S156

Biology and Molecular Epidemiology of Diphtheria Toxin and the tox Gene
Randall K. Holmes Department of Microbiology, University of Colorado

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Health Sciences Center, Denver, Colorado

Diphtheria toxin (DT) is an extracellular protein of Corynebacterium diphtheriae that inhibits

protein synthesis and kills susceptible cells. The gene that encodes DT (tox) is present in some
corynephages, and DT is only produced by C. diphtheriae isolates that harbor tox+ phages.
The diphtheria toxin repressor (DtxR) is a global regulatory protein that uses Fe2+ as co-
repressor. Holo-DtxR represses production of DT, corynebacterial siderophore, heme oxy-
genase, and several other proteins. Diagnostic tests for toxinogenicity of C. diphtheriae are
based either on immunoassays or on bioassays for DT. Molecular analysis of tox and dtxR
genes in recent clinical isolates of C. diphtheriae revealed several tox alleles that encode identical
DT proteins and multiple dtxR alleles that encode five variants of DtxR protein. Therefore,
recent clinical isolates of C. diphtheriae produce a single antigenic type of DT, and diphtheria
toxoid continues to be an effective vaccine for immunization against diphtheria.

Early History of Diphtheria Toxin
Several excellent reviews cover the early history of diphtheria
toxin [1–5]; selected highlights will be presented here without
citation of original sources.
During the 1820s, respiratory diphtheria was identified as a
distinct disease and was distinguished clinically from other
forms of sore throat. Corynebacterium diphtheriae was visual-
ized in 1883 by Klebs in stained specimens from diphtheritic
pseudomembranes, and in 1884, the organism was isolated by
Loeffler and shown to be the cause of diphtheria. In 1888, Roux
and Yersin demonstrated that filtrates from cultures of C.
diphtheriae contained a potent heat-labile toxic protein called
diphtheria toxin (DT). Very small doses of DT injected intra-
dermally into susceptible animals, such as guinea pigs or rab-
bits, cause erythema, induration, and dermonecrosis. Larger
doses of DT injected parenterally into susceptible animals cause
typical systemic lesions of diphtheria, including myocarditis,
focal necrosis in organs, such as the adrenal glands, kidneys,
and liver, and polyneuritis. In contrast, some other animals,
such as mice and rats, are highly resistant to DT. The lethal
dose of DT for humans and susceptible animals is estimated
to be ∼0.1 mg/kg of body weight.
In 1890, von Behring and Kitasato demonstrated that sus-
ceptible animals could be immunized by injection with graded
doses of diphtheria bacilli, and the serum from such immunized
Research involving experimental animals in the author’s laboratory was
conducted in accordance with guidelines of the US Department of Health
and Human Services.
Grant support: NIH (AI-14107).
Reprints or correspondence: Dr. Randall K. Holmes, Dept. of Micro-
biology, Campus Box B-175, University of Colorado Health Sciences
Center, 4200 East Ninth Avenue, Denver, CO 80262 (Randall.Holmes
@UCHSC.edu).
The Journal of Infectious Diseases 2000; 181(Suppl 1)1:S156–67
q 2000 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2000/18102S-0026$02.00
animals protected other susceptible animals from the toxic ef-
fects of DT. Only 1 year later in 1891, antitoxin prepared in
an experimental animal was used successfully to treat diphtheria
in a child. The interval from the discovery of C. diphtheriae
and DT to von Behring’s introduction of antitoxin therapy for
treatment of diphtheria was very short. In 1901, von Behring
received the first Nobel Prize in Physiology or Medicine for his
remarkable contributions to the development of serum therapy
for diphtheria and other toxin-mediated infectious diseases.
In 1909, Theobald Smith proposed that nontoxic mixtures
of DT and diphtheria antitoxin at equivalence could be used
for active immunization of humans against diphtheria. A suc-
cessful large-scale trial of toxin-antitoxin vaccine was conducted
in 1922 in New York City by W. H. Park. In 1923, Ramon
discovered that treatment with formalin eliminated the toxicity
of DT without destroying its immunogenicity. Formalin-treated
DT, now called diphtheria toxoid, rapidly became the preferred
vaccine against diphtheria, and diphtheria toxoid (usually
in combined vaccines, such as diphtheria-tetanus toxoids–
pertussis vaccine [DTP] or diphtheria-tetanus toxoids–acel-
lular pertussis vaccine [DTaP] for children and tetanus-diph-
theria toxoids vaccine for adults [Td]) is still used throughout
the world for active immunization against diphtheria. The PW8
strain of C. diphtheriae, which was isolated by Park and Wil-
liams in 1896 and produces exceptionally large amounts of DT,
is also used throughout the world to make DT for production
of diphtheria toxoid for vaccine.
