The amidase AmiC2 and its peptidoglycan substrate in filamentous cyanobacteria

Abstract

Multicellularity is an advantageous ability of cyanobacteria to successfully inhabit diverse ecological niches. A recently detected structure in the septal peptidoglycan between adjacent cells in the filaments of heterozygous cyanobacteria became evident to be indispensable for differentiation and adaptation. This structure consists of numerous perforations in the central part of septal disks and is relevant for intercellular communication. This nanopore-array is the framework for septal junction proteins, which connect neighbouring cells in the filaments and enable cell-cell communication. N acetylmuramyl-L-alanine amidases, homologous to AmiC from E. coli, are involved in nanopore formation, but differ functionally and structurally from those in unicellular bacteria. While AmiC enables the release of daughter cells during cell division in unicellular bacteria, it drills the regularly formed nanopores into septal disks of multicellular cyanobacteria. An inhibitory α-helix, which is crucial for catalytic activation of AmiC in E. coli, is missing in AmiC from heterocyst-forming cyanobacteria. This indicates a novel regulation mechanism of AmiC in filamentous cyanobacteria.

This work characterizes the interaction between the amidase AmiC2 from the multicellular cyanobacterium Nostoc punctiforme with its natural substrate, the peptidoglycan (PGN). Extensive analysis of the PGN amino acid composition, the muropeptide fragment pattern and the types of modification uncovered the complex PGN meshwork of Nostoc punctiforme with the aim to characterize the substrate of AmiC2. Interestingly, disruption of amiC2 altered the level of PGN- modifications with an effect e.g. on the abundancies of covalently bound polysaccharides on the PGN glycan strands. Furthermore, it was shown that the amidase AmiC2 was unable to hydrolyse purified wild-type cell walls but degraded the PGN derived from the amiC2 mutant, indicating that AmiC2 is sensitive to the level of PGN modifications. Amidase released peptides were identified as highly cross-linked muropeptide species by tandem mass spectrometry. Site-specific mutagenesis of AmiC2 key residues demonstrated the putative function of F564, L567 and E529 in the catalytic mechanism.