The Regulation Of Alanine Dehydrogenase By TmRNA At The Level Of Transcription And Translation In Aeromonas Veronii

The trans-translation mediated by transfer-message RNA(tmRNA)and Small protein B(Smp B)is thought to be universally present in bacteria.When ribosomes stalled in the process of translation,the trans-translation system released ribosome subunits stalled on truncated m RNAs and tags the nascent polypeptides to target them for proteolysis.Previous studies have showed that in addition to the function in trans-translation,tmRNA could act as an s RNA and regulate the expression of gene at the transcriptional level through incomplete base pairing with the target m RNA.Aeromonas veronii is a conditioned pathogen.The absence of tmRNA made Aeromonas veronii grow slower and be more sensitivity to various antibiotics,and the medial lethal concentration of tilapia reduced 6-7 times.It showed that tmRNA had an important regulatory effect on the pathogenicity,drug resistance and virulence of Aeromonas veronii.This study showed that the dual regulatory function of tmRNA on alanine dehydrogenase(ALD,EC 1.4.1.1),a trans-translation substrate,was investigated at the level of transcription and translation by means of trans-translation rescue and s RNA,which finally affects the oxidative stress tolerance of bacteria.On one hand,tmRNA acted as an antisence s RNA to negatively regulate the expression of ALD by binding to the promoter region.Firstly,we found that the expression of ald m RNA increased significantly in the stationary phase,but did not change in the logarithmic phase,by whole RT-q PCR and transcriptome sequencing analysis of wild-type and tmRNA-deleted strains in Aeromonas veronii.Secondly,the fusion vector p DH118 was constructed by fusing green fluorescent reporter(egfp)gene with ald promter,and co-transformated into E.coli Feldon stain with the expression vector for tmRNA.tmRNA acted as a negative regulator for ald promoter.Finally,point mutations were constructed according to the binding region between tmRNA and ald promoter as predicted by Inta RNA,and the results revealed that tmRNA targeted the region from-143 bp to-136 bp upstream of the transcriptional start site of ald promoter through base pairing.On the other hand,the ALD protein is identified as a trans-translation substrate tagged by tmRNA.We previously identified the ALD protein as a potential trans-translation substrate based on the results of mass spectrometry.In this study,by constructing an ALD-GST fusion expression vector and Western blot,it was proved that ALD protein was truncated and tagged by tmRNA through trans-translation.According to the prediction of the secondary structure of m RNA near the tagged site using RNAfold,in combination with the construction of the complementary mutations of key sites and Western blot,we found that the occurrence of ribosome retention was due to the stem-loop structure immediately adjacent to the tagged site.We proposed a new mechanism of ribosome stall and rescue.In accordance with the above findings showing that tmRNA regulated ALD at both transcriptional and translational levels,phenotype identification showed that tmRNA-deficient strain were more tolerant to oxidative stress than wild-type strain.This study elucidated for the first time the dual regulatory functions of tmRNA on one target,ALD,both in transcriptional and translational levels.The results of this study not only expanded the functions of tmRNA,but also provides an opportunity for further investigating the underlying mechanism of the influence of tmRNA on the oxidative tolerance,and finally provided a new target pathway for disease control of the pathogenic bacterium.