title: A general system for studying protein-protein interactions in Gram-negative bacteria.
abstract: One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged "bait" proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.
authors.affiliation: Biosciences Division, Chemical Sciences Division, Computer Science and Mathematics Division, and Physical Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. pelletierda@ornl.gov
authors.forename: Dale A
authors.initials: DA
authors.lastname: Pelletier
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Hurst
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Foote
authors.forename: Patricia K
authors.initials: PK
authors.lastname: Lankford
authors.forename: Catherine K
authors.initials: CK
authors.lastname: McKeown
authors.forename: Tse-Yuan
authors.initials: TY
authors.lastname: Lu
authors.forename: Denise D
authors.initials: DD
authors.lastname: Schmoyer
authors.forename: Manesh B
authors.initials: MB
authors.lastname: Shah
authors.forename: W Judson
authors.initials: WJ
authors.lastname: Hervey
authors.forename: W Hayes
authors.initials: WH
authors.lastname: McDonald
authors.forename: Brian S
authors.initials: BS
authors.lastname: Hooker
authors.forename: William R
authors.initials: WR
authors.lastname: Cannon
authors.forename: Don S
authors.initials: DS
authors.lastname: Daly
authors.forename: Jason M
authors.initials: JM
authors.lastname: Gilmore
authors.forename: H Steven
authors.initials: HS
authors.lastname: Wiley
authors.forename: Deanna L
authors.initials: DL
authors.lastname: Auberry
authors.forename: Yisong
authors.initials: Y
authors.lastname: Wang
authors.forename: Frank W
authors.initials: FW
authors.lastname: Larimer
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Kennel
authors.forename: Mitchel J
authors.initials: MJ
authors.lastname: Doktycz
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Morrell-Falvey
authors.forename: Elizabeth T
authors.initials: ET
authors.lastname: Owens
authors.forename: Michelle V
authors.initials: MV
authors.lastname: Buchanan
mesh_terms: D000345:Affinity Labels; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000378:metabolism*; D015335:Molecular Probes; D010957:Plasmids; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012241:Rhodopseudomonas / Q000201:enzymology; D020592:Shewanella / Q000201:enzymology
chemical_list: D000345:Affinity Labels; D001426:Bacterial Proteins; D015335:Molecular Probes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012321:DNA-Directed RNA Polymerases