level of spo0A increased by 10-fold in the ΔUTRΔΔPDE strain when compared with the ΔΔPDE strain.The intracellular c-di-AMP levels were not significantly changed upon deletion of the UTR (Fig. 4C).The results suggested that the inactivation of ydaO in B. anthracis upregulated the expression of kdp and ktr operon, which in turn restored the production of the anthrax toxin in the c-di-AMP accumulated strains.

Inactivation of potassium uptake genes affects osmotic resistance and anthrax toxin expression in B. anthracis
The function of the potassium uptake system in the B. anthracis strain was examined by creating a series of mutants with defects in potassium transport components, ΔkdpA, ΔkdpD, ΔktrC, ΔktrCΔkdpD, and ΔkdpAΔkdpD.The growth curves of these mutants were indistinguishable when compared with the growth of the parental strain in the BHI medium.We also examined cell growth in a salt medium (4.5% NaCl) and observed that the growth of ΔktrCΔkdpD and ΔktrC was inhibited in the BHI medium (4.5% NaCl) (Fig. 5A).The transcription levels of kdpA/B/C and ktrB/C/D were not significantly changed in ΔkdpD under 4.5% NaCl (Fig. 5B).qRT-PCR results showed that ktr operon was induced 2.7-to 15-fold under 4.5% NaCl treatment, whereas kdpA/B was decreased (Fig. 5C).These results suggest that ΔktrC is sensitive to NaCl due to the strongly induced ktr operon.
FIG 3 (Continued) and KdpFABC.qRT-PCR was used to measure the transcript abundance of potassium uptake genes.RNA was isolated from bacteria grown to the stationary phase in BHI (0.8% NaHCO 3 ).Expression levels of each gene were normalized to tufA.Means and SEMs are shown; n = 3. * P < 0.05; ** P < 0.01.All data were analyzed by using one-way analysis of variance followed by Tukey's post-test analysis.(C) Overexpression of KdpD enhanced the expression of the anthrax toxin.(Up) The stationary phase strains were subjected to immunoblotting with antisera raised against PA (the toxin subunit) and L6 (the loading control).The Immunoblot results demonstrated that anthrax toxin expression was enhanced in the ΔkdpAΔkdpD strain under non-stress conditions (Fig. 6A).qRT-PCR results showed that ktrB, ktrC, and ktrD expression increased significantly in the ΔkdpAΔkdpD mutant strain (approximately 5-to 20-fold), suggesting that the inactivation of kdpA and kdpD stimulates another potassium uptake system, the Ktr operon, which was consistent to the result in BHI (0.8% NaHCO 3 ) (Fig. 6B).The intracellular c-di-AMP concentration was measured in Sterne and ΔkdpAΔkdpD, and the results revealed no significant differences between them, demonstrating that the alteration in potassium absorption alone would affect anthrax toxin expression (Fig. 6C).The intracellular potassium concentra tion was measured among Sterne, ΔkdpA, ΔktrC, and ΔkdpAΔkdpD.The results showed that the intracellular potassium concentration in ΔkdpAΔkdpD increased by fourfold compared with the parental strain, whereas ΔkdpA or ΔktrC increased by twofold (Fig. 6D).Therefore, the inactivation of both kdpA and kdpD promoted potassium uptake and anthrax toxin expression in B. anthracis.We further examined kdpD expression by comparing Sterne versus ΔkdpD strains and ΔΔPDE versus ΔΔPDEΔkdpD strains.The results also showed that the inactivation of kdpD promoted kdpB/C and ktrD (Fig. S2 at http://dx.doi.org/10.6084/m9.figshare.25669923).

