PROTEIN-PHOSPHORYLATION CHAIN OF BACILLUS-SUBTILIS FRUCTOSE-SPECIFIC PHOSPHOTRANSFERASE SYSTEM AND ITS PARTICIPATION IN REGULATION OF THE EXPRESSION OF THE LEV OPERON

The proteins encoded by the fructose-inducible lev operon of Bacillus subtilis are components of a phosphotransferase system. They transport fructose by a mechanism which couples sugar uptake and phosphoenolpyr uvate-dependent sugar phosphorylation. The complex transport system co nsists of two integral membrane proteins (LevF and LevG) and two solub le, hydrophilic proteins (LevD and LevE). The two soluble proteins for m together with the general proteins of the phosphotransferase system, enzyme I and HPr, a protein phosphorylation chain which serves to pho sphorylate fructose transported by LevF and LevG. We have synthesied m odified LevD and LevE by fusing a His-tag to the N-terminus of each pr otein allowing rapid and efficient purification of the proteins. We de termined His-9 in LevD and His-15 in LevE as the sites of PEP-dependen t phosphorylation by isolating single, labeled peptides derived from P -32-labeled LevD, LevD(His)(6), and LevE(His)(6). The labeled peptides were subsequently analyzed by amino acid sequencing and mass spectros copy. Mutations replacing the phosphorylatable histidyl residue in Lev D with an alanyl residue and in LevE with a glutamate or aspartate wer e introduced in the levD and levE genes. These mutations caused strong ly reduced fructose uptake via the lev-PTS. The mutant proteins were s ynthesized with a N-terminal His-tag and purified. Mutant LevD(His)(6) was very slowly phosphorylated, whereas mutant LevE(His)6 was not pho sphorylated at all. The corresponding levD and levE alleles were incor porated into the chromosome of a B, subtilis strain expressing the lac Z gene under control of the lev promoter. The mutations affecting the site of phosphorylation in either LevD or LevE were found to cause con stitutive expression from the lev promoter of B.