The proteins encoded by the rbs operon of Escherichia coli: I. Overproduction, purification, characterization, and functional analysis of RbsA.
The nucleotide-binding component of the high-affinity ribose transport system of Escherichia coli, RbsA, was overproduced from a T7-7 expression vector, and the protein was purified. Biochemical analyses of the purified protein indicated that the ATP analogues, 5'-FSBA and 8-azido ATP, covalently labeled the protein, a reaction that was inhibited by ATP, but not by GTP or CTP. The pure protein exhibited low-level ATPase activity with a K(m) of about 140 microM. Analyses of bacterial strains carrying chromosomal deletions of rbsA and other rbs genes suggested that RbsA is important for the chemotaxis function, a surprising result that was not anticipated from previous studies. However, an inconsistency between the several results from deletion strains raises questions regarding the interpretations of the in vivo data.
Barroga
C F
CF
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Zhang
H
H
Wajih
N
N
Bouyer
J H
JH
Hermodson
M A
MA
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000345:Affinity Labels / Q000032:analysis / Q000737:chemistry; D000818:Animals; D001345:Autoradiography; D001386:Azides / Q000032:analysis / Q000737:chemistry; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002633:Chemotaxis; D002872:Chromosome Deletion; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors; D026901:Membrane Transport Proteins; D009876:Operon; D010782:Photolysis; D010957:Plasmids; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C102053:RbsA protein, E coli; D011994:Recombinant Proteins; C017040:8-azidoadenosine 5'-triphosphate; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000241:Adenosine