DISCUSSION
Our previous study suggested that c-di-AMP accumulation impaired B. anthracis virulence and toxin expression.c-di-AMP mainly regulates osmolyte homeostasis in bacteria and archaea (42).In B. subtilis, c-di-AMP modulates the uptake of potassium ions via different potassium transport systems.To analyze how c-di-AMP inhibits toxin expression, we did pull-down and DraCALA assays to screen c-di-AMP receptors.Nine c-di-AMP receptors were identified, and five of them were involved in potassium uptake.Therefore, we proposed that c-di-AMP suppress anthrax toxin expression by regulating potassium uptake.Overaccumulation of c-di-AMP reduces the intracellular potassium pool in bacteria (25).However, it is unclear whether c-di-AMP accumulation or the decreased potassium uptake impaired toxin expression.Our results demonstra ted that c-di-AMP accumulation affects anthrax toxin expression by suppressing the potassium uptake system (Fig. 7).Overexpression of the c-di-AMP receptor KdpD and KtrC enhanced the Ktr and Kdp potassium uptake system and restored the expression of the anthrax toxin.Deleting the 5′-UTR regulatory region of the kdp operon also increased both the potassium uptake system (Ktr and Kdp) expression and the produc tion of the anthrax toxin.The overexpression of KtrC or inactivation of ydaO does not alter the intracellular c-di-AMP concentration, which suggested that reduced potassium uptake might be the reason for the regulation of anthrax toxin expression.Overaccumu lation of c-di-AMP reduces the intracellular potassium pool in bacteria (25).Potassium inhibits KinC-dependent biofilm formation and stimulates Spo0A-phosphorelay histidine kinase KinB (43).In B. anthracis, phosphorylation of Spo0A promotes anthrax toxin gene expression by repressing AbrB expression (44).Further studies are required to determine whether potassium absorption system inhibition affects Spo0A phosphoryla tion.However, we cannot make a conclusion that PDE mutant is defective for anthrax toxin production because of a deficiency in potassium.Another possibility for increas ing potassium uptake restoring anthrax toxin might be compensatory.Further studies are required to analyze whether potassium is required for anthrax toxin production.B. thuringiensis possesses at least three types of K + uptake transporters, including the Trk system, Kdp system, and KimA protein, whereas B. subtilis possesses KtrAB, KtrCD, and KimA (23).The potassium uptake systems in B. anthracis differ from B. subtilis and B. thuringiensis.K + uptake in B. anthracis is mediated by two transport systems, Ktr and Kdp.For the Ktr systems, K + uptake probably occurs with the symport of sodium ions.The membrane components KtrB and KtrD probably work in tandem with the gating component KtrC as the Ktr potassium uptake components in S. aureus (45).Under high osmolarity and K + -limiting circumstances, the Ktr system is crucial for bacterial growth (33).c-di-AMP binds directly to KtrC, the membrane-associated component of the potassium transporter KtrC-KtrD and KtrC-KtrB, and prevents potassium uptake by B. anthracis.The Kdp system is required for bacterial growth in defined media under K + -limiting conditions.The main complex KdpFABC is composed of a P-type ATPase KdpB (16,46) with a channel-like subunit (KdpA) (47), the lipid-like stabilizer KdpF (48), and the unknown functional component KdpC.At low K + concentrations, the transcriptional level of KdpFABC is activated by the two-component system KdpD/KdpE (49).KdpD is located downstream of KdpC, which consists of an N-terminal domain (NTD) and a C-terminal domain (CTD).The NTD is necessary for the highest level of kdpFABC expression, whereas the CTD is critical for phosphotransfer reactions (50).After stimulation, KdpE acquires the phosphoryl group from KdpD and promotes the transcription of the kdpFABC operon.In B. anthracis, KdpD only contains the N-terminal sensor region of KdpD but lacks the C-terminal histidine kinase region.Although lacking kdpE in the B. anthracis genome, our results showed that c-di-AMP binds to KdpD, which down-regulates the expression of KdpFABC significantly.How c-di-AMP-bound KdpD repressed KdpFABC in the absence of KdpE requires further analysis.KdpFABC and KtrCB/D are probably the only two potassium uptake systems in B. anthracis because we cannot generate the ΔkdpAΔktrC double mutant strain.
Based on our result, the Ktr system appears to play a dominant role in moderate osmotic stress resistance in B. anthracis.Growth of ΔktrC was impaired under 4.5% NaCl treatment, whereas growth of ΔkdpA was not affected.The ΔktrC is sensitive to 4.5% NaCl probably because ktrC/D is strongly induced (13-15-fold) under this condition.Furthermore, our results showed that the deletion and overexpression of kdpD increased ktr operon expression.In c-di-AMP accumulated ΔΔPDE strain, kdpD over-expression increased ktr operon expression because it decreased the intracellular c-di-AMP, thus releasing the suppression of the ktr operon.The enhanced ktr potassium uptake system compensates for the repression of the kdp system in B. anthracis.Probably, inactivating one of them would stimulate the other.In the kdpA repressed strain such as ΔΔPDE and ΔkdpA, ktr operon was induced with the absence of kdpD.c-di-AMP-bound KdpD might repress ktr operon with mechanisms requiring further analysis.

Bacterial strains, plasmids, and growth conditions
The bacterial strains and plasmids are listed in Table S1 at http://dx.doi.org/10.6084/m9.figshare.25669950.The bacterial strains were grown in either Luria broth (LB) or BHI broth (Oxoid) under aerobic conditions at 37°C.
The mutants were constructed by markerless gene deletion methods, as described previously (13,51).The deletion of these genes was confirmed by DNA sequencing using the primers.The PCR confirmation results from the mutant and Sterne strain are shown in Fig. S3 at http://dx.doi.org/10.6084/m9.figshare.25669938.

Plasmid construction
The kdpA, ktrC, and kdpD alleles with the Shine-Dalgarno (SD) sequence were amplified using chromosomal DNA of B. anthracis Sterne as the template, and the fragments were cloned between specific restriction sites SphI and HindIII in pDG148.LacIQ was synthesized and inserted into the XbaI cloning site of pQE60 to create the IPTG inducible expression plasmid.The genes encoding putative potassium transporters were then introduced into the vector pQE60.For the construction of pQE60-ktrC, the ktrC gene was amplified using oligonucleotide pair pQE60-ktrCF and pQE60-ktrCR and ligated to pQE60 between EcoRI and HindIII.The ktrB and ktrD genes were amplified using oligonu cleotide pairs ktrBpQE60F/ktrBpQE60R and ktrDpQE60F/ktrDpQE60R, respectively, and fused to pQE60-ktrC by the infusion assay to create pQE60-ktrCD and pQE60-ktrCB.To construct pBAD33 cdaA lmo , the cdaA gene was amplified from L. monocytogenes EGD-e chromosomal DNA using oligonucleotides pBad33 cdaAF and pBad33 cdaAR and cloned between specific restriction sites XbaI and HindIII in pBAD33.pBAD33-cdaA lmoD171N was constructed using pBAD33-cdaA as the template, and overlapping PCR was performed with the oligonucleotides cdaA D171NF and cdaA D171NR.kdpD and 13 other potential c-di-AMP receptor genes were amplified using Sterne chromosomal DNA as the template and cloned between the NcoI and XhoI sites of the expression plasmid pET28a.The plasmid was then transformed into E.coli BL21 (DE3) cells.The DisA-6×His fusion protein was purified as described previously (13).All primers are listed in Table S2 at http:// dx.doi.org/10.6084/m9.figshare.25669959.

Differential radial capillary action of ligand assay
The 14 selected genes were cloned into the expression vector pET28a and expressed as C-terminal His-tag fusion proteins (Table 1).The expression vectors were transformed into E. coli BL21 cells, and induced protein expression was detected and confirmed, except for YugO, by immunoblotting.The protein patterns of the expression strains were analyzed by SDS-PAGE.This analysis enabled confirmation that genes were expressed after being induced with 0.8 mM IPTG.Cells were collected by centrifugation; re-sus pended in a c-di-AMP binding buffer (10 mM Tris base, 100 mM NaCl, and 5 mM MgCl 2 , pH 8.0) containing 10 µg/mL DNase, 250 µg/mL lysozyme, and 1 µM PMSF; incubated for 0.5 h; and subjected to two freeze-thaw cycles (16,24,52,53).Cell lysates were subsequently stored at -80°C.After thawing, 20 µL E. coli whole lysates were mixed with radioisotope-labeled c-di-AMP and incubated for 20 min, and 2 µL of these mixtures was spotted onto nitrocellulose membranes (PALL) using a multi-channel pipette.Membranes were allowed to dry after spotting.The radioactivity intensity was detected by a phosphor screen (PerkinElmer) and Cyclone Plus (PerkinElmer).32 P-c-di-AMP was prepared using 55 nM α 32 P-ATP incubated with 20 µM DisA from B. anthracis at 30°C overnight.Five units of calf intestine alkaline phosphatase (Fermen tas) were added to the mixture for 1 h.After incubating at 95°C for 10 min, the dena tured proteins were removed by centrifugation, and the supernatant was stored.The competition assays were performed with indicated concentrations of unlabeled ATP and c-di-AMP (Sigma).

