title: Screening for transmembrane association in divisome proteins using TOXGREEN, a high-throughput variant of the TOXCAT assay.
abstract: TOXCAT is a widely used genetic assay to study interactions of transmembrane helices within the inner membrane of the bacterium Escherichia coli. TOXCAT is based on a fusion construct that links a transmembrane domain of interest with a cytoplasmic DNA-binding domain from the Vibrio cholerae ToxR protein. Interaction driven by the transmembrane domain results in dimerization of the ToxR domain, which, in turn, activates the expression of the reporter gene chloramphenicol acetyl transferase (CAT). Quantification of CAT is used as a measure of the ability of the transmembrane domain to self-associate. Because the quantification of CAT is relatively laborious, we developed a high-throughput variant of the assay, TOXGREEN, based on the expression of super-folded GFP and detection of fluorescence directly in unprocessed cell cultures. Careful side-by-side comparison of TOXCAT and TOXGREEN demonstrates that the methods have comparable response, dynamic range, sensitivity and intrinsic variability both in LB and minimal media. The greatly enhanced workflow makes TOXGREEN much more scalable and ideal for screening, since hundreds of constructs can be rapidly assessed in 96 well plates. Even for small scale investigations, TOXGREEN significantly reduces time, labor and cost associated with the procedure. We demonstrate applicability with a large screening for self-association among the transmembrane domains of bitopic proteins of the divisome (FtsL, FtsB, FtsQ, FtsI, FtsN, ZipA and EzrA) belonging to 11 bacterial species. The analysis confirms a previously reported tendency for FtsB to self-associate, and suggests that the transmembrane domains of ZipA, EzrA and FtsN may also possibly oligomerize.
authors.affiliation: Dept of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr., Madison, WI 53706, United States.
authors.forename: Claire R
authors.initials: CR
authors.lastname: Armstrong
authors.affiliation: Dept of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr., Madison, WI 53706, United States. Electronic address: senes@wisc.edu.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Senes
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D057166:High-Throughput Screening Assays; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014734:Vibrio cholerae / Q000145:classification / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C456028:FtsB protein, E coli; D008565:Membrane Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014157:Transcription Factors; C499324:toxR protein, Vibrio cholerae; D049452:Green Fluorescent Proteins; D015500:Chloramphenicol O-Acetyltransferase
keywords: Divisome; Fluorescence; Membrane protein; TOXCAT; TOXGREEN; Transmembrane helix association