MULTIPLE PHOSPHORYLATION OF SACY, A BACILLUS-SUBTILIS TRANSCRIPTIONALANTITERMINATOR NEGATIVELY CONTROLLED BY THE PHOSPHOTRANSFERASE SYSTEM

The Bacillus subtilis SacY transcriptional antiterminator is a regulat or involved in sucrose-promoted induction of the sacB gene, SacY activ ity is negatively controlled by enzyme I and HPr, the general energy c oupling proteins of the phosphoenolpyruvate:sugar phosphotransferase s ystem (PTS), and by SacX, a membranal protein homologous to SacP, the B. subtilis sucrose-specific PTS-permease. Previous studies suggested that the negative control exerted by the PTS oil bacterial antitermina tors of the SacY family involves phosphoenolpyruvate-dependent phospho rylation by the sugar-specific PTS-permeases. However, data reported h erein show direct phosphorylation of SacY by HPr(His similar to P) wit h no requirement for SacX, Experiments were carried out to determine t he phosphorylatable residues in SacY, In silico analyses of SacY and i ts homologues revealed the modular structure of these proteins as well as four conserved histidines within two homologous domains (here desi gnated P1 and P2), present in 14 distinct mRNA- and DNA-binding bacter ial transcriptional regulators, Single or multiple substitutions of th ese histidyl residues were introduced in SacY by site-directed mutagen esis, and their effects on phosphorylation and antitermination activit y were examined. In vitro phosphorylation experiments showed that SacY was phosphorylated on three of the conserved histidines. Nevertheless , in vivo studies using cells bearing a sacB'-lacZ reporter fusion, as well as SacY mutants lacking the phosphorylatable histidyls, revealed that only His-99 is directly involved in regulation of SacY antitermi nation activity.