Our previous study demonstrated that deleting a c-di-AMP phosphodiesterase gene, pde2, abolished SpeB production at the transcriptional level in S. pyogenes (Fahmi et al., 2019). We employed transposon mutagenesis to identify potential genes involved in the regulation of SpeB production in the Δpde2 mutant. We screened ~3,000 colonies and isolated 24 transposon-generated mutants that showed SpeB activity similar to the wild type. DNA sequencing was performed using primers binding to a transposon sequence to determine the transposon insertion sites of these mutants. This process was successful for 17 mutants. Interestingly, all 17 mutants had a transposon insertion either in the promoter region of or in the dltX gene. These mutants were not clones because they all have different transposon insertion sites. The dlt operon is highly conserved in Gram-positive bacteria, encoding gene products that incorporate D-alanine ester into teichoic acid molecules (Peschel et al., 1999; Neuhaus and Baddiley, 2003). In S. pyogenes, the dlt operon comprises six genes, dltXABCDE. The first gene, dltX, encodes a membrane-associated small protein consisting of 47 amino acids ( Figure 1 ). To confirm that dltX inactivation is responsible for derepression of the SpeB production in the Δpde2 strain, we deleted dltX in the Pde2-deficient mutant and evaluated the SpeB activity of this Δpde2ΔdltX mutant. Like the transposon-generated mutants, the dltX deletion in the Δpde2 background restored SpeB activity comparable to that of the wild type ( Figures 2A, B ). To investigate further, we disrupted dltA by inserting a plasmid in the Δpde2 background and evaluated its SpeB activity. The resultant Δpde2ΩdltA mutant does not express all the genes downstream of dltA in the operon due to a polar effect. We chose dltA because dltX is too small to use this gene disruption method. The strain also produced SpeB at a level equivalent to that of the Δpde2ΔdltX mutant ( Figure 2B ). When we added the dltX gene back to the Δpde2ΔdltX, the SpeB phenotype of the Δpde2ΔdltX(pdltX) strain was almost the same as that of the Δpde2 mutant ( Figure 2D ). These results indicate that the Dlt system affects SpeB production in the Δpde2 mutants. The single gene deletion mutants, the ΔdltX mutant and ΩdltA mutant, showed SpeB activity similar to that of the wild type ( Figure 2C ).

Previous research indicates that D-alanylation defects in lipoteichoic acids (LTA) significantly alter the surface charge of GAS (Kristian et al., 2005). In this study, we investigated the role of DltX, a new member of the dlt operon in GAS, in modulating cell surface charge. The surface charges of strains were examined using the cationic protein cytochrome c. The ΔdltX and Δpde2ΔdltX mutants bound more cytochrome c than the wild-type strain ( Figure 3 ), indicating that the absence of DltX leads to an increase in negative surface charge. The amount of the cytochrome c bound to the Δpde2 mutant was a little bit higher than that bound to the wild type, but the difference was much smaller than those between the ΔdltX mutants and the wild type ( Figure 3 ), suggesting that the absence of Pde2 minimally affects the expression of the dlt operon.

D-alanylation carried out by the Dlt system decreases the affinity of cationic antimicrobial peptides (CAMPs) by reducing the negative surface charge on bacterial cells (Peschel et al., 1999; Kristian et al., 2005). We investigated the effect of dltX gene deletion in S. pyogenes on the resistance to a CAMP polymyxin B (PMB). S. pyogenes strains were cultured with varying concentrations of PMB, and the minimum inhibitory concentrations (MICs) of PMB were measured. The MIC of PMB for both the wild type and Δpde2 mutant was 50 µg/ml. In contrast, the MIC for the strains with dltX deletion, the ΔdltX and Δpde2ΔdltX mutants, decreased to 10 µg/ml. These results indicate that the dltX deletion in S. pyogenes increases the susceptibility of the bacteria to PMB.

