Mechanistic Understanding of Bacterial Signaling Proteins: Novel Insights into Their Functions, Structures, and Regulatory Mechanisms

Abstract

The increasing prevalence of antibiotic resistance represents one of the greatest threats to global health. Horizontal gene transfer through natural competence, the process by which bacteria take up extracellular DNA, is a primary route through which resistance genes and virulence factors spread among bacterial populations. Understanding the molecular mechanisms controlling natural competence could provide new targets for controlling the spread of antibiotics resistance. This work identifies ComFB as a novel c-di-NMP receptor family that controls natural competence and motility in different bacteria. Beyond its role in natural competence, ComFB's involvement in motility regulation has implications for bacterial colonization, biofilm formation, and infection establishment. Deletion of either comFB or dacA, a gene encoding cyclase enzyme responsible for synthesis of one of c-di-NMP (c-di-AMP), resulted in defects in DNA uptake and transformation efficiency in Synechocystis and Synechococcus. Both mutants showed comparable defects in type IV pilus-related proteins essential for DNA uptake machinery, establishing ComFB as a c-di-NMP receptor controlling natural competence in cyanobacteria. This discovery filled an important gap in understanding how cyclic dinucleotide signaling regulates horizontal gene transfer. Moreover, ComFB binds both c-di-AMP and c-di- GMP with comparable affinity, which raised questions about its function in organisms with different cyclic dinucleotide profiles. In Bacillus subtilis, ComFB appears to regulate motility through c-di-GMP binding, demonstrating functional versatility within the same protein family. The regulatory functions of ComFB on motility appeared to be conserved in Shewanella oneidensis, a Gram-negative bacterium, where both deletion and overexpression of ComFB impaired the motility of this bacterium. Phylogenetic analysis revealed that ComFB represents a new family of c-di-NMP receptor protein, widespread in different bacterial phyla (e.g. Cyanobacteria, Bacillota and Pseudomonadota). Despite sequence divergence across different bacterial species, ComFB proteins maintain conserved structural features and oligomerization properties. Different ComFB homologs showed varying ligand preferences, from exclusive cdi- GMP binding to genuine dual specificity to both c-di-AMP and c-di-GMP, reflecting adaptation to diverse regulatory needs while preserving core receptor architecture. This suggests ancient evolutionary origins with lineage-specific functional diversification. Thus, identifying ComFB as a c-di-GMP receptor resolved the longstanding puzzle of why bacteria possess many c-di-GMP synthesis and degradation enzymes but relatively few known receptors. ComFB represents the third widespread c-di-GMP receptor family alongside PilZ and MshEN domains, expanding our understanding of cyclic dinucleotide signaling networks. Moreover, in the frame of this work, I was able to identify serval c-di-AMP receptor proteins in cyanobacteria, including NhaS5, TrkA, KtrC and KdpD proteins, implicated in maintaining Na+/K+ homeostasis.

Die Dissertation ist gesperrt bis zum 08. April 2028 !