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J. Bacteriol. doi:10.1128/JB.01713-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Essentiality of Ribosomal and Transcription Antitermination Proteins Analyzed by Systematic Gene Replacement in E. coli.

Basic Research Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD 21702; Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702

^* To whom correspondence should be addressed. Email: court{at}ncifcrf.gov.

	Abstract

We describe here details of the method we used to identify and distinguish essential from nonessential genes on the bacterial E. 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 ribosomal RNA 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.

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