Immunity against diphtheria can be acquired either by active
immunization with diphtheria toxoid or by recovery from clin-
ical or subclinical infection with a toxinogenic strain of C.
diphtheriae. In 1913, Schick introduced a simple test, which was
based on the response to intradermal injection of a small dose
of DT, to distinguish between individuals who are susceptible
to diphtheria and those who are immune. Susceptible individ-
uals (Schick positive) develop prolonged erythema and indu-
ration at the site of toxin injection, but immune individuals
JID 2000;181 (Suppl 1) Diphtheria Toxin and the tox Gene
(Schick negative) do not. The Schick test was used both to
determine the prevalence of immunity to diphtheria among per-
sons of different ages and to assess the efficacy of different
vaccine regimens in eliciting protective antitoxic immunity. Dur-
ing the last few decades, direct measurement of anti-DT anti-
bodies in serum has replaced the Schick test as the basis
for assessing immunity against diphtheria in individuals or
populations.
Genetics of Toxinogenesis in C. diphtheriae
Understanding the genetics of toxinogenesis in C. diphtheriae
began in 1951 when Freeman reported that a nontoxinogenic
strain became toxinogenic after infection with a specific cory-
nephage [6]. Most early studies during the 1950s on phage-host
interactions and genetics of toxinogenesis used the nonlysogenic
C. diphtheriae strains C4 or C7, the tox1 corynephage b, the
tox2 corynephage g, and mutant bacteria or phages derived
from them [7–9] (and reviewed in [10, 11]). These studies dem-
onstrated that individual corynephage strains are either tox1
or tox2, there is a one-to-one correlation between lysogeniza-
tion of a nontoxinogenic strain of C. diphtheriae by a tox1
phage and acquisition of the ability to make DT, and elimi-
nation (curing) of the tox1 prophage results in loss of the ability
to produce DT. These findings established that toxinogenicity
in C. diphtheriae is determined by phage conversion (also called
lysogenic conversion, i.e., the ability of the bacterium to make
DT changes as a consequence of infection by a particular
phage).
When a genetic system became available in the late 1960s
for analysis of recombination between the closely related but
heteroimmune corynephages b and g, the tox1 marker of b and
the tox2 marker of g were found to segregate as alleles at a
discrete genetic locus [12]. Nontoxinogenic mutants of phage
b were isolated in the early 1970s, and some mutant tox alleles
were shown to determine the production of nontoxic proteins,
cross-reacting materials (CRMs), that had the same molecular
mass as DT and reacted with anti-DT antibodies [13]. This
finding demonstrated conclusively that the tox determinant of
phage b is the structural gene for DT. C. diphtheriae C7 can
produce DT not only when b is present as prophage but also
when it is present either as vegetatively replicating phage or as
a nonreplicating exogenote in a bacterium with homologous
lysogenic immunity [14–16]. These findings showed that ex-
pression of the structural gene for DT in phage b is not reg-
ulated coordinately with other phage genes that are required
only for vegetative growth or for lysogeny.
In the mid-1970s, fine-structure mapping of mutant tox al-
leles of phage b that encode amino-terminal fragments of DT
of various lengths demonstrated that the tox gene is colinear
with DT and established the orientation of transcription of the
tox gene with respect to the genetic map of phage b [17, 18].
The prophage genetic map of b was shown to be circularly
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permuted with respect to the vegetative genetic map of phage
b; and tox, which occupies a central position in the vegetative
map, was shown to be located at one end of the prophage map
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immediately adjacent to the phage attachment site (attP) [17,
19]. There are two different but functionally equivalent bacterial
attachment sites (attB) for integration of b prophage into the
chromosome of C. diphtheriae [20], and the attB sites are located
within an Arg-tRNA2 gene that is present at two different chro-
mosomal locations [21]. The fact that the tox gene is located
at the end of the prophage genetic map suggests that it might
have been incorporated into a nontoxinogenic ancestor of
phage b by an illegitimate recombination event similar to those
involved in formation of specialized transducing phages in
other phage-host systems.
During the 1980s, the restriction maps for the genomes of
phages b and g were determined [22–24], and tox alleles were
cloned and sequenced from several tox1 corynephages and mu-
tants of phage b that encode nontoxic or hypotoxic variants
of DT [25–28]. The tox1 alleles from these corynephages en-
coded DT with the same deduced amino acid sequence. These
studies set the stage for subsequent analysis of DT structure
and function by random- and site-directed mutagenesis of
cloned tox alleles, by selection in bacterial or eukaryotic systems
for tox mutants with specific phenotypes of interest, and by
purification and characterization of various recombinant forms
of DT after producing them in bacteria or cell-free expression
systems.
