Genetic analysis of cell envelope assembly and polar growth in Corynebacterium glutamicum

Abstract

Increasing our understanding of the mechanisms of bacterial cell envelope biogenesis will help identify novel enzymes for future antibiotic targets. While most current research efforts focus on understanding the mechanisms of cell envelope assembly and maintenance in classical Gram-positive and Gram-negative model systems, we studied Corynebacterium glutamicum (Cglu), a member of the Corynebacterineae suborder that has a triple-layer cell envelope resembling that of many clinically important pathogens, including Mycobacterium tuberculosis. Furthermore, C. glutamicum, like other Corynebacterineae, grows through a process of polar growth as opposed to the more well understood growth mode of dispersed cell elongation. To identify genes involved in the construction of C. glutamicum’s cell envelope and the process of polar growth, we utilized a transposon sequencing approach. We created a highly diverse transposon library in C. glutamicum which led to the identification of the essential gene set of C. glutamicum as well as genes that when mutated cause sensitivity to the drug ethambutol. Next, we performed a high-throughput phenotypic screen using a panel of drug treatments to generate a unique phenotypic fingerprint for each gene. The screen uncovered CofA as a critical co-factor of PBP1a, one of C. glutamicum’s high molecular weight penicillin binding proteins. We showed that CofA is important for the stability of PBP1a as well as its proper localization. CofA represents the first identified aPBP co-factor in the Corynebacterineae. We also took a targeted approach to understand how the three non-essential cell wall synthases in C. glutamicum work together to promote polar growth. Together, the phenotypic screen and detailed characterization of the role of cell wall synthases in polar growth have advanced our knowledge of cell surface assembly in the Corynebacterineae.