Open AccessA genome‐wide inducible phenotypic screen identifies antisense RNA constructs silencing E scherichia coli essential genes
Author(s) -
Meng Jia,
Kanzaki Gregory,
Meas Diane,
Lam Christopher K.,
Crummer Heather,
Tain Justina,
Xu H. Howard
Publication year - 2012
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2012.02503.x
Subject(s) - antisense rna , biology , gene , rna , genetics , rna silencing , gene silencing , escherichia coli , operon , rna interference
Regulated antisense RNA (as RNA ) expression has been employed successfully in G ram‐positive bacteria for genome‐wide essential gene identification and drug target determination. However, there have been no published reports describing the application of as RNA gene silencing for comprehensive analyses of essential genes in G ram‐negative bacteria. In this study, we report the first genome‐wide identification of as RNA constructs for essential genes in E scherichia coli . We screened 250 000 library transformants for conditional growth inhibitory recombinant clones from two shotgun genomic libraries of E . coli using a paired‐termini expression vector (p HN 678). After sequencing plasmid inserts of 675 confirmed inducer sensitive cell clones, we identified 152 separate as RNA constructs of which 134 inserts came from essential genes, while 18 originated from nonessential genes (but share operons with essential genes). Among the 79 individual essential genes silenced by these as RNA constructs, 61 genes (77%) engage in processes related to protein synthesis. The cell‐based assays of an as RNA clone targeting fusA (encoding elongation factor G ) showed that the induced cells were sensitized 12‐fold to fusidic acid, a known specific inhibitor. Our results demonstrate the utility of the paired‐termini expression vector and feasibility of large‐scale gene silencing in E . coli using regulated as RNA expression.