Functional expression of a Rhodospirillum rubrum gene encoding dinitrogenase reductase ADP-ribosyltransferase in enteric bacteria.
The function of the cloned draT gene of Rhodospirillum rubrum was studied by placing it under the control of the tac promoter in the vector, pKK223-3. After induction with isopropyl-beta-D-thiogalactopyranoside, dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in crude extracts of the heterologous hosts Escherichia coli and Klebsiella pneumoniae. In addition, the expression of draT produced a Nif- phenotype in the otherwise wild-type K. pneumoniae strains, the result of the ADP-ribosylation of accumulated dinitrogenase reductase (DR). DR from a nifF- background was also susceptible to ADP-ribosylation, indicating that the oxidized form of DR will serve as a substrate for DRAT in vivo. A mutation that changes the Arg-101 residue of DR, the ADP-ribose attaching site, eliminates the ADP-ribosylation of DR in vivo, confirming the necessity of this residue for modification.
Fu
H A
HA
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Wirt
H J
HJ
Burris
R H
RH
Roberts
G P
GP
D036002:ADP Ribose Transferases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005838:Genotype; D007711:Klebsiella pneumoniae / Q000235:genetics*; D008969:Molecular Sequence Data; D009591:Nitrogenase / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012247:Rhodospirillum rubrum / Q000201:enzymology / Q000235:genetics*
D011994:Recombinant Proteins; D009591:Nitrogenase; D036002:ADP Ribose Transferases; C057637:dinitrogenase reductase ADP-ribosyltransferase