Functional analysis of LCP-LytR_C-domain proteins in c-di-AMP signalling and cell differentiation in Streptomyces

The cell envelope of gram-positive bacteria comprises of peptidoglycan covalently attached to glycopolymers, and is critical for cellular integrity, shape and vitality. The soil-dwelling Streptomyces represents an important paradigm in biotechnology and pharmaceutical due to their ability to produce an array of secondary metabolites and displays a complex life-cycle transitioning from multicellular filamentous vegetative mycelia to unicellular spores. For growth, Streptomyces elongate via apical tip extension and hyphal branching, that requires localization and assembly of cell wall machinery at the growing tips, we have limited knowledge about their cell wall biogenesis, composition and functions. In their natural environment, Streptomyces often encounter osmotic stress caused by rainfall or drought. To endure such challenges, they employ the nucleotide second messenger c-di-AMP, which is a well-established regulator of ion and osmolyte homeostasis in bacteria and is produced by the diadenylate cyclase DisA in Streptomyces. An elevation in c-di-AMP upon inactivation of the phosphodiesterase ataC interferes with the growth and differentiation of the bacteria. The molecular mechanisms behind these physiological effects are not understood. Hence, the aims of this study were to identify the genetic pathways directly or indirectly regulated by c-di-AMP in Streptomyces. In this study, I identified and characterized the LCP-LytR_C domain protein CglA (Vnz_13690) as a key cell wall glycopolymer ligase in S. venezuelae. Out of four LCP-LytR_C domain encoding proteins, only deletion of cglA leads to development and sporulation defects. CglA specifically localizes in cell wall biosynthesis zones, and its mutation leads to a significant decrease of glycopolymers, enlarged vegetative hyphae with defective FtsZ-ring positioning and formation. This results in the formation of mispositioned division septum, abnormal cell compartments and spores with reduced vitality. Furthermore, this study revealed a unique physiological link between c-di-AMP signalling and cell wall glycopolymer deposition, as inactivation of cglA restores growth of the S. venezuelae disA mutant at high salinity. Besides, global RNA-seq analyses of the c-di-AMP metabolizing mutants, presented some potential elements that might be involved in the developmental defect of the ataC mutant. In summary, this study revealed CglA as a novel component of cell wall biogenesis in Streptomyces, essential for cell shape maintenance and cellular vitality in filamentous, multicellular bacteria.