The Dlt and LiaFSR systems derepress SpeB production independently in the Δpde2 mutant of Streptococcus pyogenes

The second messenger molecule, c-di-AMP, plays a critical role in pathogenesis and virulence in . We previously reported that deleting the c-di-AMP phosphodiesterase gene severely suppresses SpeB production at the transcriptional level. We performed transposon mutagenesis to gain insight into the mechanism of how Pde2 is involved in SpeB regulation. We identified one of the genes of the operon, X, as a suppressor of the SpeB-null phenotype of the mutant. The operon consists of five genes, , and in many Gram-positive bacteria, and its function is to incorporate D-alanine into lipoteichoic acids. DltX, a small membrane protein, is a newly identified member of the operon. The in-frame deletion of or insertional inactivation of in the mutant restored SpeB production, indicating that D-alanylation is crucial for the suppressor phenotype. These mutations did not affect the growth in lab media but showed increased negative cell surface charge and enhanced sensitivity to polymyxin B. Considering that mutations change cell surface charge and sensitivity to cationic antimicrobial peptides, we examined the LiaFSR system that senses and responds to cell envelope stress. The Δ mutation in the mutant also derepressed SpeB production, like the mutation. LiaFSR controls expression by regulating the expression of the transcriptional regulator SpxA2. However, the Dlt system did not regulate expression. The SpeB phenotype of the Δ Δ mutant in higher salt media differed from that of the Δ Δ mutant, suggesting a unique pathway for the Dlt system in SpeB production, possibly related to ion transport or turgor pressure regulation.