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. 2017 Feb;14(2):206-218.
doi: 10.1080/15476286.2016.1270001. Epub 2016 Dec 16.

Systematic Characterization of Artificial Small RNA-mediated Inhibition of Escherichia Coli Growth

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Free PMC article

Systematic Characterization of Artificial Small RNA-mediated Inhibition of Escherichia Coli Growth

Emiko Noro et al. RNA Biol. .
Free PMC article

Abstract

A new screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli was constructed. In this system, we used a plasmid library to express RNAs of ∼120 nucleotides, each with a random 30-nucleotide sequence that can recognize its target mRNA(s). After approximately 60,000 independent colonies were screened, several plasmids that inhibited bacterial growth were isolated. To understand the inhibitory mechanism, we focused on one sRNA, S-20, that exerted a strong inhibitory effect. A time-course analysis of the proteome of S-20-expressing E. coli and a bioinformatic analysis were used to identify potential S-20 target mRNAs, and suggested that S-20 binds the translation initiation sites of several mRNAs encoding enzymes such as peroxiredoxin (osmC), glycyl-tRNA synthetase α subunit (glyQ), uncharacterized protein ygiM, and tryptophan synthase β chain (trpB). An in vitro translation analysis of chimeric luciferase-encoding mRNAs, each containing a potential S-20 target sequence, indicated that the translation of these mRNAs was inhibited in the presence of S-20. A gel shift analysis combined with the analysis of a series of S-20 mutants suggested that S-20 targets multiple mRNAs that are responsible for inhibiting E. coli growth. These data also suggest that S-20 acts like an endogenous sRNA and that E. coli can utilize artificial sRNAs.

Keywords: Artificial small RNA; Escherichia coli; RNA-RNA interaction; growth inhibition; proteomic analysis; reporter assay; target mRNA.

Figures

Figure 1.
Figure 1.
Structure of the region inserted into the artificial sRNA expression plasmid. (A) Nucleotide sequence of the region inserted into the plasmid vector, pET-28DEL. The T7 promoter sequence is boxed in black and the transcription start site is indicated with a curved arrow. Secondary structure of the T7 terminator sequence is shown in pink. Numbers indicate the nucleotide positions from the transcription start site. Cloning site with two restriction sites is indicated in light blue. (B) Nucleotide sequence of the region inserted into the artificial sRNA expression plasmid, pASRII. ‘N’ indicates an unspecified nucleotide, and 30 Ns form a random nucleotide sequence (boxed in orange). Secondary structure of the putative Hfq-RNA-chaperone-binding region (Hfq-BR) is shown in purple. The positions of the SRA1 and SRA3 probes used for the RNA gel blotting analysis are indicated with blue lines (also see Table S1).
Figure 2.
Figure 2.
Expression of artificial sRNAs that inhibit E. coli growth. (A) Six examples of changes in E. coli growth induced by artificial sRNAs (S-10, −20, −61, −22, −7, and −8) or the empty vector pET-28DEL (Vect.). Single colonies of E. coli containing each plasmid were used to inoculate 200 μl aliquots of LB medium in 96-well plates, which were incubated at 37°C without IPTG (−) or with 40 μM IPTG (+). Cell growth was monitored by scanning the optical density at 600 nm (OD600). Data were obtained from 2 separate experiments, and the means and standard deviations of 3 cultures were calculated. (B) Northern blotting analysis of artificial sRNA expression in E. coli. E. coli containing each plasmid was grown at 37°C. After overnight incubation, the culture was diluted to OD600 = 0.3 with fresh LB medium containing 30 μg/ml kanamycin and then cultured at 37°C for 1 h. The expression of the artificial sRNAs was induced with 40 μM IPTG at 37°C. E. coli was also cultured at 37°C for 1 h without IPTG (−), as the control. The SRA1 probe was used to detect artificial sRNA and the SRA3 probe was used to detect the vector-derived sRNA (see Fig. 1 and Table S1). The arrowheads indicate the positions of the major transcripts. 5S rRNA was used as the loading control.
Figure 3.
Figure 3.
Four examples of proteins downregulated in response to S-20 induction. (A) Time point of S-20 induction and changes in E. coli growth. (B) Four examples of proteins downregulated in response to S-20 induction. Relative amounts of proteins (osmC, glyQ, ygiM, and trpB) at each induction time point were determined with a nanoLC-MS/MS analysis (see Materials and Methods). Mean (n = 3) and standard deviation for each value are shown. Vect.: vector.
Figure 4.
Figure 4.
Prediction of S-20 target mRNAs. mRNAs (operons) targeted by S-20 were predicted computationally (see Materials and Methods), and 4 examples (A-D) of the hybridization patterns between S-20 and its target mRNAs are shown. The transcription start site of each operon is indicated with a curved arrow. Numbers indicate the nucleotide positions from the translation start site of each mRNA close to the S-20 target region: (A) osmC, (B) glyQ, (C) ygiM, and (D) trpB.
Figure 5.
Figure 5.
Effects of artificial sRNAs on the translation of sRNA target-synthetic Renilla luciferase (hRluc) chimeric reporter RNAs. (A) Renilla luciferase activities of the S-20-targeted mRNA-hRluc chimeras. (B) Renilla luciferase activities of the negative control chimeras (no sRNA-binding region). An in vitro translation assay of the chimeric RNA was performed in the presence of the indicated amounts of sRNAs. These chimeric RNAs contained additional ribonucleotide sequences (approximately 30 nt) derived from the vector sequence at each 5′ terminal. Tables show the mean values and standard deviations of the relative luciferase activities from 3 experiments. *Normalized luciferase activity in the absence of sRNAs was set to 100%.
Figure 6.
Figure 6.
RNA gel shift analysis of S-20 target mRNAs. RNA-RNA interactions were examined with an RNA gel shift analysis. A FITC-labeled oligoribonucleotide, either S-20 (6 pmol) or S-8 (6 pmol), was incubated with 0-20 pmol of each target oligoribonucleotide (osmC, glyQ, ygiM or trpB) at 70°C for 7 min, and then at room temperature for 1 h. RNA-RNA interactions were analyzed by electrophoresis on a non-denaturing 4% (w/v) polyacrylamide gel. Two FITC-labeled mutant oligoribonucleotides, S-20 Ma and S-20 Mf, were examined in a similar manner (also see Fig. 7A). Similar results were obtained in at least 2 independent experiments.
Figure 7.
Figure 7.
Analysis of S-20 mutants. (A) Nucleotide sequences (shown in orange) of the regions inserted into the artificial S-20 sRNA expression plasmid pASRIIS-20 and the ribonucleotides responsible for its hybridization with each target mRNA (indicated by black circles). See also Fig. 4. (B) S-20 nucleotide sequence (insert) and its mutants. S-20 nucleotides are indicated in orange and the substituted nucleotides in the S-20 sequence are indicated in black. (*) Inhibition of E. coli growth is shown as the following 3 categories: +++, maximum OD600 < 0.6; ++, 0.6 ≤ maximum OD600 < 0.8; +, 0.8 ≤ maximum OD600. See also Fig. S5.

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