Phosphatidylinositol-specific phospholipase C of Bacillus cereus: cloning, sequencing, and relationship to other phospholipases.
The phosphatidylinositol (PI)-specific phospholipase C (PLC) of Bacillus cereus was cloned into Escherichia coli by using monoclonal antibody probes raised against the purified protein. The enzyme is specific for hydrolysis of the membrane lipid PI and PI-glycan-containing membrane anchors, which are important structural components of one class of membrane proteins. The protein expressed in E. coli comigrated with B. cereus PI-PLC in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as detected by immunoblotting, and conferred PI-PLC activity on the host. This enzyme activity was inhibited by PI-PLC-specific monoclonal antibodies. The nucleotide sequence of the PI-PLC gene suggests that this secreted bacterial protein is synthesized as a larger precursor with a 31-amino-acid N-terminal extension to the mature enzyme of 298 amino acids. From analysis of coding and flanking sequences of the gene, we conclude that the PI-PLC gene does not reside next to the gene cluster of the other two secreted phospholipases C on the bacterial chromosome. The deduced amino acid sequence of the B. cereus PI-PLC contains a stretch of significant similarity to the glycosylphosphatidylinositol-specific PLC of Trypanosoma brucei. The conserved peptide is proposed to play a role in the function of these enzymes.
Kuppe
A
A
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Evans
L M
LM
McMillen
D A
DA
Griffith
O H
OH
D000595:Amino Acid Sequence; D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics; D010738:Type C Phospholipases / Q000235:genetics* / Q000378:metabolism
D010716:Phosphatidylinositols; D010738:Type C Phospholipases