Quantitative real-time PCR
The B. anthracis Sterne strain and deletion mutants were grown overnight in BHI (0.8% NaHCO 3 ).Total mRNA was extracted with the Bacteria Extraction kit (R403-01; Vazyme Biotech Co., Ltd.) and reverse-transcribed using the QuantiTect Reverse Transcription kit (Qiagen, Valencia, CA).With a CFX96 real-time PCR detection device, cDNAs were used as the templates for real-time qRT-PCR investigation of selected genes (Bio-Rad, California).2× Universal SYBR Green Fast qPCR Mix (ABclonal) was applied for all RT-PCRs.Primers are listed in Table S2.tufA was used as the internal control.The amplification efficiency was 0.9-0.99.

Assay of anthrax toxin in supernatants
Sterne strain and deletion mutants were inoculated in 5 mL LB broth and incubated for 16 h at 37°C.Secondary inoculation (0.1%) was performed in 5 mL BHI (Oxoid) (0.8% NaHCO 3 ), and the cultures were incubated at 37°C for 18 h.The protein samples were collected and prepared as described previously (37).Membranes were blotted with antibodies against PA (1:1,000 dilution, Abcam catalog # ab13808, RRID: AB_300652) and L6 (provided by Chun-Jie Liu, Beijing Institute of Biotechnology: a ribosomal protein in the cytoplasm, 1:1,000 dilution) and then with the anti-rabbit IgG horseradish peroxi dase-conjugated secondary antibody (1:5,000 dilution).

Salt stress adaptation and dilution spot assay
The salt stress adaptation and dilution spot assay were conducted as described previously (29).Stationary-phase bacteria were diluted 1:100 in 5 mL BHI until the OD 600 reached 0.5.Secondary inoculation (1:50) was carried out into BHI supplemented with 4.5% NaCl.Growth was monitored for 8 h.At the end of the time point (8 h), the cultures were frozen at −80°C for RNA isolation.

Quantification of intracellular c-di-AMP concentration
Sterne strain and deletion mutants were inoculated in 5 mL LB broth and incubated for 16 h at 37°C.Secondary inoculation (0.1%) was performed in 5 mL BHI (Oxoid) (0.8% NaHCO 3 ), and the cultures were incubated at 37°C for 18 h.E. coli strains were used to inoculate 4 mL M9 medium (35 mM KCl) containing 0.2% (wt/vol) glycerol and 0.02% (wt/vol) glucose (20,27).The cultures were incubated overnight at 37°C and used to re-inoculate the same medium (without glucose) to an OD 600 = 0.1.Then, the cells were washed and re-suspended in M9 medium (10 mM KCl) supplemented with 0.2% (wt/vol) glycerol, 0.005% arabinose, and 80 µM IPTG.The cultures were incubated for 18 h.Five milliliters of the cultures was harvested immediately by centrifugation at 4°C, and the cell pellets were extracted by the nucleotide extraction method reported previously (54).One microliter was used for high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis, which was performed on Agilent 1260 coupled to 6240A LC/MS/MS with an RP C18 column (150 mm by 2.1 mm, Waters XSelect HSS T3).
The following buffers were used in the gradient program: buffer A: H 2 O; B: acetonitrile.
The following protocol was used for separation: 0.01 min, 5% B; 3 min, 8% B; 6 min, 90% B; 9.1 min, 5% B; and 13 min, 0% B at a flow rate of 0.3 mL min −1 .The intracellular c-di-AMP concentration was determined based on c-di-AMP (Sigma) standard plotting peak areas versus concentrations as nanogram of c-di-AMP per milligram dry weight Bacillus anthracis.Measurements were repeated in triplicates, with the same retention time as for c-di-AMP.

Quantification of intracellular potassium concentration
Sterne strain and deletion mutants were inoculated in 5 mL LB broth and incubated for 16 h at 37°C.Secondary inoculation (0.1%) was performed in 5 mL BHI (Oxoid) (0.8% NaHCO 3 ), and the cultures were incubated at 37°C for 18 h.Twenty-five-milliliter cultures were harvested, with the intracellular K + concentrations determined using an atomic absorption spectrometer (204 Duo, Agilent, USA) as described previously (20).The intracellular concentrations of K + were calculated using the following equation: in which [K] i is the intracellular K + in mg mg −1 , [K] t is the total K + in mg, and W d is the dry weight of the pellet in mg.

Statistical analysis
Data in Fig. 2, 5C, and 6A and C were analyzed by two-tailed Student's t-test.Data in Fig. 3 to 5A, 5B, and 6B and D were analyzed by using one-way analysis of variance followed by Tukey's post-test analysis.Figure 7 was created with Biorender.com.

