The Molecular Characteristics And Functional Research Of Long ASRNA AS-RpoH In Salmonella Enterica Serover Typhi

Objective:Recently,non-coding RNAs(ncRNAs)were widely discovered and researched in many different types of bacteria,which play crucial regulatory roles in the bacterial gene expression regulation.Previous work in our laboratory found a putative novel ncRNA overlap the region of gene rpo H with reverse direction by deep sequencing analysis of the Salmonella enterica serovar Typhi(S.Typhi)under different stress conditions.The ncRNA was named as AS-RpoH.In this study,we hope to discover the molecular characteristics and functions of the AS-RpoH in S.Typhi.Methods:1.Identification of the molecular characteristics of the AS-RpoH.Using RT-PCR and Northern Blot,we confirmed the expression of AS-RpoH in S.Typhi.5′Rapid-amplification of c DNA ends(5′ RACE)and RT-PCR with genome location specific primers were used to find the transcription initiation site and probable termination site of AS-RpoH,respectively.These results were used to determine the gene location and full length of the AS-RpoH.2.Investigation of the expression characteristics of AS-RpoH.The expressional levels of AS-RpoH were analyzed by Northern Blot and q RT-PCR assay in each growth phase and different environmental conditions including acidic,oxidative and hyperosmotic stress.3.The expression of AS-RpoH in rpo E and rpo S mutant.q RT-PCR assay were used to test the expression levels of AS-RpoH in rpo E and rpo S mutant under normal and different environmental conditions.4.Construction of the AS-RpoH over-expressing strain of S.Typhi.A pair of primers specific to the fragment of antisense strand of rpo H with Noc I and Xho I sites was designed for PCR.The amplicon was inserted into the vector p BAD/Myc-His A.The identified recombinant vector p BAD-AS-RpoH and empty vector were cloned into the wild type strain(WT)as AS-RpoH over-expressing strain(WT-p BAD-AS-RpoH)and control strain(WT-p BAD).5.The motility assay.Semi-solid LB plates(0.3% agar)was used to test the motile phenotype of AS-RpoH over-expression strain and the control strain.These strains were incubated to log-phase in LB broth to log-phase.Each 1 μL of bacterial liquids was inoculated into a semi-solid LB plate and incubated at 37℃ for several hours.The diameter of motility circle was measured in order to evaluate the motility of the bacteria.6.The growth curve assay.The growth of the AS-RpoH over-expression strain and the control strain were assessed under normal conditions as well as different stress conditions,such as acidic,oxidative and hyperosmotic stress.Each absorbance(OD600)of the culture at different time points was detected to draw the growth curve.7.Invasion assay with He La cells.AS-RpoH over-expression strain and the control strain were incubated to log-phase and inoculated into He La cells to compare the invasion ability between the two strains.8.The stability of rpo H m RNA in S.Typhi.AS-RpoH over-expression strain and the control strain were incubated to log-phase and compared with the rpo H m RNA level between the two strains by q RT-PCR.Results:1.The gene expression of AS-RpoH was confirmed by RT-PCR and Northern Blot assay with an approximate length of 3000 nt.5′ RACE result showed that its transcription start site was located 411 nucleotides upstream from the yhh K start codon.RT-PCR showed that the transcription termination site of the AS-RpoH was in the region of 3461~3508 nucleotides downstream.A yhh K specific probe was designed and used to do Northern Blot.The result showed the length of yhh K transcripts was same as that of AS-RpoH.2.Northern Blot and q RT-PCR results revealed that there was no obvious difference among the expression levels of AS-RpoH in different growth phases.The level of AS-RpoH decreased under acid stress and no obvious difference was observed in the other conditions.3.Under normal condition,the relative expression level of the AS-RpoH decreased in the rpo S mutant strains compared with the wild type strain.The expression of AS-RpoH decreased in the rpo E mutant strain under acid stress.4.AS-RpoH over-expression strain(WT-p BAD-AS-RpoH)and the control strain(WT-p BAD)were successfully constructed.5.By the motility assay,no significant difference was found between AS-RpoH over-expression strain and the control strain.6.There was no significant difference of growth between AS-RpoH over-expression strain and wild-type strain under different stress conditions.7.Compared with the control strain,the invasive ability to He La cells was attenuated after over-expressing AS-RpoH in S.Typhi.8.The result of q RT-PCR revealed that the over-expression of AS-RpoH may increase the stability of rpo H m RNA.Conclusions:A long as RNA AS-RpoH was confirmed in S.Typhi in this study,with a full length of 3461~3508 nt,and the AS-RpoH is the extension of the 3′-UTR of the yhh K gene.AS-RpoH may influence the gene expression of S.Typhi at the acid stress condition,and this process may need rpo S.Over-expression of as RNA AS-RpoH can attenuate the invasive capacity to He La cells and can also increase the stability of rpo H m RNA.