Bacteriocin production in Erwinia carotovora, subspecies carotovora, strain 379
Creators: name: Ward, Leonard John
Publisher: name: University of British Columbia
Year: 2010

description: Erwinia carotovora subsp. carotovora strain 379 (Ecc 379) produced a particulate bacteriocin called carotovoricin-379 which resembled a bacteriophage tail. Carotovoricin-379, producing both clear and diffuse zones of growth inhibition, was active against several other Erwinia carotovora strains. It was detected in the filter-sterilized supernatant of Ecc 379 under standard cultural conditions. Its concentrations in the supernatant fraction of cultured Ecc 379 were increased by induction with mitomycin C (0.2 ug/ml). Induction was followed by cell lysis, which was reflected by a sharp reduction in culture turbidity. Growth of Ecc 379 at 37 C with or without mitomycin C resulted in the loss of particulate carotovoricin-379 production. Under these conditions, a low molecular weight, highly diffusible bacteriocin component was detected which produced large diffuse zones of inhibition with three of the four standard Erwinia carotovora indicator strains used. Cell lysis following induction and a wild-type resistance to erythromycin and chloramphenicol were also temperature sensitive. Carotovoricin-379 production was investigated by isolating intermediates at several times after induction. These intermediates were analysed by electron microscopy (EM) and Sephacryl S-300 column chromatography. EM showed a subunit-like arrangement of bacteriocin components around a central core. Carotovoricin-379 particles were initiated as fimbrae-like projections which matured by the gradual external addition of bacteriocin components. This maturation process was accompanied by a general increase in molecular weight, bioactivity and ultrastructural appearance of particulate carotovoricin-379. By using a modified negative staining protocol, cell projections which resembled carotovoricin-379 intermediates were seen physically attached to intact producing cells. On the basis of these results a hypothetical model for carotovoricin-379 production was outlined. Genetic analysis of a temperature sensitive, particulate bacteriocin production in Erwinia carotovora subsp. carotovora strain 379 (Ecc 379) was performed with and without mobilization vector R68.45. E. coli transconjugants which both produced particulate carotovoricin-379 and degraded crystal violet pectate medium (CVP) were produced only when R68.45 was used. In addition, the transfer frequencies obtained were indicative of chromosomally derived determinants. Erythromycin-and chloramphenicol-resistant transconjugants were obtained regardless of R68.45 mediation. DNA analysis by agarose gel electrophoresis showed that Ecc contained a resident megaplasmid which was self-transmissible. This plasmid coded for erythromycin and chloramphenicol resistance along with the production of the low molecular weight carotovoricin-379 component similar to that produced by heat treated Ecc 379. Analysis of transconjugants by electron microscopy showed a protuberance of surface "blebs" or vesicles. In addition, alkaline phosphatase (a periplasmic enzyme) was detected in supernatants of transconjugants. These facts implied that the megaplasmid may also be involved in the release of many exo-proteins of Erwinia. Polyclonal rabbit antiserum was developed against particulate carotovoricin-379. This antiserum was tested against culture supernatants from wild-type Erwinia and several Erwinia x E. coli transconjugants using radial immunodiffusion (RID), immunosensitive electron microscopy (ISEM) and Western blotting. The polyclonal antiserum against particulate carotovoricin-379 reacted with supernatant fractions from all transconjugants. CVP+ transconjugants consistently showed a wider range of reactive bacteriocin components than CVP transconjugants. A low molecular weight partially active carotovoricin-379 component was detected in CVP- transconjugants by antiserum raised against particulate carotovoricin. This suggested a relatedness between the two major bacteriocin components of carotovoricin-379.