Large-scale identification of trans-translation substrates targeted by tmRNA in Aeromonas veronii

https://doi.org/10.1016/j.micpath.2020.104226Get rights and content

Highlights

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    Construction of a mutant tmRNA which labels the trans-translation substrates by a His6 tag.

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    Identification of totally 24 potential trans-translation substrates with various functions.

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    Conformation of the truncations of PspA and AsmA labeled by tmRNA mutant with His6 tag.

Abstract

Transfer-messenger RNA (tmRNA) is ubiquitous in bacteria, acting as the core component for the trans-translation system that contributes to label the aberrantly synthesized peptides for degradation and to release the stalled ribosomes. Deletion of tmRNA causes a variety of phenotypes related to important physiological processes in bacteria. To illustrate the molecular mechanism of the versatility of tmRNA in aquatic pathogen Aeromonas veronii, we mutated the C-terminal nucleotides of tmRNA (MutmRNA) for encoding a tag containing six histidine residues (His6tag), so as to capture and enrich the trans-translation substrates from the cell lysates through a Ni2+-NTA affinity chromatograph. The results showed that the concentrated substrates were detected as distinct and specific bands in western blotting using anti-His antibody, demonstrating that specific defective mRNAs were frequently and intensively rescued by trans-translation during the translation process in A. veronii. The substrates were analyzed by LC-MS/MS and further identified by searching a theoretically constructed database specific for A. veronii. Total of 24 potential substrates were identified, with various functions involved in metabolism, as well as structure and signal-based cellular events. Among the identified substrates, PspA and AsmA were labeled by Flag, and expressed in the presence of the modified trans-translation system in E. coli. Their labelings with MutmRNA were validated by purification through Ni2+-NTA column followed by western blotting using anti-Flag antibody. This study provided the most abundant set of endogenous targets for tmRNA in A. veronii, and facilitated further investigations about the molecular mechanism and signal pathway of tmRNA-mediated trans-translation.

Section snippets

Author Statement

Muzhi Peng: Investigation, Xin Cao: Investigation, Visualization, Yanqiong Tang: Funding acquisition, Hong Li: Data curation, Xiang Ma: Conceptualization, Writing - original draft, Writing - review & editing, Funding acquisition, Zhu Liu: Conceptualization, Methodology, Supervision, Project administration, Funding acquisition, Writing - review & editing.

Strains and culture conditions

Aeromonas veronii C4 was grown aerobically at 30 °C and BL21 strain of Escherichia coli was at 37 °C in Luria-Bertani (LB) medium. The medium was supplemented with 50 mg/mL ampicillin for regular growth of A. veronii, and 50 mg/mL kanamycin for the positive transformants carrying the derivative vectors of pBBR-MCS-2. Bacterial growth was monitored by determining the optical density at 600 nm (OD600).

Determination of growth curve

Bacteria were grown in 5 mL LB medium at 30 °C overnight. The overnight culture was transferred

The variant MutmRNA maintained the biological functions as wild type tmRNA

The predicted tag encoded by tmRNA of A. veronii was ANDENYALAA, wherein C-terminus of the coding sequence was paired with the adjacent sequence to form a stem-loop structure (Fig. 1A). According to the previous strategy [23], the wild-type tmRNA gene, ssrA, was mutated as the variant MutmRNA, wherein the reading frame encoding the degradation tag (YALAA) and the stop code (TAA) was mutated to encode the sequence of ANDENHHHHHH (Fig. 1A). Therefore, the trans-translation products were not

Discussion

tmRNA is discovered in 1979, as a segment of sRNA ranging from 230 to 400 nucleotides in size (Ray and Apirion, 1979). In the past 40 years, tmRNA was found to act in not only the quality control of protein synthesis, but also the regulations of essential physiological processes in bacteria [14,30], although the physiological roles of the tmRNA-mediated tagging remains unknown. Identification of tmRNA substrates is certainly an effective way to gain insight into the physiological roles and

Declaration of competing interest

No conflicts of interest statement for this article.

Acknowledgements

This work was supported by the Hainan Natural Science Foundation No. 319QN161 (to H.L.), the National Science and Technology Major Project No. 2019ZX08010004 (to Z.L.), and the grants from the National Natural Science Foundation of China Nos. 31772887 (to Z.L.) and 31860676 (to Y.T.) and 31960027(to X.M.), and the funding of 111 Project (D20010).

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    The authors contributed equally to this study.

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