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Journal of Bacteriology, April 2007, p. 2844-2853, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01713-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Essentiality of Ribosomal and Transcription Antitermination Proteins Analyzed by Systematic Gene Replacement in Escherichia coli{triangledown}

Mikhail Bubunenko,1,2 Teresa Baker,2 and Donald L. Court2*

Basic Research Program, SAIC-Frederick, Inc., National Cancer Institute—Frederick, Frederick, Maryland 21702,1 Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute—Frederick, Frederick, Maryland 217022

Received 6 November 2006/ Accepted 18 January 2007

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.

* Corresponding author. Mailing address: Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702. Phone: (301) 846-5940. Fax: (301) 846-6988. E-mail: court{at}ncifcrf.gov.

{triangledown} Published ahead of print on 2 February 2007.

Journal of Bacteriology, April 2007, p. 2844-2853, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01713-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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