An extended ToxR POSSYCCAT system for positive and negative selection of self-interacting transmembrane domains.
Assay systems based on the ToxR protein are widely used to investigate interaction of transmembrane domains that come from natural proteins or are isolated from combinatorial libraries. The principle of this method is that self-interaction of any given transmembrane domain, which is expressed within a ToxR chimeric protein, drives ToxR-ToxR assembly in a bacterial inner membrane. In current versions of the system, ToxR-ToxR interaction drives transcription activation of the cholera toxin (ctx) promoter and thereby induces expression of downstream reporter genes in appropriately constructed bacterial strains. Here, we describe the application of other known ToxR-regulated promoters. We show that interacting transmembrane domains also promote ToxR-driven activation of the ompU promoter. Conversely, these interactions efficiently repress transcription from the constitutively active ompT promoter. We present novel Escherichia coli strains whose chromosomes harbor fusions of ompU or ompT promoters with different reporter genes. Depending on the used promoter, self-interaction of transmembrane domains induces or represses reporter enzyme expression in these cells. These strains extend current applications of the ToxR protein and may find use in mapping transmembrane helix-helix interfaces and selection of transmembrane domains with medium affinities.
Lindner
Eric
E
Technische Universität München, Lehrstuhl für Chemie der Biopolymere, TU München, Weihenstephaner Berg 3, D-85354 Freising-Weihenstephan, Germany.
Unterreitmeier
Stephanie
S
Ridder
Anja N J A
AN
Langosch
Dieter
D
D018829:Adhesins, Bacterial / Q000235:genetics*; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D020650:Combinatorial Chemistry Techniques; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D018272:Porins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012641:Selection, Genetic; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation; D014170:Transformation, Genetic
D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C096349:OmpU protein, Vibrio cholerae; D018272:Porins; D014157:Transcription Factors; C405564:ompT protein, bacteria; C499324:toxR protein, Vibrio cholerae