2a) with the IC50 value of 28 μM. Subsequently, the inducer concentrations for sensitizing PT44 clone against fusidic acid (which targets EF-G) were further optimized (Fig. 2b). Our results indicated that at 45 μM IPTG, the asRNA clone exhibits 12-fold increase (IC50 at 0 μM divided by IC50 at 45 μM) in MK-2206 mw sensitivity to the specific inhibitor (Fig. 2b). The optimized cell-based assay was performed against serial dilutions of nine other antibiotics (Fig. 2c). Results showed that the fusA asRNA clone was the most sensitive to
fusidic acid (12-fold), followed by erythromycin (fivefold) and tetracycline (fourfold), both are well-known antibiotics targeting protein synthesis (Fig. 2c). It was recognized that conditional silencing by introduced asRNAs in Gram-negative
bacteria is less efficient than in Gram-positive bacteria (Wagner & Flardh, 2002). Specifically, while global essential genes in S. aureus (Ji et al., 2001; Forsyth et al., 2002) and S. mutans (Wang & Kuramitsu, 2005) have Crizotinib datasheet been identified by regulated asRNAs, the adoption of such approach in Gram-negative bacteria has not been reported (Good & Stach, 2011). Although the reasons for such discrepancy are not well defined, one possible explanation lies in the reduced stability of plasmid-borne artificial asRNAs in E. coli probably due to the presence of RNase E in this bacterium (Xu et al., 2010), but not in S. aureus. For this reason, Nakashima and colleagues (Nakashima et al., 2006) designed a series of E. coli plasmid vectors which produce RNA molecules with paired-termini to increase the asRNA stability G protein-coupled receptor kinase and conditional gene silencing. Targeted antisense fragment cloning using such paired-termini vectors has produced asRNA constructs which have shown to knock-down
or silence the expression of a number of essential genes in E. coli (Nakashima et al., 2006). In this communication, we report a genome-wide application of regulated asRNA expression in E. coli using the vector pHN678. Here, we demonstrated that employing this paired-termini vector indeed identified a large number of asRNA constructs targeting E. coli essential genes and, to a lesser extent, some nonessential genes which share operons with essential genes. While asRNA constructs targeting essential genes of a number of cellular processes in E. coli were identified (Table 1 and Table S1), particularly striking was the observation that the asRNAs predominately silence the expression of essential genes (77% of total genes) involved in protein synthesis processes (tRNAs, tRNA synthetases, transcription, ribosomal proteins, and translation factors) (Table S1). We speculate that this bias may have been caused by high basal level (leaky) promoter (Ptrc) activity from the vector in the absence of IPTG (Nakashima & Tamura, 2009) during the library transformation process.