Identification And Characterization Of Cis-Encoded Antisense RNAs Associated With The Replication Process Of Salmonella Enterica Serovar Typhi

BackgroundBacterial non-coding RNAS (ncRNAs) are key players in reprogramming protein expression upon environmental change, in particular under stress conditions. They are involved in a number of cellular processes in bacteria, including acid resistance, iron homeostasis, quorum sensing, Mg+2/Ca+2transport and virulence, ABC transport systems, global repression of OMP synthesis and control of expression of global transcription factors. The trans-encoded RNAs are the most extensively studied of the known non-coding RNAs. Genome-wide transcriptomic analyses now indicate that bacteria make large numbers of cis-acting antisense RNAs, the functional relevance of most of the antisense transcripts however remain unknown.ObjectiveThe bacterial replisome is composed of a large number of enzymes, which work in exquisite coordination to accomplish chromosomal replication. By deep sequencing analysis of transcriptome of Salmonella enterica serovar Typhi (S. Typhi), antisense RNA sequences encoded in-cis to the genes associated with the replication processes were discovered. The purpose of this work was to investigate the regulatory roles these antisense RNAs play as far as bacteria replication is concerned.Methods1. Determination of transcription initiation and termination sites of the antisense RNAs (asRNAs):The5’-and3’-rapid amplification of cDNA ends (RACE) analysis were used to determine the transcription initiation site and transcription termination site of asRNAs respectively. The results of RACE and Northern blot analysis were used to determine the full length of the asRNAs.2. Strain constructions:Strains overexpressing the asRNAs from arabinose inducible pBAD promoter were constructed. This was done by amplifying the full length fragments of the asRNAs, cloning them into pBAD myc/His A and transforming S. Typhi wild type strain with these recombinant vectors. RNase III and RNase E mutants of S. Typhi were constructed by homologous recombination mediated by suicide plasmid. Fur mutant was also constructed by the lambda red recombinase method. 3. Analysis of asRNAs expression under different growth conditions:Quantitative real time PCR (qRT-PCR), and Northern blot analysis was used to determine the expression of the asRNAs at different OD values representing the various phases of bacteria growth, namely the lag phase, log phase, and the stationary phase. Same analysis was also used to determine the expression of the asRNAs under different stress conditions namely, acid stress, oxidative stress, osmotic stress and iron limitation, which are reminiscence of the conditions pathogenic S. Typhi goes through in the gut or within the cells of the host.4. Analysis of effect asRNAs overexpression have on mRNA levels of targets:Total RNA was isolated from the overexpressing strains grown to an OD600of0.6at0,5,10, and20min after addition of L-arabinose (0.02%final concentration) to cultures. mRNA levels of target sense genes were monitored by qRT-PCR and Northern blot upon full length overexpression of the asRNAs from arabinose inducible pBAD promoter.5. Analysis of effect of overexpressing the asRNAs on the growth of S. Typhi:the growth of wild type strain S. Typhi harbouring an empty plasmid and that of strains overexpressing the asRNAs were assessed. These strains were subjected to growth under normal as well as stress conditions and the absorbances of the culture media were taken hourly over a time period to determine the growth curve. Further experiments were carried out based on the outcome of the above experiments.Results1. Identification of the asRNAs:Transcription initiation and termination sites of a series of asRNAs that were found by deep sequencing analysis to be involved in replication of S. Typhi were determined by5□and3□-RACE. Two of the antisense RNAs, AsdA (antisense RNA of dnaA gene) and AspC (antisense RNA of parC gene) were selected for in-depth analysis. AsdA, which is540nucleotides long is located514nucleotides downstream of dnaA start codon and extends to16nucleotides upstream of dnaA start codon. The871nucleotides AspC is complimentary to the sequences410nucleotides downstream of parC start codon and extends to the mid portion of the gene. Northern blot confirmed the in vivo expression of the asRNAs.2. Characterisation of the asRNAs:Consistent with literature, Northern blot and qRT analysis of expression showed that the asRNAs are highly expressed in the stationary phase of the bacterial growth and also under stress conditions such iron starvation, acid and osmotic stress. 3. Construction of bacterial strains:Strains that express AsdA and AspC from a pBAD inducible plasmid, as well as strains that have deletions in the genes encoding RNase III, RNase E and ferric uptake regulator (fur) were successfully constructed, which was proved by sequencing assay. These isogenic strains, together with other already constructed strains were used to investigate the role of AsdA and AspC expression in S. Typhi.4. Functional analysis:Pulse expression analysis showed that overexpression of the antisense RNA resulted in a significant increase in the mRNA levels of both dnaA and parC. Stability experiments and the expression of the asRNAs in RNase III and RNase E mutant background revealed that interaction between AsdA or AspC and their sense mRNA increases the stability of the later by protecting it from endoribonucleotic-dependent cleavage.5. Effect on growth of S. Typhi:Expression of AsdA and AspC was found to enhance the growth of S. Typhi, as strains expressing the asRNAs grew better than the wild type. This enhanced growth was also observed in stress conditions in which the antisense asRNA was expressed.6. Effect on the motility of S. Typhi:Experiments to verify whether expression of the asRNA affects the physical characteristics of S. Typhi revealed that the motility, as observed on swim agar plate, of the bacteria enhanced significantly in the asRNA overexpressing strains as compared to the wild type. This, for pathogenic bacteria like S. Typhi is vital for invasion and colonisation of the host.ConclusionIt has been established by this study that AsdA and AspC antisense RNAs associated with the replication processes of S. Typhi are indeed transcribed not merely as a by-product of the cell’s transcription machinery, but plays a vital role as far as the stability of the target mRNAs are concerned. This increased in stability will ultimately result in efficient expression of genes by increasing translation of mRNAs of genes involved in replication, and enable the bacteria to survive effectively, especially under unfavourable conditions.