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Identification of novel genes including rpmF and yjjQ critical for Type II persister formation in Escherichia coli

By Shuang Liu, Nan Wu, Shanshan Zhang, Yumeng Zhang, Wenhong Zhang, Ying Zhang

Posted 30 Apr 2018
bioRxiv DOI: 10.1101/310961

Persister cells, which are characterized by inactive metabolism and tolerance to antibiotics or stresses, pose a significant challenge to the treatment of many persistent infections. Although multiple genes have been reported to be involved in persister formation through transposon mutant library screens, how persisters are formed during the natural process of persister formation as the culture transitions from log phase to stationary phase is unclear. Here, using E. coli as a model, we performed a comprehensive transcriptome analysis of gene expression profiles of successive cultures of an E. coli culture at different critical time points, starting from persister-free S1-nonexistence phase (3h) to persister appearing S2-emergence phase (4h), and persister abundant stage S3-abundance phase (5h). The differentially expressed genes (≥2-fold) in persister appearing stage (S1 to S2 transition) and persister abundant stage (S1 to S3) were compared, and 51 and 29 genes were identified to be up-regulated, respectively. Importantly, 13 genes (gnsA, gnsB, ybfA, yjjQ, ymdF, yhdU, csgD, yncN, rpmF, ydcX, yohJ, ssrA, rbsD) overlap in both persister S2-emergence phase and S3-abundance phase, including a member of the trans-translation pathway (ssrA) as well as an orphan toxin (ydcX), which are two well-known persister genes while the remaining 11 novel genes (gnsA, gnsB, ybfA, yjjQ, ymdF, yhdU, csgD, yncN, rpmF, yohJ, rbsD) have not been reported previously. Persister levels of 7 constructed knockout mutants (ΔgnsA, ΔybfA, ΔyjjQ, ΔyhdU, ΔcsgD, ΔyohJ and ΔrpmF) and 10 overexpression strains (gnsA, gnsB, ybfA, yjjQ, ymdF, yhdU, csgD, rpmF, yohJ, rbsD) in E. coli uropathogenic strain UTI89 were determined upon treatment with different cidal antibiotics (ampicillin, levofloxacin and gentamicin). Additionally, ranking of these overlapping genes according to their impact on persister levels were also performed. Two genes (rpmF encoding 50S ribosomal subunit protein L32, and yjjQ encoding a putative LuxR-type transcription factor) showed the most obvious phenotype on persister levels in both knockout and overexpression studies, which suggests they are broad and key factors for persister formation. While previous studies cannot distinguish if a given persister gene is involved in persister formation or persister survival, our findings clearly identify novel persister forming genes and pathways involving a ribosome protein and a LuxR type transcription factor during the bona fide persister formation process and may have implications for developing improved treatment of persistent infections.

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