Widespread ribosome stalling in a genome-reduced bacterium and the need for translational quality control

Abstract

Summary Trans-translation is a ubiquitous bacterial mechanism of ribosome rescue mediated by a transfer-messenger RNA (tmRNA) that adds a degradation tag to the truncated nascent polypeptide. Here, we characterize this quality control system in a genome-reduced bacterium, Mycoplasma pneumoniae (MPN), and perform a comparative analysis of protein quality control components in slow and fast-growing prokaryotes. We show in vivo that in MPN the sole quality control cytoplasmic protease (Lon) degrades efficiently tmRNA-tagged proteins. Analysis of tmRNA-mutants encoding a tag resistant to proteolysis reveals extensive tagging activity under normal growth. Unlike knockout strains, these mutants are viable demonstrating the requirement of tmRNA-mediated ribosome recycling. Chaperone and Lon steady-state levels maintain proteostasis in these mutants suggesting a model in which co-evolution of Lon and their substrates offer simple mechanisms of regulation without specialized degradation machineries. Finally, comparative analysis shows relative increase in Lon/Chaperone levels in slow-growing bacteria suggesting physiological adaptation to growth demand.

Development of a Serum-Free Medium To Aid Large-Scale Production of Mycoplasma -Based Therapies

“…RNA was isolated using the miRNeasy kit (Qiagen) following the manufacturer’s instructions, including the in-column DNase I treatment. The quality of RNA (amount and integrity) was assessed using a BioAnalyzer device (Agilent), and the transcriptome sequencing (RNA-seq) libraries were prepared at the CRG Genomics Unit using the TruSeq stranded mRNA sample prep kit v2 ( 55 ). Sequencing was performed using a HiSeq 2500 instrument (Illumina) with HiSeq v4 chemistry and 2 × 50-bp paired-end reads.…”

“…Processing of sequencing reads was performed as described ( 55 ) using the wild-type reference genome of M. pneumoniae M129 (NCBI accession no. NC_000912.1 ).…”