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Research has been conducted on the macromolecules which parasite Escherichia coli envelope proteins and infect bacteria. Results suggest that these macromolecules affect E. coli via different interactions between the proteins and between colicin translocation domain and a protein.

A study concerning Tol-peptidoglycan-associated lipoprotein (PAL) system of Escherichia coli is presented. Results indicate that PAL and periplasmic protein TolB interact with OmpA and the major lipoprotein.

Research was conducted on the TolR protein of Escherichia coli and the interactions of TolR with TolA and TolQ were confirmed. TolR was found to dimerize and the resulting dimer was shown to interact with TolQ. The TolR transmembrane domain was observed to be implicated in the TolA-TolR and TolQ-TolR interactions, while TolR central and C-terminal domains seemed to be involved in TolR dimerization. The role of the TolR C-terminal in the TolA-TolR interaction and its association with the membranes were also demonstrated.

Colicins are toxins encoded by plasmids that are formed by and are active against Escherichia coli and similar bacteria. Their structure is composed of a domain for the recognition of a specific receptor, a domain for translocation and a domain for the lethal activity. Colicins can cross the formidable physical barrier of the outer membrane either through the passive diffusion pathway or the energy-dependent pathway. In addition to porins, a group of proteins called Tol is needed for colicin import.

Research was conducted to examine the relationship between the trimeric porins of Escherichia coli and TolB, a periplasmic protein of the cell envelope Tol complex. The TolB protein was determined on Ni-nitrilotri-acetic acid-agarose beads while membrane extracts were enriched with OmpA and lipopolysaccharide-associated OmpC, OmpF, and PhoE. Results indicated that TolBHis could interact with the trimeric forms of proteins.

A research on the binding of colicins A and E1 and their interaction with ToIA soluble derivatives was conducted. Research methodologies included plasmid constructs, purification of ToIA derivatives and N-terminal domains of colicins A and E1, Western blotting and peptide analysis. Results showed that the N-terminal domain of colicin E1 and A can interact with the C terminal of TolA. Furthermore, the binding of colicins A and E1 to ToIA were estimated by surface plasmon resonance and were shown to be similar.

Tagging of the TolB proteins of the tolB gene by insertion into a C-terminal region reveals that the 43 kDa protein TolB* is the functional protein and that the 47.5 kDa protein form TolB is the precursor of the functional protein. Both the protein and precursor are present on the periplasm and are distinct in their N-terminal regions, with the N-terminal sequence inducing a bacterial signal for TolB protein. Use of an antibody against the TolB protein supports the structure and function of the tagged TolB and indicates that only on TolB* overexpression the precursor form develops.

Lymphokine IL-2, a specific receptor that is produced by induced T. lymphocytes, aids immune response, and generates recombinant IL-2 (met-IL-2) in Escherichia coli. An E. coli construct that can generate and export IL-2 is identified to aid the purification of recombinant proteins. The IL-2 gene is targeted at DNA fragments encoding signal peptides. Positively charged amino acids are replaced with glutamic acid to analyze the effect of charge disposition at the N-terminus of the mature portion of the protein.

The effects of the pore-forming domain of colicin A on acyl chain order and lipid organization of model membrane system derived from Escherichia coli were investigated using nuclear magnetic resonance (NMR) methods. A fatty acid auxotrophic mutant of E. coli was grown in the presence of deuterium-labeled oleic acid and was used for the NMR study. The colicin A pore-forming domain was inserted in the membrane and catalyzed at acidic pH. A major perturbing effect on acyl chain order was observed upon insertion of the pore-forming colicins but bilayer organization remained unaffected and exhibited stability.

Hybrid colicins were constructed to identify the specific determinants in pore-forming colicin A and colicin B that are responsible for recognition by the integral membrane immunity proteins. These ColA/ColB and ColB/ColA chimeric proteins were generated by homologous DNA recombination. The results show that the hydrophobic alpha-helical hairpin of colicin pore-forming domains is the major determinant for immunity specificity.