Multiple phosphorylation of SacY, a Bacillus subtilis transcriptional antiterminator negatively controlled by the phosphotransferase system.
The Bacillus subtilis SacY transcriptional antiterminator is a regulator involved in sucrose-promoted induction of the sacB gene. SacY activity is negatively controlled by enzyme I and HPr, the general energy coupling proteins of the phosphoenolpyruvate:sugar phosphotransferase system (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 on bacterial antiterminators of the SacY family involves phosphoenolpyruvate-dependent phosphorylation by the sugar-specific PTS-permeases. However, data reported herein show direct phosphorylation of SacY by HPr(His approximately P) with no requirement for SacX. Experiments were carried out to determine the phosphorylatable residues in SacY. In silico analyses of SacY and its homologues revealed the modular structure of these proteins as well as four conserved histidines within two homologous domains (here designated P1 and P2), present in 14 distinct mRNA- and DNA-binding bacterial transcriptional regulators. Single or multiple substitutions of these histidyl residues were introduced in SacY by site-directed mutagenesis, and their effects on phosphorylation and antitermination activity 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 antitermination activity.
Tortosa
P
P
Laboratoire de Génétique des Microorganismes, Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique ERS 567, F-78850 Thiverval-Grignon, France.
Aymerich
S
S
Lindner
C
C
Saier
M H
MH
Reizer
J
J
Le Coq
D
D
D000483:Alleles; D000595:Amino Acid Sequence; D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D019143:Evolution, Molecular; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism; D010766:Phosphorylation; D016601:RNA-Binding Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; C076894:SacY protein, Bacillus subtilis; D014157:Transcription Factors; D006639:Histidine; D010728:Phosphoenolpyruvate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System