This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Merlin, C.
Right arrow Articles by Masters, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Merlin, C.
Right arrow Articles by Masters, M.

 Previous Article  |  Next Article 

Journal of Bacteriology, August 2002, p. 4573-4581, Vol. 184, No. 16
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.16.4573-4581.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Tools for Characterization of Escherichia coli Genes of Unknown Function

Christophe Merlin, Sean McAteer, and Millicent Masters*

Institute for Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland

Received 17 January 2002/ Accepted 14 May 2002

Despite the power of sequencing and of emerging high-throughput technologies to collect data rapidly, the definitive functional characterization of unknown genes still requires biochemical and genetic analysis in case-by-case studies. This often involves the deletion of target genes and phenotypic characterization of the deletants. We describe here modifications of an existing deletion method which facilitates the deletion process and enables convenient analysis of the expression properties of the target gene by replacing it with an FRT-lacZ-aph-Plac-FRT cassette. The lacZ gene specifically reports the activity of the deleted gene and therefore allows the determination of the conditions under which it is actively expressed. The aph gene, encoding resistance to kanamycin, provides a selectable means of transducing a deleted locus between strains so that the deletion can be combined with other relevant mutations. The lac promoter helps to overcome possible polar effects on downstream genes within an operon. Because the cassette is flanked by two directly repeated FRT sites, the cassette can be excised by the Flp recombinase provided in trans. Removing the cassette leaves an in-frame deletion with a short scar which should not interfere with downstream expression. Replacements of yacF, yacG, yacH, yacK (cueO), yacL, ruvA, ruvB, yabB, and yabC made with the cassette were used to verify its properties.

* Corresponding author. Mailing address: Institute of Cell and Molecular Biology, University of Edinburgh, Darwin Building, King's Buildings, Mayfield Rd., Edinburgh EH9 3JR, Scotland. Phone: 44 (0)131 650 5355. Fax: 44 (0)131 650 8650. E-mail: M.Masters{at}ed.ac.uk.

Journal of Bacteriology, August 2002, p. 4573-4581, Vol. 184, No. 16
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.16.4573-4581.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Aberg, A., Fernandez-Vazquez, J., Cabrer-Panes, J. D., Sanchez, A., Balsalobre, C. (2009). Similar and Divergent Effects of ppGpp and DksA Deficiencies on Transcription in Escherichia coli. J. Bacteriol. 191: 3226-3236 [Abstract] [Full Text]  
  • Sengupta, S., Nagaraja, V. (2008). YacG from Escherichia coli is a specific endogenous inhibitor of DNA gyrase. Nucleic Acids Res 36: 4310-4316 [Abstract] [Full Text]  
  • Darmon, E., Lopez-Vernaza, M. A., Helness, A. C., Borking, A., Wilson, E., Thacker, Z., Wardrope, L., Leach, D. R. F. (2007). SbcCD Regulation and Localization in Escherichia coli. J. Bacteriol. 189: 6686-6694 [Abstract] [Full Text]  
  • Zahra, R., Blackwood, J. K., Sales, J., Leach, D. R. F. (2007). Proofreading and Secondary Structure Processing Determine the Orientation Dependence of CAG{middle dot}CTG Trinucleotide Repeat Instability in Escherichia coli. Genetics 176: 27-41 [Abstract] [Full Text]  
  • Klumpp, J., Fuchs, T. M. (2007). Identification of novel genes in genomic islands that contribute to Salmonella typhimurium replication in macrophages. Microbiology 153: 1207-1220 [Abstract] [Full Text]  
  • Thacker, Z., Darmon, E., Keppel, F., Masters, M. (2006). The Escherichia coli rpoS-Dependent htrC Gene Is Not Involved in the Heat Shock Response. J. Bacteriol. 188: 8317-8320 [Abstract] [Full Text]  
  • Dudley, E. G., Abe, C., Ghigo, J.-M., Latour-Lambert, P., Hormazabal, J. C., Nataro, J. P. (2006). An IncI1 Plasmid Contributes to the Adherence of the Atypical Enteroaggregative Escherichia coli Strain C1096 to Cultured Cells and Abiotic Surfaces. Infect. Immun. 74: 2102-2114 [Abstract] [Full Text]  
  • Sharma, R., Tesfay, S., Tomson, F. L., Kanteti, R. P., Viswanathan, V. K., Hecht, G. (2006). Balance of bacterial pro- and anti-inflammatory mediators dictates net effect of enteropathogenic Escherichia coli on intestinal epithelial cells. Am. J. Physiol. Gastrointest. Liver Physiol. 290: G685-G694 [Abstract] [Full Text]  
  • Benders, G. A., Powell, B. C., Hutchison, C. A. III (2005). Transcriptional Analysis of the Conserved ftsZ Gene Cluster in Mycoplasma genitalium and Mycoplasma pneumoniae. J. Bacteriol. 187: 4542-4551 [Abstract] [Full Text]  
  • Hidalgo, A. A., Trombert, A. N., Castro-Alonso, J. C., Santiviago, C. A., Tesser, B. R., Youderian, P., Mora, G. C. (2004). Insertions of Mini-Tn10 Transposon T-POP in Salmonella enterica sv. typhi. Genetics 167: 1069-1077 [Abstract] [Full Text]  
  • Merlin, C., Masters, M., McAteer, S., Coulson, A. (2003). Why Is Carbonic Anhydrase Essential to Escherichia coli?. J. Bacteriol. 185: 6415-6424 [Abstract] [Full Text]  
  • Merlin, C., Gardiner, G., Durand, S., Masters, M. (2002). The Escherichia coli metD Locus Encodes an ABC Transporter Which Includes Abc (MetN), YaeE (MetI), and YaeC (MetQ). J. Bacteriol. 184: 5513-5517 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»