Most toxinogenic strains of C. diphtheriae contain DNA se-
quences related to phage b [29, 30]. Furthermore, among a
collection of six tox1 corynephages with different phenotypes,
including b, all were found by DNA hybridization to be related
to b except for the toxinogenic phage d that appears to represent
a different phage lineage [31, 32]. Current evidence supports
the hypothesis that all toxinogenic isolates of C. diphtheriae
contain converting phages, although some of the converting
phages may be defective or unable to form plaques on available
C. diphtheriae indicator strains.
Most nontoxinogenic isolates of C. diphtheriae and most tox2
corynephages do not contain any detectable tox-related DNA
sequences. A few studies have investigated the uncommon tox2
isolates of C. diphtheriae or corynephages from them that have
tox-related DNA sequences but do not encode functional DT
[29, 33, 34]. In the case of the tox2 corynephage g, an ∼1.5-kb
insertion is present that interrupts the coding region for the DT
signal sequence and prevents tox translation [33]. The nucle-
otide sequence of the ends of this element indicate that it is
probably an insertion sequence, but transposition of the pre-
sumed insertion sequence has not been demonstrated directly.
Use of the insertion sequence as a DNA probe for Southern
hybridization experiments demonstrates that it is present in
variable copy numbers among different isolates of C. diph-
theriae [35]. Phage g differs from phage b in immunity speci-
ficity, but it is co-immune with the tox1 corynephage p [31].
S158 Holmes
Among 14 nontoxinogenic isolates of C. diphtheriae with tox-
related DNA sequences that were isolated in the United States
in the late 1970s, 12 from South Dakota produce enzymatically
active but nontoxic amino-terminal fragments of DT and ap-
pear to be repetitive isolates of a single strain with a chain-
terminating point mutation in its tox allele. Phage Adp from
1 of these 12 isolates is co-immune with g and p, and restriction
fragments of its DNA are identical in size to those from cor-
ynephage p. On the basis of these findings, phage Adp appears
to be a tox mutant of phage p. The 2 other nontoxinogenic C.
diphtheriae isolates in this set were isolated in Alaska and Flor-
ida [34]. The tox2 phage 787 isolated from the Alaskan isolate
787 contains tox-related DNA sequences and is also co-immune
with g and p. Neither C. diphtheriae 787 nor a C. diphtheriae
C7(787) lysogen prepared in the laboratory produced any frag-
ment of DT detectable by very sensitive Western blots with
diphtheria antitoxin, and the restriction fragments of DNA
from phage 787 were not identical in size with those from either
g or p. No infective corynephage was recovered from the non-
toxinogenic C. diphtheriae isolate 788 from Florida, although
a defective phage genome may be present in that isolate. The
nontoxinogenic phenotypes of phages Adp and 787 and of C.
diphtheriae 788 were cis-dominant, suggesting that mutations
either in the DT coding region or in the tox promoter deter-
mined the phenotypes, but additional data will be needed to
define the precise molecular defects in the tox loci from these
strains of C. diphtheriae and their phages. The existence of
cryptic tox genes in clinical isolates of C. diphtheriae raises the
theoretical possibility, however, that functional tox1 alleles
might arise occasionally in nature by recombination between
genetically related corynephages that contain tox-related DNA
sequences but different tox2 alleles with nonoverlapping
mutations.
Mode of Action and Structure of Diphtheria Toxin
DT is one of the best studied of all bacterial toxins (reviewed
in [4, 36, 37]). Both native and recombinant forms of DT are
available in highly purified form. The amino acid sequence of
DT is determined. The crystal structure of DT is established
by x-ray diffraction analysis. Most aspects of the mode of ac-
tion of DT, including its binding to cellular receptors, its entry
into eukaryotic cells, and the enzymatic mechanism for its in-
tracellular toxicity, are characterized at the molecular level and
interpreted in terms of the known structure. Many variants of
DT with amino acid substitutions that affect its biologic activity
are characterized. This section will briefly review selected high-
lights of these extensive studies.
