The aerobactin-mediated iron uptake system in Escherichia coli K-12. Karl Graham. Wooldridge 2381/34375 https://figshare.le.ac.uk/articles/thesis/The_aerobactin-mediated_iron_uptake_system_in_Escherichia_coli_K-12_/10135772 Some strains of E. coli secrete a low molecular weight compound, aerobactin, with a high affinity for ferric iron. After complexing ferric iron aerobactin may be transported back into the cell and used as a source of iron. The ability to secrete aerobactin and utilize iron derived from this source has been demonstrated to be an important virulence determinant for bacteria causing extraintestinal disease. The genes encoding the aerobactin system are often associated with large plasmids and are arranged in an operon consisting of five genes. Four genes encode enzymes responsible for the biosynthesis of aerobactin, while the fifth encodes an outer membrane receptor specific for ferric aerobactin. Antibodies against the outer membrane aerobactin receptor protein were raised in an attempt to evaluate the prospect of using the aerobactin receptor as a protective antigen. Antiserum was shown to react specifically in ELISA assays with whole cells of E. coli K-12 expressing the receptor and to inhibit uptake of [14C]aerobactin. The antiserum did not, however, react with the aerobactin receptor protein in a range of E. coli isolates from representative E. coli serotypes isolated from human extraintestinal infections. This was shown to be due to shielding of the receptor protein by lipopolysaccharide molecules on the surface of these strains. The presumed role of a number of chromosomally encoded gene products in the uptake of aerobactin across the outer and cytoplasmic membranes of E. coli has never been directly demonstrated. Their functions were confirmed experimentally, and entry of aerobactin into the cytoplasm of an uptake proficient cell was demonstrated for the first time. Inhibition of uptake into the cytoplasm of aerobactin, but not aerobactin-derived iron, was demonstrated in the presence of endogenous aerobactin. The aerobactin receptor protein also acts as a receptor for the bacteriocin cloacin DF13. No other envelope-associated proteins were known to be involved in sensitivity to cloacin DF13. The major outer membrane protein OmpF was demonstrated to be required in addition to the aerobactin receptor for sensitivity to cloacin DF13. It was shown not to be required for initial binding of the bacteriocin to the receptor and a role in subsequent translocation across the outer membrane was suggested. Studies on a number of mutant aerobactin receptor proteins revealed that structural information necessary for stable association with the outer membrane was present throughout most of the length of the polypeptide. A region involved in binding and/or translocation of aerobactin was identified and another region was shown to be involved in cloacin DF13 binding and/or translocation. A mutation which altered the signal sequence such that it more closely resembled the consensus structure for signal peptides increased the efficiency of processing of the precursor protein. 2015-11-19 08:52:53 IR content