Biochemical and Biophysical Research Communications
Knockdown of recA gene expression by artificial small RNAs in Escherichia coli
Highlights
► Artificial small RNAs (sRNAs) targeting RecA were engineered. ► Artificial sRNAs were able to knockdown endogenous RecA expression. ► sRNA expression inhibited swarming motility and increased antibiotic sensitivity.
Introduction
Bacterial RecA is a highly conserved multifunctional protein that plays a central role in DNA repair and regulation of the SOS response to DNA damage [1], [2]. RecA has also been implicated in pathogenic processes such as swarming motility [3], activation of toxin biosynthesis [4], [5], competence [6], [7], virulence factor production [8] and genetic diversification of biofilms [9]. Thus, RecA has multiple cellular functions that make it vital for an organism’s survival and pathogenicity. More recently, the Collins group showed that all the major bactericidal antibiotics lead to the production of toxic hydroxyl radicals which trigger the bacteria to counteract by activating the SOS response [10]. Their work provides an explanation for the increased antibiotic sensitivity of the recA mutants that are not capable of activating the SOS response [11], [12], and suggests RecA or other genes involved in the SOS response as potential novel drug targets to combat the ever-increasing problem of antibiotic resistance.
The goal of the present work is to develop “artificial small RNAs (sRNAs)” that can posttranscriptionally repress RecA expression. In contrast to the permanent recA knockout strains that have been used to study the gene functions, our trans-acting artificial sRNAs can dynamically and finely modulate the RecA expression level. Such artificial sRNAs may enable a more accurate emulation of RecA-targeting drugs whose effects are often transient and imperfect, and could be used to study temporal and dosage effects of RecA-targeting drugs.
Our artificial sRNAs are inspired by the natural noncoding bacterial sRNAs that posttranscriptionally regulate their target mRNAs in trans [13]. This regulation occurs via imperfect Watson–Crick base pairing between the sRNA and its target mRNA and is facilitated by the chaperone protein Hfq [14]. Pairing occurs over a seed region of 6–8 base-pairs although longer interactions have been discovered [15]. Several recent studies have highlighted the modular nature of these sRNAs to possess three discrete domains: (1) mRNA target interacting (antisense) domain, (2) Hfq binding domain, and (3) transcriptional terminator domain [16], [17], [18], [19]. Taking advantage of this modular nature, our group constructed a library in which a randomized antisense domain is fused to an Hfq-binding/transcription terminator domain (sRNA scaffold) derived from natural sRNAs. Using a reporter plasmid that contains the 5′ leader sequence from the targeted mRNA, we isolated a number of artificial sRNAs that efficiently repressed expression of an outer-membrane porin (OmpF) and flagellin (FliC) in Escherichia coli [20].
In the present study, we performed a high-throughput screen to identify artificial anti-recA sRNAs and demonstrate that the wild-type E. coli expressing the sRNAs exhibit phenotypes consistent with RecA knockdown. The artificial sRNAs targeting RecA can be useful for studying the diverse functions of RecA as well as for developing new antibiotic strategies.
Section snippets
Bacterial strains and culture conditions
E. coli TOP10 cells (Invitrogen, F- mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG λ−) were used in all experiments, unless specified otherwise. Western blot analysis, swarming motility assay and ciprofloxacin sensitivity assay were performed using E. coli K-12 MG1655 cells (F− λ− ilvG− rfb-50 rph-1) obtained from Coli Genetic Stock Center (CGSC). The cells were grown at 37 °C (unless specified otherwise) from single-colony isolates or
Results and discussion
In this work, a plasmid library was designed to constitutively express artificial sRNAs with a randomized antisense domain (20–30 nt) fused to the Spot42 sRNA scaffold as previously described (Supplementary Fig. S1A) [20]. This library was subsequently transformed into TOP10 E. coli cells containing the reporter plasmid pKP33-RecA-GFPuv which transcribes the 5′ UTR and the first 12 codons derived from the recA mRNA fused to gfpuv coding region from a synthetic constitutive promoter (
Acknowledgements
This work was supported by National Science Foundation (1016357). Y.S. was supported by the JSPS International Training Program (ITP).
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