Understanding the molecular basis for the intracellular ac-
tion of DT began with the observation in 1959 that inhibition
of protein synthesis is the first effect of DT on cultures of
susceptible eukaryotic cells [38]. DT also inhibits protein syn-
thesis in cell-free extracts of eukaryotic cells, and such cell-free
JID 2000;181 (Suppl 1)
protein synthesizing systems were ideal for dissecting the bio-
chemical requirements for and the target of DT action. Nico-
tinamide adenine dinucleotide (NAD) was shown to be required
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for inhibition of protein synthesis in cell extracts [39], and the
component of the protein synthesis machinery inhibited by DT
was identified as elongation factor 2 (EF-2) [40, 41]. Finally,
DT was demonstrated to act catalytically by transferring the
adenosine diphosphate ribose (ADPR) moiety from NAD to
EF-2, thereby inactivating EF-2 and inhibiting chain elongation
during protein synthesis [42].
Studies during the 1970s showed that DT is a proenzyme
that must be processed to activate its latent NAD : EF-2
ADPR-transferase activity. Mature extracellular DT produced
by C. diphtheriae is an ∼58-kDa polypeptide consisting of 535
amino acid residues. It contains four cysteine (C) residues, and
it has two internal disulfide bonds that link C186 to C201 and
C461 to C471. Treatment of DT with trypsin selectively cleaves
the peptide bond located on the carboxyl-terminal side of the
surface-exposed arginine (R) residue R190, R192, or R193 to
generate an amino-terminal fragment A (DT-A) and a car-
boxyl-terminal fragment B (DT-B) that remain covalently
linked by the disulfide bond between C186 and C201 [43, 44].
Reduction of that disulfide bond generates free DT-A, which
corresponds with the catalytic domain (C-domain) of DT, and
free DT-B, which corresponds with the translocation and re-
ceptor-binding domains (T-domain and R-domain, respec-
tively) of DT [45, 46]. Intoxication of cells is a direct conse-
quence of the ADP ribosylation and inactivation of EF-2 in
the cytoplasm catalyzed by DT-A. When a single molecule of
DT-A is introduced directly into the cytoplasm, it is sufficient
to kill a eukaryotic cell [47]. DT-A must therefore be quite stable
in the cytoplasm of eukaryotic cells.
Studies during the 1980s showed that the NAD : EF-2
ADPR-transferase reaction catalyzed by DT-A involves an
inversion of configuration from the b-anomer of NAD to
the a-anomer in ADP ribosylated EF-2 [48]. The acceptor
site for ADP-ribosylation by DT-A is a posttranslationally
modified histidine residue, 2-[3-carboxyamido-3-(trimethylam-
monio)propyl]histidine, called diphthamide, which occurs only
at one conserved position in EF-2 from eukaryotes and Archea
[49, 50]. Kinetic analysis of the NAD : EF-2 ADPR-transferase
reaction demonstrates a sequential mechanism in which DT-A
must bind first to NAD before it can interact with EF-2 [51].
The diphthamide residue in EF-2 is not essential for viability
of eukaryotic cells, and mutants of DT-susceptible cells that
cannot make diphthamide [52–54] or that have variant forms
of EF-2 that cannot be ADP-ribosylated [55] are resistant to
the toxic effects of DT.
Although cells from various animal species differ dramati-
cally in susceptibility to DT, protein synthesis in cell-free ex-
tracts from all of them is inhibited by DT-A. This finding in-
dicates that cells from resistant species do not take up and
internalize DT in the same manner as cells from susceptible
JID 2000;181 (Suppl 1) Diphtheria Toxin and the tox Gene
species. Studies during the 1970s demonstrated that highly sus-
ceptible cells have larger numbers of high-affinity DT receptors
on their plasma membranes than do cells with lower suscep-
tibility and that DT-resistant cells lack high-affinity receptors
for DT [56–58]. Studies in the 1990s resulted in cloning of the
gene encoding the DT receptor and identification of the gene
product as the heparin-binding epidermal growth factor pre-
cursor (HB-EGF precursor) [59, 60], purification of the DT
receptor protein [61], localization of DT receptor activity to the
extracellular EGF domain of the HB-EGF precursor [62, 63],
and demonstration that association of the receptor with
DRAP27/CD9 in the plasma membrane increases receptor ac-
tivity and susceptibility to DT [64, 65]. Analysis of differences
between the EGF domains of the HB-EGF precursors from
susceptible human or monkey cells and resistant mouse cells,
together with construction and analysis of chimeric and mutant
forms of the HB-EGF precursor, showed that residue E141 is
essential and that residues R115 and L127 are important for
function of the HB-EGF precursor as the DT-receptor [66, 67].
The binding of DT to the HB-EGF precursor triggers re-
ceptor-mediated endocytosis of the toxin via clathrin-coated
pits, and the DT-receptor complexes are directed to the en-
dosomal pathway [56, 68]. Most of the DT that enters cells by
this pathway is directed to lysosomes for degradation, and only
a small proportion is responsible for intoxication [68, 69].
