Abstract
Proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus subtilis were overexpressed, purified to near homogeneity, and characterized. The proteins isolated include Enzyme I, HPr, the glucose-specific IIA domain of the glucose-specific Enzyme II (IIAglc), and the mannitol-specific IIA protein, IIAmtl. Site specific mutant proteins of IIAglc and HPr were also overexpressed and purified, and their properties were compared with those of the wild type proteins. These proteins and their phosphorylated derivatives were characterized with respect to their immunological cross-reactivities employing the Western blot technique and in terms of their migratory behavior during sodium dodecyl sulfate-gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing. The interactions between homologous and heterologous Enzymes I and HPrs, between homologous and heterologous HPrs and the IIAglc proteins, and between homologous and heterologous IIAglc proteins and IIBCscr of B. subtilis as well as IICBglc of Escherichia coli were defined and compared kinetically. The mutant HPrs and IIAglc proteins were also characterized kinetically as PTS phosphocarrier proteins and/or as inhibitors of the phosphotransferase reactions of the PTS. These studies revealed that complexation of IIAglc with the mutant form of HPr in which serine 46 was replaced by aspartate (S46D) did not increase the rate of phosphoryl transfer from phospho Enzyme I to S46D HPr more than when IIAmtl was complexed to S46D HPr. These findings do not support a role for HPr(Ser-P) in the preferential utilization of one PTS carbohydrate relative to another. Functional analyses in E. coli established that IIAglc of B. subtilis can replace IIAglc of E. coli with respect both to sugar transport and to regulation of non-PTS permeases, catabolic enzymes, and adenylate cyclase. Site-specific mutations in histidyl residues 68 and 83 (H68A and H83A) inactivated IIAglc of B. subtilis with respect to phosphoryl transfer and its various regulatory roles.
Highlights
Proteins of the phosphoeno1pyruvate:sugarphosphotransferase system (PTS) of Bacillus subtilis were overexpressed,purifiedtonear homogeneity, and characterized
We describe the over- as well as Mg2+ and PEP-dependent, association has been expression and purification of all of the soluble proteins of reported for Enzyme I of S. typhimurium (Kukuruthe B. subtilis glucose and mannitol PTS including the IIAglC zinska et al, 1982, 1984)
We further carry out the requisite kinetic analyses to define the protein-protein interactions, both homologous and heterologous, between components of the B. subtilis and E. coli PTS constituents and show that IIA domain of the glucosespecific Enzyme I1 (IIAg)'" of B. subtilis can substitute for IIAg'"of E. coli with respect to all of its known regulatory functions
Summary
We further carry out the requisite kinetic analyses to define the protein-protein interactions, both homologous and heterologous, between components of the B. subtilis and E. coli PTS constituents and show that IIAg'" of B. subtilis can substitute for IIAg'"of E. coli with respect to all of its known regulatory functions. These studies suggest that contrary to a previous proposal, complexation ofIIAglC with HPr(Ser-P) does not allow preferential glucose phosphorylation relative to mannitol phosphorylation when IIAm" is complexed with HPr(Ser-P). Clude that approximately 18%of the totalcellular protein in crude extracts of E. coli MZ1 bearing the overexpression
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