The LiaFSR gene regulatory system, which is composed of a membrane-bound repressor protein (LiaF), a sensor kinase (LiaS), and a response regulator (LiaR), functions to detect and responds to cell envelope stress induced by CAMPs (Arias et al., 2011; Lin et al., 2020). Given that the degree of D-alanylation of teichoic acids affects cell envelope charge and both the Dlt and LiaFSR systems respond to CAMPs, we investigated the role of the LiaFSR system in SpeB regulation in the Δpde2 mutant. We deleted the response regulator liaR in the Δpde2 background and measured SpeB activity. The Δpde2ΔliaR mutant derepressed SpeB production like the Δpde2ΔdltX mutant, although at a lower level ( Figure 4 ). This result indicates that LiaFSR also influences SpeB production in the Δpde2 mutant.

Since SpeB production was also influenced by LiaFSR in the Δpde2 mutant ( Figure 4 ), and defects in D-alanylation may result in the cell envelope stress by altering cell surface charge, we investigated if SpeB activity restoration by dlt mutation in the Δpde2 background was through the LiaFSR system. LiaFSR responds to cell envelope stressors by regulating the expression of spxA2 in Gram-positive bacteria (Baker et al., 2020; Sanson et al., 2021). For example, the treatment of the cell wall stressor vancomycin (0.5 µg/ml) stimulates LiaFSR, which enhances the transcription of spxA2 in S. pyogenes (Lin et al., 2020). We measured spxA2 transcript levels in cells with or without vancomycin treatment ( Figure 5 ). As expected, all the strains, except the liaR deletion mutants, showed an increased amount of spxA2 transcript when treated with vancomycin ( Figure 5A ). Moreover, significant changes of the spxA2 transcript level in the Δpde2, ΔdltX, and Δpde2ΔdltX mutants compared to that of the wild type was not observed when they were treated with vancomycin ( Figure 5B ). A negative control, tetracycline (1 µg/ml), a protein synthesis inhibitor, showed no significant change (less than twofold) of the spxA2 transcript level. These results suggest that neither c-di-AMP levels in cells nor D-alanylation of teichoic acids affect the expression of spxA2.

The amounts of c-di-AMP levels of strains were measured. Both the dltX and liaR deletion in the wild type did not change cellular c-di-AMP level, but all pde2 deletion mutants, the Δpde2, Δpde2ΔdltX, and Δpde2ΔliaR mutants produced increased amounts of c-di-AMP ( Figure 6A ). These increases agree to previous studies since the deletion of a c-di-AMP phosphodiesterase gene, pde2 or gdpP increases cellular c-di-AMP level in S. pyogenes (Fahmi et al., 2019). Interestingly, dltX deletion in the Δpde2 mutant increased c-di-AMP amount further, but liaR deletion in the Δpde2 mutant did not affect c-di-AMP production.

We investigated if the impaired capability of K⁺ transport affects cellular c-di-AMP levels. The ΔktrB strain, the mutant with the deletion of the high-affinity K⁺ transporter gene, produced a high amount of c-di-AMP, even more than that of the Δpde2 mutant ( Figure 6B ). The ΔktrA strain, the mutant with the deletion of K⁺ transport inhibitor gene, produced a lower amount of c-di-AMP level than that of the wild type. These results indicate that the relationship between K⁺ transport capability and c-di-AMP production is inversely proportional in S. pyogenes.

The SpeB phenotype of the Δpde2ΔdltX mutant was not changed in high salt media, unlike the wild type and Δpde2ΔliaR mutant. We examined the SpeB activities of strains in high-salt media. Regular C medium used to make protease detection plates contains 10 mM K₂HPO₄. To increase salt concentration, we doubled or tripled the amount of K₂HPO₄. In these higher salt C media, the SpeB activities of the wild type and Δpde2ΔliaR mutants were reduced ( Figure 7 ). However, the SpeB activities of the Δpde2ΔdltX mutant did not change in higher salt media. Thus, DltX and LiaR respond differently to a high osmolarity condition in the Δpde2 background. The ΔdltX and ΔliaR mutants still showed SpeB activity in high salt conditions.

