PhoP induces RyjB expression under acid stress in Escherichia coli

Abstract

Bacterial small RNAs (sRNAs) play a pivotal role in post-transcriptional regulation of gene expression and participate in many physiological circuits. An ~80-nt-long RyjB was earlier identified as a novel sRNA, which appeared to be accumulated in all phases of growth in Escherichia coli. We have taken a comprehensive approach in the current study to understand the regulation of ryjB expression under normal and pH stress conditions. RpoS was not necessary for ryjB expression neither at normal condition nor under acid stress. Hfq also emerged to be unnecessary for RyjB accumulation. Interestingly, RyjB was detected as a novel acid stress induced sRNA. A DNA binding protein PhoP, a component of PhoP/Q regulon, was found to regulate ryjB expression at low pH, as the elimination of phoP allele in the chromosome exhibited a basal level of RyjB expression under acid stress. Ectopic expression of PhoP in ΔphoP cells restored the overabundance of RyjB in the cell. Overexpression of RyjB increased the abundance of sgcA transcripts, with which RyjB shares a 4-nt overlap. The current study increases our knowledge substantially regarding the regulation of ryjB expression in E. coli cell.

Keywords: Hfq; acid stress; sRNA-mediated gene regulation; small RNAs.

Graphical Abstract

Figure 1

Accumulation of RyjB at different growth phases of E. coli cells. A. Schematic illustration of genomic location and orientation of ryjB gene. B. Relative expression of ryjB gene was analysed through RT-qPCR analysis in a 24-h growth cycle. RyjB quantity at each point was normalized by comparison to RT-qPCR of 5S rRNA. Each point in the graph represents the relative amount of RyjB when compared with 1.5-h sample. Data at each point were presented as the average ± SD from three separate experiments. Inset, growth curve of MG1655 cells. Each point on the curve represents the point of sample withdrawal.

Figure 2

Role of Hfq in RyjB expression. A. Structure of RyjB predicted by Mfold web server. The sequence above the bold line in the structure is complementary to the 5′-end of sgcA mRNA. B. Comparison of the accumulation of RyjB in wild type and Δhfq cells in a 24-h growth cycle analysed by RT-qPCR analysis. 5S rRNA was taken as internal control. Relative amount of RyjB was presented at each point compared to the RyjB accumulation at 1.5 h, which was set as 1. Data were presented as mean ± S.D. from three independent experiments.

Figure 3

Amount of RyjB in WT and ΔrpoS cells under pH stress. A. Wild type cells were grown up to mid-exponential phase (OD₆₀₀–0.4) at 37°C and pH of the medium was adjusted to either 5.0 (adjusted by 50-mM HOMOPIPES) or 9.0 (adjusted by 50 mM Tris–HCl). Samples were removed at the indicated time and total RNA was isolated for RT-qPCR analysis. 5S rRNA gene was taken as internal control. RyjB accumulation at 0 min was set as 1. B. 15 μg of total RNAs isolated from the samples withdrawn at indicated time after acid stress (pH adjusted by 50-mM sodium acetate) were analysed by northern blot. RyjB transcribed in vitro was run in the last lane (Std) as size standard. The number under each lane represents the relative amount of RyjB when compared with 0-min sample, which was set as 1. 5S rRNA gene was taken as loading control. C. Accumulation of RyjB in ΔrpoS cells under acid stress analysed by RT-qPCR. 5S rRNA gene was used as internal control.

Figure 4

RyjB expression is regulated by PhoP. A. Amount of phoP mRNA upon acid stress (pH adjusted by 50-mM HOMOPIPES) was estimated by RT-qPCR analysis. 5S rRNA gene was taken as internal control for the normalization of the amount of phoP mRNA at each time point. Accumulation of phoP mRNA at 0 min was set as 1 and relative amount of phoP at other time point was calculated. B. PhoP accumulation after acid stress (pH adjusted by 50 mM sodium acetate) in wild type cells was measured by western blotting. The number under each lane represents the relative amount of PhoP protein when compared with 0-min sample, which was set as 1. Ribosomal protein S1 was taken as loading control. C. Acid stress was given to mid-exponential phase (OD₆₀₀ = 0.4) wild type and ΔphoP cells. Total RNA was isolated from the cells at indicated times after acid stress and was utilized for RT-qPCR analysis. Expression of 5S rRNA gene was utilized as internal control.

Figure 5

Ectopic expression of PhoP upregulates ryjB expression. A. RT-qPCR analysis was performed to estimate the amount of phoP mRNA in wild type (WT) and ΔphoP cells harbouring empty vector or pPhoP recombinant plasmid. 5S rRNA gene was taken as internal control for the normalization of the amount of phoP mRNA at each time point. Accumulation of phoP mRNA in WT cells was set as 1 and relative amount of phoP at other time point was calculated. B. RyjB abundance was quantified through RT-qPCR analysis in WT and ΔphoP cells harbouring either empty vector or pPhoP recombinant plasmid. 5S rRNA gene was taken as reference gene for the normalization of the amount of RyjB at each time point. Accumulation of RyjB in WT cells was set as 1 and relative amount of RyjB at other time point was calculated.

Figure 6

Effect of RyjB overexpression on sgcA accumulation in the cell. Cells with empty vector or pRyjB growing at mid-exponential phase (OD₆₀₀ = 0.4) were treated with 1-mM IPTG for an hour. Cells were harvested and total RNA was isolated from each sample. Accumulation of RyjB (A) or sgcA (B) was measured by RT-qPCR analysis. 5S rRNA gene was taken as internal control for the normalization of the amount of ryjB or sgcA transcript at each point. Accumulation of RyjB or sgcA in the cells with empty vector was set as 1 and relative amount of RyjB or sgcA in the cells with pRyjB was calculated. C. RT-qPCR analysis was performed to measure the accumulation of sgcA mRNA under acid stress as described earlier. Expression of 5S rRNA gene was utilized as internal control.