A general system for studying protein-protein interactions in Gram-negative bacteria.
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.
Pelletier
Dale A
DA
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
Hurst
Gregory B
GB
Foote
Linda J
LJ
Lankford
Patricia K
PK
McKeown
Catherine K
CK
Lu
Tse-Yuan
TY
Schmoyer
Denise D
DD
Shah
Manesh B
MB
Hervey
W Judson
WJ
McDonald
W Hayes
WH
Hooker
Brian S
BS
Cannon
William R
WR
Daly
Don S
DS
Gilmore
Jason M
JM
Wiley
H Steven
HS
Auberry
Deanna L
DL
Wang
Yisong
Y
Larimer
Frank W
FW
Kennel
Stephen J
SJ
Doktycz
Mitchel J
MJ
Morrell-Falvey
Jennifer L
JL
Owens
Elizabeth T
ET
Buchanan
Michelle V
MV
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
D000345:Affinity Labels; D001426:Bacterial Proteins; D015335:Molecular Probes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012321:DNA-Directed RNA Polymerases