S. pyogenes HSC5 (emm genotype 14) (Hanski et al., 1992; Port et al., 2013) was employed for all experiments, including strain construction. Molecular cloning experiments utilized Escherichia coli DH5α or TOP10 (Invitrogen), which was cultured in Luria-Bertani broth. The routine culture of S. pyogenes employed Todd-Hewitt medium (BBL) supplemented with 0.2% yeast extract (Difco) (THY medium), and cells were grown at 37°C in sealed tubes without agitation. Unless otherwise indicated, C medium (Lyon et al., 1998) was used to grow S. pyogenes for SpeB activity assay and RNA preparation for real-time qRT-PCR. Bacto agar (1.4%, w/v; Difco) was added to make solid media. Cultures on solid media were incubated under the anaerobic condition created by a commercial product (e.g., GasPak; catalog no. 260678; BBL). When appropriate, antibiotics were added to the media at the following concentrations if they are not specified: kanamycin, 50µg/ml for E. coli and 500µg/ml for S. pyogenes; erythromycin, 500µg/ml for E. coli and one µg/ml for S. pyogenes.

Plasmid DNA was isolated via a commercial kit (e.g., Gene Elute plasmid miniprep kit; Sigma) and used to transform S. pyogenes or E. coli as described previously (Caparon et al., 1991). Enzymes for DNA cloning and PCR were used according to the manufacturers’ recommendations. Chromosomal DNA was purified from S. pyogenes using a commercial kit (e.g., GenElute bacterial genomic DNA kit; Sigma).

For the transposon mutagenesis, TnΩKm2, a Tn4001 derivative containing a kanamycin resistance determinant, was employed (Kang et al., 2012). Briefly, the purified plasmid containing TnΩKm2 was introduced to the Δpde2 mutant using electroporation (voltage: 2100V, Capacitor: 25 uF, Resistance: 200 Ohms). The colonies with kanamycin resistance were patched on the protease indicator plates, and the strains showing the wild-type level protease activity were selected. The transposon insertion sites in those strains were identified by sequencing of chromosomal DNA with a primer binding to a transposon sequence. The sequencing data were compared with the NCBI genomic database (http://www.ncbi.nlm.nih.gov/BLAST/) to identify transposon-insertion sites.

The generation of the pde2 deletion mutant has been described elsewhere (Fahmi et al., 2019). Other gene deletion mutations, ΔdltX and ΔliaR on chromosomal loci were generated by employing the shuttle vector with a temperature-sensitive replication origin, pJRS233 (Cho and Kang, 2013). Briefly, the target gene and ~ 1000 bp sequences immediately upstream and downstream were amplified by PCR. This PCR product was inserted into pJRS233 using the fast-cloning method (Li et al., 2011). The plasmid with a target gene deletion allele was obtained by inverse PCR. The target gene deletion plasmids, pΔdltX and pΔliaR, were created using this method. The primers used to generate the PCR products are listed in Table 1 . pΔdltX or pΔliaR was used to replace each target gene in the wild type or the Δpde2 mutant by the gene deletion method that employs the temperature-sensitive replication origin, as described previously (Cho and Kang, 2013). The ΩdltA or Δpde2ΩdltA mutant was constructed by insertional disruption of the dltA gene through single homologous recombination. An internal region of the dltA gene was amplified by PCR and then inserted into the suicide vector pCIV2 (Okada et al., 1993; Lyon et al., 2001). The resultant plasmid, pΩdltA, was used to transform HSC5 or the Δpde2 mutant into resistance to kanamycin. Since the suicide vector pCIV2 lacks the replication origin of S. pyogenes, the plasmid can only exist in S. pyogenes by integrating into the chromosome through homologous recombination. The fidelity of all genetic constructs was confirmed by PCR and/or DNA sequencing.

The dltX-complemented Δpde2ΔdltX(pdltX) strain was generated using the plasmid p7INT that inserts into the streptococcal chromosome (McShan et al., 1998). The DNA containing the dltX gene and its promoter region was amplified by PCR and inserted into p7INT through the fast-cloning method (Li et al., 2011). The resulting plasmid pdltX was transferred into the Δpde2ΔdltX mutant to make the Δpde2ΔdltX(pdltX) strain.

