No abstract

Era GTPase is universally present in microbes including Mycobacterium tuberculosis (Mtb) complex bacteria. While Era is known to regulate ribosomal assembly in Escherichia coli and predicted to be essential for in vitro growth, its function in mycobacteria remains obscured. Herein, we show that Era ortholog in the attenuated Mtb H37Ra strain, MRA_2388 (annotated as EraMT) is a cell envelope localized protein harbouring critical GTP-binding domains, which interacts with several envelope proteins of Mtb. The purified Era from M. smegmatis (annotated as EraMS) exhibiting ~90 % sequence similarity with EraMT, exists in monomeric conformation. While it is co-purified with RNA upon overexpression in E. coli , the presence of RNA does not modulate the GTPase activity of the EraMS as against its counterpart from other organisms. CRISPRi silencing of eraMT does not show any substantial effect on the in vitro growth of Mtb H37Ra, which suggests a redundant function of Era in mycobacteria. Notably, no effect on ribosome assembly, protein synthesis or bacterial susceptibility to protein synthesis inhibitors was observed upon depletion of EraMT in Mtb H37Ra, further indicating a divergent role of Era GTPase in mycobacteria.

Ribosome hibernation is a key survival strategy bacteria adopt under environmental stress, where a protein, hibernation promotion factor (HPF), transitorily inactivates the ribosome and slows down its overall protein synthesis. The mechanism is well studied in enteric bacteria, which mainly hibernate its ribosome in 100S disome form through a dual domain, long HPF (HPFlong) or a single domain, short HPF (HPFshort) in concert with another ribosome modulation factor. Mycobacteria under hypoxia (low oxygen) stress overexpresses RafH protein regulated under DosR regulon, a critical factor for its survival. The RafH, a dual domain HPF, an orthologue of bacterial HPFlong, hibernates ribosome in 70S monosome form only. Here we report the cryo- EM structure ofMycobacterium smegmatis, a close homologue ofM. tuberculosis, 70S ribosome in complex with the RafH factor at an overall 2.8 Angstrom resolution. The RafH N- terminus domain (NTD) is conserved and binds to the decoding center of the ribosomal small subunit, a similar binding site of HPFlongNTD, but additionally it also interacts with the inter subunit bridge, B2a. Contrary to the HPFlongCTD, the RafH CTD, which is larger, binds to a unique site at the platform binding center of the ribosomal small subunit and sandwiches between bS1 and uS11 ribosomal proteins. The two domain connecting linker regions, which remain mostly disordered in earlier reported HPFlongstructures, interacts mainly with the anti-Shine Dalgarno sequence of the 16S rRNA. The helix H54a of 23S rRNA, unique to the mycobacterial ribosome, adopts a different conformation and come close to RafH CTD, suggesting its role in ribosome hibernation. RafH inhibitsin-vitroprotein synthesis in a concentration dependent manner. Further, the modeling studies provided the structural basis for the incompatibility of mycobacterial ribosomes forming 100S like hibernating ribosomes.

Ribosome hibernation is a key survival strategy bacteria adopt under environmental stress, where a protein, hibernation promotion factor (HPF), transitorily inactivates the ribosome and slows down its overall protein synthesis. The mechanism is well studied in enteric bacteria, which mainly hibernate its ribosome in 100S disome form through a dual domain, long HPF (HPFlong) or a single domain, short HPF (HPFshort) in concert with another ribosome modulation factor. Mycobacterium tuberculosis encounters hypoxia (low oxygen) as a major stress in the host macrophages, and it overexpresses RafH protein, which is critical for its survival. The RafH, a dual domain HPF, an orthologue of bacterial HPFlong, hibernates ribosome in 70S monosome form only. Here we report the cryo- EM structure of Mycobacterium smegmatis, a close homologue of M. tuberculosis, 70S ribosome in complex with the RafH factor at an overall 2.8 Å resolution. The RafH N- terminus domain (NTD) is conserved and binds to the decoding center of the ribosomal small subunit, a similar binding for HPFlong NTD, but additionally it also interacts with the inter subunit bridge, B2a. Contrary to the HPFlong C- terminus domain (CTD), the RafH CTD, which is larger, binds to a unique site at the platform binding center of the ribosomal small subunit and sandwiches between bS1 and uS11 ribosomal proteins. The two domain connecting linker regions, which remain mostly disordered in earlier reported HPFlong structures, interacts mainly with the anti-Shine Dalgarno sequence of the 16S rRNA. The helix H54a of 23S rRNA, unique to the mycobacterial ribosome, adopts a different conformation and come close to RafH CTD, suggesting its role in ribosome hibernation. RafH inhibits in-vitro protein synthesis in a concentration dependent manner. Further, the modeling studies provided the structural basis for the incompatibility of mycobacterial ribosomes to form 100S like disome architecture.

Survival response of the human tuberculosis pathogen, Mycobacterium tuberculosis (Mtb) to a diverse environmental cues is governed through its versatile transcription regulatory mechanisms with the help of a large pool of transcription regulators (TRs). Rv1830 is one such conserved TR, which remains uncharacterized in Mtb. It was named as McdR based on an effect on cell division upon its overexpression in Mycobacterium smegmatis. Recently, it has been implicated in antibiotic resilience in Mtb and reannotated as ResR. While Rv1830 affects cell division by modulating the expression of M. smegmatis whiB2, the underlying cause of its essentiality and regulation of drug resilience in Mtb is yet to be deciphered. Here we show that ResR/McdR, encoded by ERDMAN_2020 in virulent Mtb Erdman, is pivotal for bacterial proliferation and crucial metabolic activities. Importantly, ResR/McdR directly regulates ribosomal gene expression and protein synthesis, requiring distinct disordered N-terminal sequence. Compared to control, bacteria depleted with resR/mcdR exhibit delayed recovery post-antibiotic treatment. A similar effect upon knockdown of rplN operon genes further implicates ResR/McdR-regulated protein translation machinery in attributing drug resilience in Mtb. Overall, findings from this study suggest that chemical inhibitors of ResR/McdR may be proven effective as adjunctive therapy for shortening the duration of TB treatment.