Assembly of colicin A in the outer membrane of producing Escherichia coli cells requires both phospholipase A and one porin, but phospholipase A is sufficient for secretion.
Three oligomeric forms of colicin A with apparent molecular masses of about 95 to 98 kDa were detected on sodium dodecyl sulfate (SDS)-polyacrylamide gels loaded with unheated samples from colicin A-producing cells of Escherichia coli. These heat-labile forms, called colicins Au, were visualized both on immunoblots probed with monoclonal antibodies against colicin A and by radiolabeling. Cell fractionation studies show that these forms of colicin A were localized in the outer membrane whether or not the producing cells contained the cal gene, which encodes the colicin A lysis protein responsible for colicin A release in the medium. Pulse-chase experiments indicated that their assembly into the outer membrane, as measured by their heat modifiable migration in SDS gels, was an efficient process. Colicins Au were produced in various null mutant strains, each devoid of one major outer membrane protein, except in a mutant devoid of both OmpC and OmpF porins. In cells devoid of outer membrane phospholipase A (OMPLA), colicin A was not expressed. Colicins Au were detected on immunoblots of induced cells probed with either polyclonal antibodies to OmpF or monoclonal antibodies to OMPLA, indicating that they were associated with both OmpF and OMPLA. Similar heat-labile forms were obtained with various colicin A derivatives, demonstrating that the C-terminal domain of colicin A, but not the hydrophobic hairpin present in this domain, was involved in their formation.
Cavard
Daniele
D
Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Centre National de la Recherche Scientifique, 13402 Marseille Cedex 20, France. cavard@ibsm.cnrs-mrs.fr
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D003087:Colicins / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D007553:Isotope Labeling; D010741:Phospholipases A / Q000378:metabolism*; D054466:Phospholipases A1; D018272:Porins / Q000235:genetics / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D001425:Bacterial Outer Membrane Proteins; D003087:Colicins; C113724:OmpC protein; C109237:OmpF protein; D018272:Porins; D013462:Sulfur Radioisotopes; D010741:Phospholipases A; D054466:Phospholipases A1; C412876:outer membrane phospholipase A