Translocation requires exposure of the DT to acidic conditions
comparable to acidified endosomes [70, 71], which induces a
conformational change and permits the translocation domain
of DT to insert into membranes and form a channel through
which translocation of the A fragment occurs [72–75]. If cells
with DT-receptor complexes on the plasma membrane are ex-
posed to acidic extracellular pH, translocation occurs directly
across the plasma membrane with intoxication of the target cell
[70, 71]. Recent evidence indicates that translocation of DT
occurs preferentially from early endosomes by an ATP-depen-
dent process that is facilitated by cellular proteins including
bCOP, whereas DT that reaches late endosomes is degraded by
the lysosomal pathway [69, 76]. Single amino acid replacements
at various positions throughout the translocation domain of
DT have dramatic effects on the activity of membrane channels
formed by DT in Vero cells, indicating that the channel activity
is an inherent property of DT and not caused by DT activation
of endogenous cellular channels [77]. Intoxication of cells by
DT can be prevented by interfering with any of the early steps
in this endocytic pathway, including interfering with endocy-
tosis via clathrin-coated pits (by overexpression of dynamin)
or blocking acidification of endosomes (by various pharma-
cologic agents) [69, 78, 79]. In addition, mutant cells that are
deficient in the ability to acidify endosomes are resistant to
intoxication by DT [80].
Structures have now been determined by x-ray crystallo-
graphy for free DT, DT in complex with the endogenous nu-
cleotide ApUp, DT in complex with NAD, and the catalytic
S159
domain of DT [81–86]. DT has three distinct domains: the
amino-terminal C-domain responsible for ADP ribosylation of
EF-2; the centrally located T-domain responsible for insertion
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into membranes at acidic pH, formation of channels, and trans-
location of the C-domain across endosomal membranes for
delivery to the cytosol; and the carboxyl-terminal R-domain
responsible for binding of DT to the HG-EGF precursor that
functions as the DT receptor on susceptible cells. DT and
several other well-characterized ADP ribosylating toxins share
a common NAD-binding motif with highly conserved residues
that are essential for catalytic activity, and a detailed model for
the mechanism of catalysis of ADP ribosylation by DT has
been proposed [85, 87, 88]. The structural basis for binding of
NAD by DT is well defined, and a model for interaction of
the NAD-DT complex with EF-2 that is consistent both with
the sequential enzymatic mechanism and the crystal structure
involves displacement by NAD binding of the loop sequence
from residue 32 to 54 followed by interaction of the re-oriented
loop sequence with EF-2 [85]. Site-directed mutagenesis and
analysis of the resulting mutant forms of DT are now being
used extensively to test specific models for the catalytic, trans-
locating, and receptor-binding functions of DT that are based
on the crystal structures, but analysis of this exciting and
rapidly growing area of research is beyond the scope of the
present review.
Regulation of Diphtheria Toxin Production
Production of DT by toxinogenic isolates of C. diphtheriae
is affected dramatically by the composition of the culture me-
dium and the conditions of cultivation (reviewed in [89–91]).
The best-studied effect, which will be considered in detail here,
is the role of iron in toxinogenesis. Studies during the 1930s
demonstrated that DT production is greatest when C. diph-
theriae is grown under iron-limiting conditions and is severely
inhibited under high-iron growth conditions [92]. The extensive
early investigations on phenotypic differences between C.
diphtheriae grown under low- and high-iron conditions are re-
viewed elsewhere [2, 3].
Molecular studies on regulation of DT synthesis began in
the 1970s. DNA from the tox1 corynephage b was shown to
direct the synthesis of DT and other phage-encoded proteins
in an in vitro transcription/translation system from Escherichia
coli, and addition of extracts from C. diphtheriae to this system
inhibited the synthesis of DT without affecting production of
other phage-encoded proteins [93]. Subsequent studies identi-
fied mutants of C. diphtheriae C7(b) that were insensitive to
the inhibitory effect of iron on DT production and demon-
strated that this phenotype was determined by the bacterial
genome and not the genome of phage b [94]. Mutant b phages
were also isolated that enabled C. diphtheriae lysogens con-
taining them to produce DT under high-iron conditions, al-
though they exhibited pleiotropic phenotypes that affected the
S160 Holmes
maximum amounts of DT produced as well as the regulation
of DT production by iron [95–97]. These phage mutants ex-
hibited a cis-dominant phenotype [96, 97], and fine-structure
mapping revealed that the tox-201 mutation selected for de-
tailed analysis was located immediately adjacent to the struc-
tural gene for DT on the side corresponding to its transcrip-
tional origin [95]. Analysis of the residual DT biosynthetic
capacity of C. diphtheriae C7(b) following addition of rifampin
or iron to DT-producing cultures, as a measure of functional
DT-specific mRNA, provided evidence that iron regulation of
DT production occurred at the level of transcription and in-
hibited production of DT-specific mRNA [98]. Taken together,
these data supported the hypothesis that the chromosome of
C. diphtheriae encodes a repressor that can utilize iron as a co-
repressor to inhibit transcription of the tox gene and decrease
production of DT under high-iron growth conditions [99, 100].
