Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation.

The order Corynebacteriales includes major industrial and pathogenic Actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis. These bacteria have multi-layered cell walls composed of the mycolyl-arabinogalactan-peptidoglycan complex and a polar growth mode, thus requiring tight coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the coiled-coil protein Wag31. Here, using C. glutamicum, we report the discovery of two divisome members: a gephyrin-like repurposed molybdotransferase (Glp) and its membrane receptor (GlpR). Our results show how cell cycle progression requires interplay between Glp/GlpR, FtsZ and Wag31, showcasing a crucial crosstalk between the divisome and elongasome machineries that might be targeted for anti-mycobacterial drug discovery. Further, our work reveals that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis, similar to the gephyrin/GlyR system that mediates synaptic signalling in higher eukaryotes through network organization of membrane receptors and the microtubule cytoskeleton.

FtsEX-independent control of RipA-mediated cell separation in Corynebacteriales.

The bacterial cell wall is a multi-layered mesh, whose major component is peptidoglycan (PG), a sugar polymer cross-linked by short peptide stems. During cell division, a careful balance of PG synthesis and degradation, precisely coordinated both in time and space, is necessary to prevent uncontrolled destruction of the cell wall. In Corynebacteriales, the D,L endopeptidase RipA has emerged as a major PG hydrolase for cell separation, and RipA defaults have major implications for virulence of the human pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. However, the precise mechanisms by which RipA mediates cell separation remain elusive. Here we report phylogenetic, biochemical, and structural analysis of the Corynebacterium glutamicum homologue of RipA, Cg1735. The crystal structures of full-length Cg1735 in two different crystal forms revealed the C-terminal NlpC/P60 catalytic domain obtruded by its N-terminal conserved coiled-coil domain, which locks the enzyme in an autoinhibited state. We show that this autoinhibition is relieved by the extracellular core domain of the transmembrane septal protein Cg1604. The crystal structure of Cg1604 revealed a (ß/α) protein with an overall topology similar to that of receiver domains from response regulator proteins. The atomic model of the Cg1735-Cg1604 complex, based on bioinformatical and mutational analysis, indicates that a conserved, distal-membrane helical insertion in Cg1604 is responsible for Cg1735 activation. The reported data provide important insights into how intracellular cell division signal(s), yet to be identified, control PG hydrolysis during RipA-mediated cell separation in Corynebacteriales.

The Antibacterial Type VII Secretion System of Bacillus subtilis: Structure and Interactions of the Pseudokinase YukC/EssB.

Type VIIb secretion systems (T7SSb) were recently proposed to mediate different aspects of Firmicutes physiology, including bacterial pathogenicity and competition. However, their architecture and mechanism of action remain largely obscure. Here, we present a detailed analysis of the T7SSb-mediated bacterial competition in Bacillus subtilis, using the effector YxiD as a model for the LXG secreted toxins. By systematically investigating protein-protein interactions, we reveal that the membrane subunit YukC contacts all T7SSb components, including the WXG100 substrate YukE and the LXG effector YxiD. YukC's crystal structure shows unique features, suggesting an intrinsic flexibility that is required for T7SSb antibacterial activity. Overall, our results shed light on the role and molecular organization of the T7SSb and demonstrate the potential of B. subtilis as a model system for extensive structure-function studies of these secretion machineries. IMPORTANCE Type VII secretion systems mediate protein extrusion from Gram-positive bacteria and are classified as T7SSa and T7SSb in Actinobacteria and in Firmicutes, respectively. Despite the genetic divergence of T7SSa and T7SSb, the high degree of structural similarity of their WXG100 substrates suggests similar secretion mechanisms. Recent advances revealed the structures of several T7SSa cytoplasmic membrane complexes, but the molecular mechanism of secretion and the T7SSb architecture remain obscure. Here, we provide hints on the organization of T7SSb in B. subtilis and a high-resolution structure of its central pseudokinase subunit, opening new perspectives for the understanding of the T7SSb secretion mechanism by using B. subtilis as an amenable bacterial model.

Population genomics and antimicrobial resistance in Corynebacterium diphtheriae.

BACKGROUND: Corynebacterium diphtheriae, the agent of diphtheria, is a genetically diverse bacterial species. Although antimicrobial resistance has emerged against several drugs including first-line penicillin, the genomic determinants and population dynamics of resistance are largely unknown for this neglected human pathogen. METHODS: Here, we analyzed the associations of antimicrobial susceptibility phenotypes, diphtheria toxin production, and genomic features in C. diphtheriae. We used 247 strains collected over several decades in multiple world regions, including the 163 clinical isolates collected prospectively from 2008 to 2017 in France mainland and overseas territories. RESULTS: Phylogenetic analysis revealed multiple deep-branching sublineages, grouped into a Mitis lineage strongly associated with diphtheria toxin production and a largely toxin gene-negative Gravis lineage with few toxin-producing isolates including the 1990s ex-Soviet Union outbreak strain. The distribution of susceptibility phenotypes allowed proposing ecological cutoffs for most of the 19 agents tested, thereby defining acquired antimicrobial resistance. Penicillin resistance was found in 17.2% of prospective isolates. Seventeen (10.4%) prospective isolates were multidrug-resistant (≥ 3 antimicrobial categories), including four isolates resistant to penicillin and macrolides. Homologous recombination was frequent (r/m = 5), and horizontal gene transfer contributed to the emergence of antimicrobial resistance in multiple sublineages. Genome-wide association mapping uncovered genetic factors of resistance, including an accessory penicillin-binding protein (PBP2m) located in diverse genomic contexts. Gene pbp2m is widespread in other Corynebacterium species, and its expression in C. glutamicum demonstrated its effect against several beta-lactams. A novel 73-kb C. diphtheriae multiresistance plasmid was discovered. CONCLUSIONS: This work uncovers the dynamics of antimicrobial resistance in C. diphtheriae in the context of phylogenetic structure, biovar, and diphtheria toxin production and provides a blueprint to analyze re-emerging diphtheria.

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum.

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.