The RipA and RipB Peptidoglycan Endopeptidases Are Individually Nonessential to Mycobacterium smegmatis

Mycobacteria possess a series of Rip peptidoglycan endopeptidases that have been characterized in various levels of detail. The RipA and RipB proteins have been extensively studied and are DL-endopeptidases, and RipA has been considered essential to Mycobacterium smegmatis and Mycobacterium tuberculosis We show here that the ripA and ripB genes are individually dispensable in M. smegmatis and that at least one of the genes must be expressed for viability. We characterized strains carrying in-frame deletion mutations of ripA and ripB and found that both mutant strains exhibited increased susceptibility to a limited number of antibiotics and to detergent but that only the ΔripA mutant displayed hypersusceptibility to lysozyme. We also constructed and characterized ΔripD and ΔripAΔripD mutants and found that the single mutant had only an intermediate lysozyme hypersusceptibility phenotype compared to that of wild-type cells while loss of ripD in the ΔripA background partially rescued the antibiotic and lysozyme phenotypes of the ΔripA mutant. We show that the RipA endopeptidase, which has been considered essential for cell division in certain mycobacteria, is not essential but that at least it or a similar protein, RipB, must be expressed by the bacteria for viability. This work is the first description of strains carrying single deletion mutations of RipA, RipB, and a novel endopeptidase-like protein, RipD.