IV. INVESTIGATION OF C. DIPHTHERIAE
79
infected with certain tox+-carrying bacteriophages, they produced a typical
diphtherial toxin.
(c) Tox: structural gene, for toxin biosynthesis. Genetic crosses between
toxigenic and related non-toxigenic diphtherial phages have revealed that
the capacity to convert to toxigeny, the tox character, maps in a single
region of the phage genome. Until 1971 it was not established, however,
whether this region contains the structural information for toxin synthesis,
or whether tox acts indirectly to permit the expression of a host structural
gene.
Having this in mind, Holmes and Barksdale (1969) claimed the need for
phages which would code for altered toxins. At an earlier date Matsuda and
Barksdale (quoted by Barksdale and Arden, 1974) had screened a large
number of tox- wild diphtheria bacilli, for altered toxin, but found none
(unpublished data).
Uchida et al. (1971) and Matsuda, et al. (1972) succeeded in obtaining
nitrosoguanidine mutants of phage , i 3 which induced production of altered
toxin proteins (cross-reacting materials), thus furnishing the first certain
evidence that tox is the structural gene for diphtherial toxin. The protein
thus obtained, while not toxic, had many of the characteristics of toxin. It
was immunologically identical and cross-reacted with pure and specific
diphtherial toxin.
As regards the cellular site of toxin production the data of Uchida and
Yoneda (1967) suggested that it is the cytoplasmic membrane.
B. Physiology of diphtherial toxin
1. Molecular structure
Diphtherial toxin, the product of gene tox, is released extracellularly
as a single polypeptide chain of molecular weight of 62 000 daltons (Gill
and Dinius, 1971 ; Collier and Kandel, 1971). This is lethal for man, guinea
pigs, rabbits and birds in doses of 130 ng per kilogram body weight (Barksdale, 1970).
In rabbits only a few picograms will give a visible skin reaction. Mice
and rats are singularly resistant to toxin. The only known biological
activities of the intact toxin molecule are its toxicity and its ability to fix to
mammalian cells.
Intact, newly synthesised diphtherial toxin is enzymically inactive in
oitro. When subjected to a brief treatment with trypsin in the presence of a
thiol the molecule is “nicked” by hydrolysis of a single peptide bond
located in the loop, formed by the N-terminal end of its two disulphide
bridges. The resulting enzymically active “nicked” and reduced toxin
consists of two fragments-A, amino-terminal and B, carboxyl-terminal of
79
infected with certain tox+-carrying bacteriophages, they produced a typical
diphtherial toxin.
(c) Tox: structural gene, for toxin biosynthesis. Genetic crosses between
toxigenic and related non-toxigenic diphtherial phages have revealed that
the capacity to convert to toxigeny, the tox character, maps in a single
region of the phage genome. Until 1971 it was not established, however,
whether this region contains the structural information for toxin synthesis,
or whether tox acts indirectly to permit the expression of a host structural
gene.
Having this in mind, Holmes and Barksdale (1969) claimed the need for
phages which would code for altered toxins. At an earlier date Matsuda and
Barksdale (quoted by Barksdale and Arden, 1974) had screened a large
number of tox- wild diphtheria bacilli, for altered toxin, but found none
(unpublished data).
Uchida et al. (1971) and Matsuda, et al. (1972) succeeded in obtaining
nitrosoguanidine mutants of phage , i 3 which induced production of altered
toxin proteins (cross-reacting materials), thus furnishing the first certain
evidence that tox is the structural gene for diphtherial toxin. The protein
thus obtained, while not toxic, had many of the characteristics of toxin. It
was immunologically identical and cross-reacted with pure and specific
diphtherial toxin.
As regards the cellular site of toxin production the data of Uchida and
Yoneda (1967) suggested that it is the cytoplasmic membrane.
B. Physiology of diphtherial toxin
1. Molecular structure
Diphtherial toxin, the product of gene tox, is released extracellularly
as a single polypeptide chain of molecular weight of 62 000 daltons (Gill
and Dinius, 1971 ; Collier and Kandel, 1971). This is lethal for man, guinea
pigs, rabbits and birds in doses of 130 ng per kilogram body weight (Barksdale, 1970).
In rabbits only a few picograms will give a visible skin reaction. Mice
and rats are singularly resistant to toxin. The only known biological
activities of the intact toxin molecule are its toxicity and its ability to fix to
mammalian cells.
Intact, newly synthesised diphtherial toxin is enzymically inactive in
oitro. When subjected to a brief treatment with trypsin in the presence of a
thiol the molecule is “nicked” by hydrolysis of a single peptide bond
located in the loop, formed by the N-terminal end of its two disulphide
bridges. The resulting enzymically active “nicked” and reduced toxin
consists of two fragments-A, amino-terminal and B, carboxyl-terminal of