Characterization of additional mutants of C. diphtheriae
C7(b) that produce DT during growth in medium containing
enough iron to repress DT production by wild type C7(b) led
to the discovery of a siderophore-dependent system for iron
uptake in C. diphtheriae [101, 102]. The ability of these mutants
to produce DT during growth under high-iron conditions is due
to their inability to assimilate siderophore-bound iron, which
presumably causes the intracellular environment to be iron-
deficient even when the growth medium is iron-replete. The
iron-uptake system was shown to be a ferric ion–specific, high-
affinity, siderophore-dependent, energy-requiring system that
was dependent on the proton-motive force [101]. A mutant of
C. diphtheriae that is unable to produce the siderophore (cory-
nebactin) was isolated and characterized, and corynebactin was
partially purified [101, 103]. The structure of corynebactin from
C. diphtheriae has not been determined, and it is unclear
whether the recently determined structure of a siderophore (also
called corynebactin) from Corynebacterium glutamicum [104] is
related to corynebactin from C. diphtheriae. The Park-Williams
8 (PW8) strain that produces very large amounts of DT and is
used worldwide for production of diphtheria toxoid is a natu-
rally occurring corynebactin-deficient variant of C. diphtheriae
[105]. This finding is of considerable interest because of the
possibility that the siderophore deficiency might contribute to
the highly toxinogenic phenotype of the PW8 strain. Recent
studies demonstrated that C. diphtheriae can also utilize exoge-
nous heme or hemoglobin as a source of iron by a pathway
that uses corynebacterial heme oxygenase to degrade heme and
is independent of the siderophore-dependent iron uptake path-
way [106–108].
Cloning of the structural gene for diphtheria toxin and the
immediately contiguous upstream regulatory sequences was ac-
complished during the 1980s [25–28]. Shortly thereafter, the tox
promoter was characterized, and transcription of DT-specific
mRNA was shown by S1 nuclease mapping to originate both
in C. diphtheriae and in E. coli at nucleotide positions 241 and
240 upstream of the GTG translational start for the tox struc-
JID 2000;181 (Suppl 1)
tural gene [33]. A sequence similar to the consensus for the 235
region of j70 promoters in E. coli was located starting at po-
sition 274 from the tox structural gene, and two possible 210
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sequences were located starting at positions 254 and 248. Sub-
sequent studies using site-directed mutagenesis demonstrated
that the sequence starting at position 248 was the primary 210
promoter region, although the sequence starting at position
254 could function with moderate efficiency as a 210 promoter
region if the primary sequence was inactivated [109].
Direct characterization of the diphtheria toxin repressor
(DtxR), which has been predicted to exist since the 1970s, has
progressed rapidly during the past decade. First, crude extracts
from C. diphtheriae grown under high- but not low-iron con-
ditions were shown to contain a factor that bound to the pre-
sumed tox operator and protected a specific nucleotide sequence
from digestion by DNase I [110]. Shortly thereafter, 2 groups
independently cloned the gene (dtxR) that encodes DtxR by
screening libraries of chromosomal genes from C. diphtheriae
C7 in E. coli for iron-dependent inhibition of a reporter gene
expressed under control of the tox promoter from corynephage
b [111, 112]. The predicted product of dtxR is a 226–amino
acid polypeptide, and transcription of the dtxR gene in C.
diphtheriae occurs constitutively at a low level under low- and
high-iron growth conditions [112]. Although the physiologic
function of DtxR in C. diphtheriae is similar to that of the ferric
uptake regulator Fur from E. coli and other gram-negative
bacteria [113], Fur does not regulate the tox promoter, and
DtxR does not regulate Fur-dependent promoters. DtxR and
Fur are therefore the prototypes for two different types of bac-
terial iron-dependent regulatory proteins. Sequencing of the
dtxR allele from the C7(b)hm723 mutant that makes DT under
high-iron conditions [94] demonstrated that it contains a single
nucleotide substitution resulting in substitution of histidine for
arginine at residue 47 of DtxR (designated R47H) [114, 115].
