Identification of new components of the RipC-FtsEX cell separation pathway of Corynebacterineae

Author summary The pathways involved in bacterial surface assembly are critical for cell morphogenesis and serve as attractive targets for antibiotic development. Bacteria in the suborder Corynebacterineae, which includes important pathogens like Mycobacterium tuberculosis, possess a unique multilayered surface structure. In addition to the common peptidoglycan cell wall, they have an attached polysaccharide layer called arabinogalactan and an outer membrane made of mycolic acids. To enhance our understanding of cell surface biogenesis in these bacteria, we performed a global genetic analysis of gene function in the model system Corynebacterium glutamicum (Cglu) using transposon sequencing. In addition to defining the essential gene set in this organism, our analysis also identified SteA and SteB as components of the cytokinetic ring. These factors are conserved among the Corynebacterineae, and our results reveal that they play a critical role in the final stages of cytokinesis by promoting remodeling of the peptidoglycan layer at the division site. Several important human pathogens are represented in the Corynebacterineae suborder, including Mycobacterium tuberculosis and Corynebacterium diphtheriae. These bacteria are surrounded by a multilayered cell envelope composed of a cytoplasmic membrane, a peptidoglycan (PG) cell wall, a second polysaccharide layer called the arabinogalactan (AG), and finally an outer membrane-like layer made of mycolic acids. Several anti-tuberculosis drugs target the biogenesis of this complex envelope, but their efficacy is declining due to resistance. New therapies are therefore needed to treat diseases caused by these organisms, and a better understanding of the mechanisms of envelope assembly should aid in their discovery. To this end, we generated the first high-density library of transposon insertion mutants in the model organism C. glutamicum. Transposon-sequencing was then used to define its essential gene set and identify loci that, when inactivated, confer hypersensitivity to ethambutol (EMB), a drug that targets AG biogenesis. Among the EMBs loci were genes encoding RipC and the FtsEX complex, a PG cleaving enzyme required for proper cell division and its predicted regulator, respectively. Inactivation of the conserved steAB genes (cgp_1603-1604) was also found to confer EMB hypersensitivity and cell division defects. A combination of quantitative microscopy, mutational analysis, and interaction studies indicate that SteA and SteB form a complex that localizes to the cytokinetic ring to promote cell separation by RipC-FtsEX and may coordinate its PG remodeling activity with the biogenesis of other envelope layers during cell division.