Identification Of Noncoding RNA As-RpoH In Salmonella Enterica Serover Typhi And Phenotype Research

Objective: In recent years, a lot of bacterial non-coding RNAs have been found, which play a very important role in gene expression and regulation. We have found some putative nc RNAs including Asr H in Salmonella enterica serovar Typhi(S. Typhi) by RNA sequencing. In this research, we investigated the whole molecular sequence and gene location in S. Typhi and derermined the expression of the Asr H. We will try to investigate the function of Asr H in S. Typhi.Methods:1. Identification of Asr H. Northern Blot and quantitative real time PCR(q RT-PCR) were used to confirm the existence of Asr H and to investigate the Asr H expressional levels in different growth phases.2. Assessment of the total length of Asr H. 5′ Rapid Amplification of c DNA Ends(RACE) were used to find the transcription initiation site of Asr H, and tiling PCR was used to find the transcription termination region. Results were used to determine the full length of Asr H.3. Preparation of the gene deleted mutant of S. Typhi. The asr H promoter deleted mutant of S. Typhi was prepared by the homologous recombination method mediated by the suicide plasmid p GMB151, which was confirmed by final sequence analysis.4. RT-PCR assay of the asr H expression of the mutant. RT-PCR analysis was performed to determine the asr H and rpo H expressional levels in S. Typhi and the asr H promoter mutant.5. The growth curves assay. Growth curves of the wild type and asr H promoter deleted mutant were performed to observe the growth of S. Typhi in different stress conditions.6. The motility assay. Different strains of S. Typhi was inoculated on LB plate with 0.3% argarose and incubated at 37℃ overnight, the size of each bacterial zone on the plate was measured to represent the bacterial motility.7. Biofilm formation test. Crystal violet dyeing test was used to compare the biofilms of wild-type strain and asr H promoter deleted mutant.Results:1. We confirmed the existence of the non-coding RNA Asr H in S. Typhi by Northern Blot. Northern Blot and q RT-PCR results also showed that Asr H is highly expressed in the late log-phase(OD 600 1.2) of S. Typhi.2. We determined the transcription initiation and termination sites of Asr H by 5′ RACE and Tiling PCR respectively, and found that the full length of Asr H was 846 bp-1007 bp approximately. The transcription start point of Asr H is located 119 bp downstream of the rpo H gene and transcriptional direction is opposite to the gene. It covers most of the rpo H gene coding region, with no complete ORF structure in the sequence. Therefore, Asr H is a long non coding RNA.3. Using the homologous recombination mediated by suicide plasmid p GMB151, a mutant was successfully created, in which the 120 bp of asr H promoter region was deleted.4. RT-PCR confirmed the expression of asr H and rpo H in the wild strains. In contrast, rpo H was expressed only in the asr H promoter deleted mutant.5. The growth of the asr H mutant was slower under the acidic and hyper osmotic stress conditions, but was faster under oxidative stress condition than the wild-type strain.6. The motility assay revealed a markedly reduced motility in the asr H mutant.7. Results of the biofilm formation test showed that the biofilm formation ability of the asr H promoter deleted mutant was stronger than the wild strain.Conclusions: 1. This study confirmed the existence of the non-coding RNA Asr H in S. Typhi and the gene location of Asr H.2. The non-coding RNA AsrH is beneficial to bacterial growth in acidic and hyper osmotic stress conditions, it may be essential to bacterial motility and inhibition of biofilm formation in S. Typhi.