Colicin M is inactivated during import by its immunity protein

Abstract

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.

Distinct colicin M-like bacteriocin-immunity pairs in Burkholderia

“…Inhibitory action is mediated by phosphatase activity that cleaves lipid II peptidoglycan precursors, releasing undecaprenol and pyrophosphoryl groups 21 22 . Immunity is provided by a downstream-located gene, transcribed in opposite direction, that encodes a cytoplasmic membrane-anchored immunity protein ( C ol M immunity protein, Cmi), facing the periplasmic space and protecting the producer cells via an unknown mechanism 23 24 . The Cmi monomer is composed of four β strands and four α helices and requires an intramolecular disulfide bond to be functional 25 .…”

Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST

“…The Pf3 transmembrane domain allows insertion into the inner membrane of bacteria with the protein facing the cytoplasm 23–25 . In the second construction, FAST was fused at the C-terminus of the transmembrane domain of the specific immunity protein Cmi, which inhibits the action of colicin M in the periplasm of E. coli 26 . The transmembrane domain of Cmi also allows insertion into the inner membrane, but unlike Pf3, Cmi exposes the residues fused to its C-terminus in the periplasm 26 .…”

“…In the second construction, FAST was fused at the C-terminus of the transmembrane domain of the specific immunity protein Cmi, which inhibits the action of colicin M in the periplasm of E. coli 26 . The transmembrane domain of Cmi also allows insertion into the inner membrane, but unlike Pf3, Cmi exposes the residues fused to its C-terminus in the periplasm 26 . Western blot on whole cell extracts using anti-E-tag primary antibodies demonstrated that both proteins were produced (Supplementary Fig.…”

Activation of Colicin M by the FkpA Prolyl Cis-Trans Isomerase/Chaperone

“…Cmi renders cells resistant to imported Cma (31). Cmi is located in the periplasm and anchored to the cytoplasmic membrane by its N-terminal hydrophobic sequence (32, 33). Membrane fixation brings Cmi to the site where Cma cleaves its substrate and prevents release of Cmi by osmotic shock.…”

“…Membrane fixation brings Cmi to the site where Cma cleaves its substrate and prevents release of Cmi by osmotic shock. We used the previously constructed plasmid pPG773, in which the translocation and membrane anchor of the penicillin-binding protein PBP3 was fused to the periplasmic activity domain of Cmi (33). Transformants carrying this plasmid confer immunity without induction of T7 polymerase synthesis because it contains an E. coli polymerase promoter.…”