, 2002; Ji et al., 2004) and to discover novel antibacterial inhibitors

(Young et al., 2006; Wang et al., 2007). Furthermore, hundreds of S. aureus asRNA strains have been configured into a TargetArray, which was employed to study mechanisms of Tanespimycin action of antibacterial inhibitors (Donald et al., 2009; Xu et al., 2010). Thus, regulated asRNA expression has a great potential for antibiotic drug discovery. However, the regulated asRNA approach has seen limited success in Gram-negative bacteria, including E. coli. There have been no published reports describing the adoption of the regulated asRNA approach for comprehensive genome-wide essential gene determination and/or silencing in Gram-negative bacteria. It has been recognized that asRNA-mediated down-regulation of gene expression in E. coli is inefficient for reasons not yet clearly understood (Wagner & Flardh, 2002). Attempts to improve the efficiency were rather frustrating initially (Engdahl et al., 2001). Several years ago, a series of expression vectors were designed such that expressed asRNA molecules have paired-termini to enhance their stability and hence gene knock-down efficiency Selleckchem Ku 0059436 in E. coli (Nakashima et al., 2006). In this report, we present a first genome-wide attempt to obtain cell growth inhibitory E. coli

asRNA constructs through phenotypic screening two shotgun genomic libraries based on a paired-termini expression vector, pHN678 (Nakashima et al., 2006). Our results will stimulate further studies of gene functions, coordinated gene expression on operons and interactions of cellular processes via regulated asRNA in E. coli. Furthermore, the collection of the E. coli asRNA clones generated using this approach will be a valuable tool in the antibiotic drug discovery, especially for therapeutics targeting Gram-negative bacterial pathogens. Genomic cAMP DNA was extracted from E. coli MG1655 cells (American Type Culture Collection, Manassas, VA) using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) followed by partial digest with Sau3AI

or CviKI-1 (NEB, Ipswich, MA). The resulting DNA fragments (200–800 bp) were purified from agarose gels using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, CA). Plasmid vector was digested with BamHI (if Sau3AI was used to digest the genomic DNA) or SnaBI (if CviKI-1 was used), dephosphorylated using Antarctic Phosphatase (NEB) and then ligated with the inserts using the T4 DNA ligase (Life Technologies, Carlsbad, CA). Ligation mixtures were transformed into E. coli DH5α competent cells (Life Technologies) and plated onto LB agar plates plus 34 μg mL−1 chloramphenicol. Cloning efficiency of the pHN678 library was determined by colony PCR using the following primers: 5′-CGACATCATAACGGTTCTGGCAAAT-3′ (forward) and 5′-GACCGCTTCTGCGTTCTGATTT-3′ (reverse) (Eurofins MWG Operon, Huntsville, AL).