I. TYPING METHODS FOR CLOSTRIDIUM
9
active against the producing strain. Some are sensitive to proteolytic enzymes, high temperatures and extremes of pH, while others are resistant.
One type appears to be dialysable, the other is not. Attempts to purify the
bacteriocins of C. perfringens define them as proteins or protein-saccharide
complexes. T h e bacteriocins seem to represent a heterogeneous group of
molecules and more experimental work is required to adequately describe
their chemistry and mode(s) of action.
B. Bacteriocins of C. botulinum
Bacteriocins of C. botulinum were first reported in 1966 by two different
groups of investigators. Kautter et al. (1966) described bacteriocins produced by non-toxigenic strains of C. botulinum (resembling toxigenic type
E organisms) which were bacteriolytic for vegetative cells and prevented
germination of spores of C. botulinum type E. Of the species examined only
C. botulinum type E and, to a lesser extent, C. perfringens and C. acetobutylicum were sensitive to this type of bacteriocin which was named
boticin E. Boticin E was heat stable, dialysable, unaffected by chloroform
and inactivated by trypsin. Ethyl alcohol and acetone precipitates of the
bacteriocin were fully active but trichloroacetic acid precipitates were only
partially so.
One boticin E ( S 5 ) was composed of two components (Ellison and
Kautter, 1970) : a lower molecular weight boticin ranging between 5000
and 30,000 daltons and accounting for about 80% of the inhibitory activity
and a high molecular weight component in excess of 40 x 106 daltons.
Both components were resistant to boiling and sensitive to trypsin. The lower
molecular weight fraction was most susceptible to trypsin. The lower
molecular weight boticin was stable over a p H range of 1.1-9-5. Both boticins prevented the outgrowth of spores and had bactericidal activity for
vegetative cells of the indicator strain of C. botulinum type E, 070. The
bacteriocin stopped cell growth and caused an extreme drop in viability
within 9 min. The action of the small bacteriocin could not be reversed
by subsequent addition of trypsin suggesting rapid uptake of the bacteriocin. Methods of purification and handling of the boticins described in
this Chapter should be of value in further studies of these molecules.
Electron microscopy of the indicator strain after treatment with the
lower molecular weight boticin (now named S5) (Ellison et al., 1971)
revealed aggregation of DNA, apparent structural rearrangement of mesosomes and eventual dissolution of cell contents leaving bacterial ghosts
with intact cell walls and remnants of the cytoplasmic membrane and
internal structures.
Only a few strains of those clostridia which are non-toxigenic but otherwise identical to C. botulinum type E produce boticins (Anastasio et al.,
9
active against the producing strain. Some are sensitive to proteolytic enzymes, high temperatures and extremes of pH, while others are resistant.
One type appears to be dialysable, the other is not. Attempts to purify the
bacteriocins of C. perfringens define them as proteins or protein-saccharide
complexes. T h e bacteriocins seem to represent a heterogeneous group of
molecules and more experimental work is required to adequately describe
their chemistry and mode(s) of action.
B. Bacteriocins of C. botulinum
Bacteriocins of C. botulinum were first reported in 1966 by two different
groups of investigators. Kautter et al. (1966) described bacteriocins produced by non-toxigenic strains of C. botulinum (resembling toxigenic type
E organisms) which were bacteriolytic for vegetative cells and prevented
germination of spores of C. botulinum type E. Of the species examined only
C. botulinum type E and, to a lesser extent, C. perfringens and C. acetobutylicum were sensitive to this type of bacteriocin which was named
boticin E. Boticin E was heat stable, dialysable, unaffected by chloroform
and inactivated by trypsin. Ethyl alcohol and acetone precipitates of the
bacteriocin were fully active but trichloroacetic acid precipitates were only
partially so.
One boticin E ( S 5 ) was composed of two components (Ellison and
Kautter, 1970) : a lower molecular weight boticin ranging between 5000
and 30,000 daltons and accounting for about 80% of the inhibitory activity
and a high molecular weight component in excess of 40 x 106 daltons.
Both components were resistant to boiling and sensitive to trypsin. The lower
molecular weight fraction was most susceptible to trypsin. The lower
molecular weight boticin was stable over a p H range of 1.1-9-5. Both boticins prevented the outgrowth of spores and had bactericidal activity for
vegetative cells of the indicator strain of C. botulinum type E, 070. The
bacteriocin stopped cell growth and caused an extreme drop in viability
within 9 min. The action of the small bacteriocin could not be reversed
by subsequent addition of trypsin suggesting rapid uptake of the bacteriocin. Methods of purification and handling of the boticins described in
this Chapter should be of value in further studies of these molecules.
Electron microscopy of the indicator strain after treatment with the
lower molecular weight boticin (now named S5) (Ellison et al., 1971)
revealed aggregation of DNA, apparent structural rearrangement of mesosomes and eventual dissolution of cell contents leaving bacterial ghosts
with intact cell walls and remnants of the cytoplasmic membrane and
internal structures.
Only a few strains of those clostridia which are non-toxigenic but otherwise identical to C. botulinum type E produce boticins (Anastasio et al.,