UBC Theses and Dissertations
Partial characterization of the molecular basis for carotovoricin-379 sensitivity in Erwinia carotovora Smith, Donna Susan
In the evaluation of several methods of quantifying the activity of carotovoricin-379, the tetrazolium assay and the spot-plate assay were rejected because of insensitivity and irreproducibility, respectively. An adaption of the critical dilution method, called the microtitre plate assay, overcame the weaknesses of the other methods. Found to be acceptably rapid, sensitive, and reproducible, this new method facilitated further investigations of carotovoricin-379 sensitivity in Erwlnia carotovoza subspecies atroseptica (Eca). Representative strains of the serogroups of Eca displayed different levels of sensitivity to carotovoricin-379. Because all sensitive test strains adsorbed carotovoricin-379, these differences were attributed to tolerance rather than resistance. This implied that although less sensitive strains were capable of adsorbing carotovoricin-379, the events leading to the death of the cell were not triggered. A linear relationship between the carotovoricin-379 dosage and the log of the proportion of surviving cells in test cultures indicated that carotovoricin-379 followed single-hit killing kinetics. This result showed that only one adsorbed particle which is able to reach its target of action is required to kill a cell. In less sensitive strains, therefore, a high proportion of adsorbed particles are unable to reach the site of action. Increased relative fluorescence of 8-anilino-1-naphthalene sulfonic acid (ANS) was observed upon treatment of sensitive cells with carotovoricin-379. These increases were not observed when non-sensitive cells were treated in an identical manner. Because ANS fluorescence is a qualitative indicator of membrane polarization, these results indicated that carotovoricin-379 acts by effecting a collapse of the proton motive force. The cytoplasmic membrane, therefore, was tentatively identified as the target of action of carotovoricin-379. Sodium dodecylsulfate polyacryamide gel electrophesis of membrane proteins extracted from sensitive cells revealed that these proteins were distributed into either the Triton-soluble or insoluble fractions. Carotovoricin-379 neutralization activity, however, was distributed evenly between them, correlating with the distribution of lipopolysaccharide (LPS). Membrane protein failed to neutralize carotovoricin-379 activity. LPS from sensitive cells, however, exhibited neutralization activity. Neutralization activity was sensitive to periodate, suggesting that the receptor site is carbohydrate. Direct binding of carotovoricin-379 to the LPS was demonstrated with an indirect enzyme-linked immunosorbent assay using polyclonal antiserum raised against carotovoricin-379. Strong positive reactions were obtained with LPS from sensitive strains; however the reaction obtained with non-sensitive strains were either extemely weak or negative. From these results it was concluded that the carotovoricin-379 receptor site in Erwinia carotovora is located on the LPS.