Colicin M is inactivated during import by its immunity protein.
Colicin M (Cma) displays a unique activity that interferes with murein and O-antigen biosynthesis through inhibition of lipid-carrier regeneration. Immunity is conferred by a specific immunity protein (Cmi) that inhibits the action of colicin M in the periplasm. The subcellular location of Cmi was determined by constructing hybrid proteins between Cmi and the TEM-beta-lactamase (BlaM), which confers resistance to ampicillin only when it is translocated across the cytoplasmic membrane with the aid of Cmi. The smallest Cmi'-BlaM hybrid that conferred resistance to 50 micrograms/ml ampicillin contained 19 amino acid residues of Cmi; cells expressing Cmi'-BlaM with only five N-terminal Cmi residues were ampicillin sensitive. These results support a model in which the hydrophobic sequence of Cmi comprising residues 3-23 serves to translocate residues 24-117 of Cmi into the periplasm and anchors Cmi to the cytoplasmic membrane. Residues 8-23 are integrated in the cytoplasmic membrane and are not involved in Cma recognition. This model was further tested by replacing residues 1-23 of Cmi by the hydrophobic amino acid sequence 1-42 of the penicillin binding protein 3 (PBP3). In vivo, PBP3'-'Cmi was as active as Cmi, demonstrating that translocation and anchoring of Cmi is not sequence-specific. Substitution of the 23 N-terminal residues of Cmi by the cleavable signal peptide of BlaM resulted in an active BlaM'-'Cmi hybrid protein. The immunity conferred by BlaM'-'Cmi was high, but not as high as that associated with Cmi and PBP3'-'Cmi, demonstrating that soluble Cmi lacking its membrane anchor is still active, but immobilization in the cytoplasmic membrane, the target site of Cma, increases its efficiency. Cmi delta 1-23 remained in the cytoplasm and conferred no immunity. We propose that the immunity protein inactivates colicin M in the periplasm before Cma can reach its target in the cytoplasmic membrane.
Gross
P
P
Universität Tübingen, Germany.
Braun
V
V
D000595:Amino Acid Sequence; D000668:Ampicillin Resistance; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins; D002462:Cell Membrane / Q000737:chemistry; D003087:Colicins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D006602:Hexosyltransferases / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000235:genetics; D002267:Muramoylpentapeptide Carboxypeptidase; D046915:Penicillin-Binding Proteins; D046928:Peptidoglycan Glycosyltransferase; D010458:Peptidyl Transferases / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D001618:beta-Lactamases / Q000032:analysis / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; C478704:FtsI protein, E coli; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C488856:cmI protein, E coli; C007104:colicin immunity proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D046928:Peptidoglycan Glycosyltransferase; D002267:Muramoylpentapeptide Carboxypeptidase; D001618:beta-Lactamases