The dtxR allele from PW8 is identical to that from C7, but
dtxR from C. diphtheriae 1030 biotype belfanti is only 91.6%
identical with the allele from C7 and encodes a DtxR variant
that differs from C7 DtxR by 6 amino acid substitutions in the
carboxyl-terminal region [115].
The recombinant DtxR protein was expressed in E. coli, pu-
rified by various methods, including metal ion affinity chro-
matography, and tested in vitro for interaction with DNA frag-
ments containing the tox promoter/operator region by gel shifts,
DNase I and hydroxyl radical footprinting, and other methods
[116–119]. These studies demonstrated that binding of DtxR
to the tox promoter/operator requires divalent cations (Cd21,
Co21, Fe21, Mn21, Ni21, or Zn21), that the DtxR-DNA com-
plexes exhibit slower electrophoretic mobility than the free
DNA fragments, that a specific sequence of ∼30 bp is protected
by DtxR from DNase I digestion, that the protected sequence
contains a 27-bp sequence with 9-bp inverted repeats at each
end, and that the protein interacts symmetrically with the two
halves of the palindromic promoter/operator sequence. They
JID 2000;181 (Suppl 1) Diphtheria Toxin and the tox Gene
also showed that DtxR does not bind to a DNA fragment
containing the fur promoter/operator, the tox-201 allele that
exhibits cis-dominant resistance to repression of DT synthesis
by iron, or a mutant tox promoter/operator from which one
arm of the palindrome is deleted. Subsequent studies demon-
strated that the common feature of DtxR-regulated operators
is a palindromic 19-bp core with the consensus sequence
TTAGGTTAGCCTAACCTAA [120–122], and the nucleotide
substitutions responsible for the operator-constitutive pheno-
types of the mutant alleles tox-201 and tox-202 are located
within this core sequence [123]. Binding of Ni21 to DtxR has
an apparent dissociation constant of 9 3 1027 M to 2 3 1026
M, is cooperative, and results in quenching of the intrinsic
fluorescence of the W104 in DtxR [124, 125]. Monomeric apo-
DtxR is in weak equilibrium with dimers, but the dimers are
stabilized by interaction with the divalent cations that activate
repressor activity [125].
The structures of wild type holo-DtxR in complex with Cd21,
Co21, Fe21, Mn21, Ni21, or Zn2; wild type apo-DtxR; and the
C102D variant of holo-DtxR in complex with Ni21 have been
determined by x-ray crystallography [126–131]. The highest res-
olutions currently available are 1.85 Å for holo-DtxR in com-
plex with Co21 [130] and 2.2 Å for apo-DtxR [131]. Both apo-
DtxR and holo-DtxR dimers can crystallize in either of two
forms [131]. In crystal form 1, the asymmetric unit is the DtxR
monomer and the dimer axis corresponds with the crystallo-
graphic two-fold; in crystal form 2, the DtxR dimer is the
asymmetric unit and the dimer axis is not a crystallographic
axis.
Each DtxR monomer has three domains. Domain 1 (residues
1–73) is located at the amino terminus and contains a helix-
turn-helix motif that is the DNA-binding site. Domain 2 (resi-
dues 74–144) is the dimerization and metal-binding domain. It
has two binding sites for the divalent cations that can activate
DtxR, and high resolution structures reveal that site 1 binds a
sulfate or phosphate anion in addition to the divalent cation
[129, 130]. At site 1, the ligands for metal binding by wild type
DtxR are the side chains of H79, E83, and H98 and the anion,
and the additional ligands for anion binding are the side chains
of R80, S126, and N130. At site 2, the ligands for metal binding
by wild type DtxR are the carbonyl oxygen of C102, the side
chains of E105 and H106, and a solvent molecule. Domain 3
(residues 145–226) exhibits greater flexibility than domains 1
and 2 and is disordered in structures of DtxR at lower reso-
lutions. At high resolution, domain 3 exhibits an SH3-like fold
[129, 130], but the function of domain 3 is not known. In the
C102D variant of DtxR, the side chains of residues D102 and
M10 are reported to serve as additional metal-binding ligands
at site 2 [128].
On the basis of small differences in structure between apo-
DtxR and holo-DtxR dimers observed with structures at low
resolution, a model was proposed that binding of metal ions
activates DtxR by changing its quaternary structure and in-
S161
ducing a caliper-like rotation of the monomers relative to each
other [127, 128]. High-resolution structures of apo-DtxR and
holo-DtxR in both crystal forms 1 and 2, which permit analysis
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of the structural differences in DtxR caused by metal binding
as well as crystal packing, have recently been determined [131].