The susceptibility of mutant strains to the cell membrane-targeting antibiotic polymyxin B was monitored as follows. S. pyogenes cells grown in THY medium overnight were inoculated into fresh THY medium containing polymyxin B (100, 50, 10, 5, 1, 0.5, and 0 µg/ml). Cells were then grown in a 96-well plate overnight at 37°C, and the OD₆₀₀ of the overnight cultures (~18 h post-inoculation) was measured to determine the final cell density. This experiment was performed in triplicate.

THY media (50 ml) were inoculated with 2-3% overnight cultures and cultivated to the early exponential phase. Cells were collected by centrifugation at 7000 g for 10 min, resuspended in 50 ml of chemically defined media (DMEM), and incubated overnight. Then, the cells were washed twice with 20 ml of morpholino propanesulfonic acid (MOPS) buffer (20 mM, pH 7), adjusted to the final cell OD₆₀₀ to 3 in 2 ml MOPS buffer with 0.2 mg/ml cytochrome c (Sigma-Aldrich, St. Louis, MO), and incubated for 10 minutes in a shaker at room temperature. As a control, 0.2 mg/ml cytochrome c was incubated in MOPS buffer under the same conditions without bacteria. After 10 min, cells were removed by centrifugation, and the cytochrome c content of the supernatants was quantified photometrically by measuring OD₅₃₀.

Real-time qRT-PCR was conducted as described elsewhere (Cho and Kang, 2013). The primers for qRT-PCR are listed in Table 1 . The gyrase A subunit gene (gyrA) was used as the internal reference gene to normalize the expression level of a specific transcript between samples (Kang et al., 2010). The reported data represent the means and standard errors from three independent assays performed on different days with new RNA samples.

Cells at the exponential phase (0.3 – 0.4 of OD₆₀₀) were collected and incubated in a fresh medium with an antibiotic at 37°C for an hour. Then, cells were collected and lysed for RNA purification.

Overnight cultures in THY medium were serially diluted with fresh THY medium, and the diluted cells (2µl) were spotted onto protease indicator agar plates (C medium agar plates containing 2% skim milk). The plates were then incubated anaerobically at 37°C for 24 h to 48 h, and SpeB activity that displays a clear zone around the spotted cells was observed.

The measurement of c-di-AMP concentration in cell extract was conducted as previously described (Fahmi et al., 2019). Briefly, S. pyogenes strains were grown to the exponential phase (OD600 = ~0.4) in 10 ml THY medium, washed three times with PBS, resuspended in 1 ml PBS, and lysed through PlyC treatment. The clear supernatant of the culture was collected in a fresh tube after centrifugation at 7,000 relative centrifugal force (rcf) at 4°C for 10 min. The cell lysates were then boiled for 10 min. Clear supernatants were collected after centrifugation and stored at -20°C until used to measure c-di-AMP concentration. The purified CabP protein was diluted to 50 µg/ml in coating buffer (50 mM Na2CO3, 50 mM NaHCO3, pH 9.6), and 100 µl of the solution was added to each well to coat the wells of a 96-well flat-bottom plate. The plates were sealed with plastic wrap and incubated overnight at 4°C. The coated wells were then washed three times with PBS containing 0.05% Tween 20 (PBST) and blocked with 5% bovine serum albumin (BSA). The cell extract samples were diluted (5 times) with 50 mM Tris buffer (pH 8). 100µl of controls, standards, and samples were added to the coated wells (in triplicate). The plates were incubated for 2 hrs at room temperature. Each well of the plates was washed three times with 200 µl PBST. Next, 100 µl of 0.1 µg/ml high-performance streptavidin (Thermo Fisher Scientific) in PBS was added and incubated for 1 hr. After wells were washed three times with PBST, 100 µl of the substrate (0.5 mg of o-phenylenediamine dihydrochloride [Sigma-Aldrich] in citrate buffer [pH 5] containing 20 µl H₂O₂) was added to each well and incubated for 30 min at room temperature. Finally, the reactions were stopped with 100 µl of 2M H₂SO₄. The OD₄₉₂ of each well was measured using a plate reader. A standard curve was generated and used to measure the levels of c-di-AMP in samples.

Each statistical test applied to the experiments was described in the figure legends.