FIG 2 FIG 3
FIG 2 Inhibition of KtrCB/CD and KdpFABC transport activity by c-di-AMP.(A) E. coli LB2003 carrying relevant plasmids were cultivated in M9 medium supplemented with 80 µM IPTG for KtrCB/CD and KdpFABC induction and 0.005% arabinose for the induction of the cdaA alleles.The different potassium transport systems (KtrCB/CD/FABC) are expressed via pQE60 and CdaA/CdaA D171N via pBAD33.(B) Optical density (OD) 600 of 22 h growth in M9 medium.Means and standard errors of the means (SEMs) are shown.n = 3. **** P < 0.0001 by two-tailed Student's t-test.The different potassium transport systems (KtrCB/CD/ABC) are expressed via pQE60 and CdaA/CdaA D171N via pBAD33.(C) Intracellular c-di-AMP concentration.Means and SEMs are shown; n = 3. * P < 0.05.All data were analyzed by two-tailed Student's t-test.
blots are representative of three replicates.(Down) Gray values of the bands.Means and SEMs are shown; n = 3. * P < 0.05 by two-tailed Student's test.(D) Intracellular c-di-AMP concentration.Means and SEMs) are shown; n = 3. * P < 0.05.All data wereanalyzed by using one-way analysis of variance followed by Tukey's post-test analysis.

FIG 4 (
FIG 4 (Continued)(Down) Gray values of the bands.Means and SEMs are shown; n = 3. * P < 0.05 by using one-way analysis of variance followed by Tukey's post-test analysis.(B) qRT-PCR was used to measure transcript abundance of kdp operon genes, toxin gene regulator, and spo0A kinase genes.RNA was isolated from bacteria grown to the stationary phase in BHI (0.8% NaHCO 3 ).Expression levels of each gene were normalized to tufA.Means and SEMs are shown; n = 4. # P < 0.1; * P < 0.05; *** P < 0.001.All data were analyzed by using one-way analysis of variance followed by Tukey's post-test analysis.(C) Intracellular c-di-AMP concentration.Means and SEMs) are shown; n = 3. * P < 0.05.All data were analyzed by using one-way analysis of variance followed by Tukey's post-test analysis.

FIG 5
FIG 5 Inactivation potassium uptake genes affect osmotic resistance in B. anthracis.(A) Representative growth kinetics of B. anthracis and potassium uptake gene mutants in BHI or BHI broth supplemented with 4.5% NaCl.OD 600 at the indicated time points was measured.(B) qRT-PCR was used to measure the transcript abundance of kdp operon and ktr operon genes.RNA was isolated from bacteria grown to the stationary phase in BHI (4.5% NaCl).Expression levels of each gene were normalized to tufA.Means and SEMs are shown; n = 4. * P < 0.05.All data were analyzed by using one-way analysis of variance followed by Tukey's post-test analysis.(C) qRT-PCR was used to measure the transcript abundance of kdp operon and ktr operon genes in Sterne strain.RNA was isolated from bacteria grown to the stationary phase in BHI (0.8% NaHCO 3 or 4.5% NaCl).Expression levels of each gene were normalized to tufa.Means and SEMs are shown; n = 4. * P < 0.05.All data were analyzed by two-tailed Student's t-test.

FIG 6 (
FIG 6 (Continued) phase strains were subjected to immunoblotting with antisera raised against PA (the toxin subunit) and L6 (the loading control).The blots are representative of three replicates.(Down) Gray values of the bands.Means and SEMs are shown; n = 3. * P < 0.05 by two-tailed Student's test.(B) Inactivation of kdpA and kdpD stimulated ktrBCD expression.RNA was isolated from bacteria grown to the stationary phase in BHI (0.8% NaHCO 3 ).Expression levels of each gene were normalized to tufA.Means and SEMs are shown; n = 4. * P < 0.05; ** P < 0.01; *** P < 0.001.All data were analyzed by using one-way analysis of variance followed by Tukey's post-test analysis.(C) Intracellular c-di-AMP concentration.Means and SEMs) are shown; n = 3.All data were analyzed by two-tailed Student's t-test.(D) Intracellular potassium concentration.Means and SEMs) are shown; n = 3. * P < 0.05; *** P < 0.001.All data were analyzed by two-tailed Student's t-test.

TABLE 1
Expression plasmids used in the DraCALA assay a The bold values mean the positive candidates in the DraCALA assay.