title: Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli.
abstract: BACKGROUND
  Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library.
  
  
  RESULTS
  Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport.
  
  
  CONCLUSIONS
  Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the potential of functional clones interacting with host proteins to inhibit growth would make this approach most relevant for the study of prokaryotic genomes.
authors.affiliation: Department of Infectious Disease, Wyeth Research, 401 N, Middletown Rd, Pearl River, NY 10965, USA. shaney@wyeth.com
authors.forename: Steven A
authors.initials: SA
authors.lastname: Haney
authors.forename: David
authors.initials: D
authors.lastname: Keeney
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.forename: Soraya
authors.initials: S
authors.lastname: Moghazeh
authors.forename: Steve
authors.initials: S
authors.lastname: Projan
authors.forename: Beth
authors.initials: B
authors.lastname: Rasmussen
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D016680:Genome, Bacterial; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D014170:Transformation, Genetic; D020798:Two-Hybrid System Techniques / Q000592:standards; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins