Genetical and physiological studies into the basis of Colicin E2 susceptibilty in Escherichia coli K12.

In recent years a significant amount of biological research has been focussed on the cytoplasmic membrane of the cell. This has emerged as a complex structure, participating in cell biosynthetic activities in addition to osmoregulation. However the functional organisation of the membrane is little understood, and in this may lie the key to several biological problems. An approach to the study of the coordination of biosynthetic processes by the cell membrane may be through the use of agents interacting specifically with the membrane. One such class of compounds may well be the natural protein antibiotics, colicins. Colicins may be distinguished from conventional antibiotics by their unique mode of action with sensitive cells, whereby lethality is promoted from specific fixation sites in the bacterial cell wall. The colicin molecule neither penetrates the cell surface layer, nor causes gross cell membrane damage, and it has been postulated that interaction of the colicin protein with proteins of the cell membrane lead eventually to cell death. The studies described in this Thesis have been concerned in particular with the interaction of colicin E2, one of the E-group of colicins, with the bacterial cell, and the mode of action of this colicin. Colicin E2 is characterised by the induction of a rapid degradation of cellular DNA, and the inhibition of cell division in sensitive bacteria. It was hoped that the genetical and physiological characterisation of bacterial mutants specifically refractive to colicin E2 would indicate the nature of the altered component, normally participating in the transmission of the effect of the extracellular colicin to the intracellular DNA, and/or the cell division machinery. Mutants refractive to colicin E2, designated Ref-II, were therefore isolated from a number of strains of E. coli K12. These mutants were systematically examined, and it was found that although the majority were relatively insensitive to UV irradiation, approximately 33% did display significantly increased sensitivity. Further-more, these Ref-II UVs strains were sensitive to the detergent, Sodium Deoxycholate, and grew slowly in complex medium, properties indicative in the first instance of an altered membrane component in this class of mutant, and in the second, of a normal role of the component in some aspect of cellular metabolism. These Ref-II UVs mutants could also be distinguished from Ref-II UVr mutants by possession of various other pleiotropic characters. For example, the majority of Ref-II UVs mutants were defective in recombination, some were "resistant" to bacteriophage lambda, one grew in long filaments, and one produced excessive polysaccharide particularly when cultured on minimal agar, properties which may all be related to defective DNA metabolism in the mutants. Genetic analysis revealed that pleiotropic characteristics shown by the Ref-II UVs mutants were 100% co-transducible with the refII locus. Moreover, if two or more cistrons were present, mutations in these producing either Ref-II or Ref-II UVs mutants respectively, they must be adjacent on the chromosome. The refII locus was mapped and found to be co-transducible with serB, and to lie within four genes of the hsp locus on the bacterial chromosome. Reversion studies confirmed that the complex pleiotropy displayed by Ref-II UVs mutants did in fact arise from mutation in a single gene, and not from a series of closely linked, multisite mutations arising from the action of the mutagen employed. Moreover, the site of the reversion mutation proved to be at the refII locus, and not at a second locus, mapped and found to be closely linked to thr, potentiating the expression of colicin E2-refractivity. Different hypotheses may account for the occurrence of each individual pleiotropic character of the Ref-II UVs mutants. However, collectively, it becomes apparent that the altered component in this class of mutants normally participates in DNA metabolism in the cell, and either (a) the enzymes participating in the repair and recombination of bacterial DNA, and possibly even the DNA molecule itself, may be bound to the altered component; or (b) the altered component may be one of an aggregate of proteins concerned specifically with chromosomal metabolism. Direct evidence that the altered component, at least in Ref-II UVs mutants, is in fact structural, and presumably membrane, is received from the delayed expression of UV resistance after the receipt of the UVr gene in Ref-II UVr/UVs heterogenotes. Future experiments involving the isolation and comparison of membrane fractions from mutant and non-mutant strains will now be critical in the confirmation of this hypothesis