We describe here details of the method we used to identify and distinguish essential from nonessential genes on the bacterial Escherichia coli chromosome. Three key features characterize our method: high-efficiency recombination, precise replacement of just the open reading frame of a chromosomal gene, and the presence of naturally occurring duplications within the bacterial genome. We targeted genes encoding functions critical for processes of transcription and translation. Proteins from three complexes were evaluated to determine if they were essential to the cell by deleting their individual genes. The transcription elongation Nus proteins and termination factor Rho, which are involved in rRNA antitermination, the ribosomal proteins of the small 30S ribosome subunit, and minor ribosome-associated proteins were analyzed. It was concluded that four of the five bacterial transcription antitermination proteins are essential, while all four of the minor ribosome-associated proteins examined (RMF, SRA, YfiA, and YhbH), unlike most ribosomal proteins, are dispensable. Interestingly, although most 30S ribosomal proteins were essential, the knockouts of six ribosomal protein genes, rpsF (S6), rpsI (S9), rpsM (S13), rpsO (S15), rpsQ (S17), and rpsT (S20), were viable.A gene may either be essential or nonessential for viability of a cell. An essential gene encodes a function which is required under all growth conditions, and so its elimination is lethal to the cell. Hence, this is generally the most interesting, yet difficult, type of genes to identify and characterize. In an era when many genomes have been sequenced and their coding regions identified, the ability to distinguish essential from nonessential genes still requires careful experimental assessment (23,24,29,48). Here, we create gene replacements to distinguish these two classes of genes via direct selection in Escherichia coli using phage Red-mediated homologous recombination, termed recombineering (12,20,69).Recombineering uses the Red recombination functions of phage to manipulate the DNA on chromosomes or plasmids of enteric bacteria (12,18). In this work, recombineering was adapted to directly identify essential genes in E. coli. The procedure is not intended for a high-throughput identification of essential genes, although it could certainly be upgraded to a larger-scale analysis. It should be most useful for a targeted, knowledge-based analysis of cellular systems and their individual components, as we have demonstrated in this paper. Our method was developed using three features: (i) an extremely high efficiency of recombineering (69); (ii) the ability of recombineering to provide precise replacement of just the open reading frame (ORF) of a chromosomal gene with the orf of an antibiotic resistance cassette, taking care to design and express such a replacement orf from the regulatory regions of the replaced gene (69) so as to avoid polar effects of the insert on transcription and translation; and (iii) naturally occurring duplications of regions in th...