These studies demonstrate that binding of metal ions induces
a change in tertiary rather than quaternary structure of DtxR,
with reorientation of domain 1 and a 20-residue segment at the
carboxyl terminus of domain 2 of each monomer with respect
to an invariant dimer core formed by the remainder of domain
2 of both monomers. As a consequence, the distance between
certain reference points in the helix-turn-helix motifs is shorter
by up to 1.7 Å, and the angle between the axes of the DNA-
binding helices in these motifs is greater by up to 67 in holo-
DtxR than in apo-DtxR. These structural changes provide in-
sights into the probable mechanism of activation of DtxR by
Fe21 or other divalent cations. However, in most structures of
holo-DtxR, the occupancy of site 1 by metal ions is high, site
2 is unoccupied or exhibits low occupancy by metal ions, and
the sulfhydryl group of C102 is modified by oxidation or prob-
able formation of a mixed disulfide [126, 127, 129–131]. In
contrast, in crystals of the C102D variant of holo-DtxR, site
2 exhibits higher occupancy by metal ions than does site 1 [128].
Additional information is provided by a recently reported
partial structure, at 3 Å resolution, of activated DtxR-C102D
in complex with a 33-bp DNA segment containing the tox
operator [132]. Surprisingly, two dimers of DtxR that do not
interact with each other bind to opposite faces of the DNA at
positions that are 5 bp apart but symmetrically disposed with
respect to the center of the tox operator. Residues 3–120 of the
DtxR dimers exhibit conformational differences from apo-
DtxR that are similar in kind but somewhat greater in mag-
nitude than those described above for holo-DtxR. In addition,
residues 3–6 at the amino terminus have undergone a helix-to-
coil transition that may further facilitate the binding of DtxR
to DNA. The helix-turn-helix motifs in each monomer of the
activated DtxR are oriented so that the side chains of Gln43
can interact with bases in the major groove and multiple other
amino acids can interact with phosphate groups in the DNA
backbone.
The function of DtxR has also been analyzed by introducing
mutations into the cloned dtxR gene and analyzing their effects
on expression of DtxR-regulated reporter genes in E. coli. An
important role for C102 was established by demonstrating that
all possible amino acid substitutions for C102 except aspartate
abolished DtxR activity, while the C102D variant was signifi-
cantly less active than wild type DtxR [133]. Random bisulfite
mutagenesis of dtxR followed by phenotypic screening and nu-
cleotide sequencing identified 20 mutations that affected single
codons, of which 18 resulted in single amino acid substitutions
in DtxR and 2 were chain terminating [124]. Two DtxR variants
with amino acid substitutions in domain 1 had the wild type
S162 Holmes
phenotype, and all other DtxR variants exhibited decreased
repressor activity varying from slight to dramatic.
Among the DtxR variants with decreased activity, ten sub-
stitutions and one truncation were located in domain 1, 5 sub-
stitutions and one truncation occurred in domain 2, and only
1 substitution was in domain 3. Most of these substitutions
affected residues in or immediately adjacent to the DNA-bind-
ing motif, the dimer interface, or metal-binding site 2 [124, 126].
Single alanine substitutions for each of the amino acids that
serve as ligands for metal binding at site 1 (H79, E83, and H98)
and site 2 (C102, E105, and H106) demonstrated that substi-
tutions at site 1 had little or no effect on repressor activity but
that substitutions at site 2 abolished repressor activity [128,
133]. In addition, single alanine substitutions for residues E6,
D9, M10, R13, and E17 in domain 1, which interact directly
or indirectly with amino acids in domain 2 that participate in
metal binding, resulted in decreased repressor activity. The in-
vestigators concluded that site 2 is the primary metal-binding
site that functions directly in activation of DtxR and that site
1 has at best a minor role in DtxR activation [128].
A recent study reinvestigated single alanine substitutions for
metal-binding residues at sites 1 and 2 and extended the analysis
to include substitutions for the anion-binding residues R80,
S126, and N130 as well as residue E20 in domain 1, which
interacts directly with R80 in domain 2 via two hydrogen bonds
[134]. The results confirmed that substitutions for metal-binding
residues at site 1 did not decrease DtxR activity as much as
substitutions at site 2, but in contrast, replacement of R80,
S126, N130, or E20 by alanine decreased DtxR activity almost
as much as the site 2 substitutions. These findings demonstrate
that residues involved in anion binding at site 1 and the R80-
E20 interaction are essential for repressor activity and support
the conclusion that both the anion-cation binding site 1 and
the cation-binding site 2 are important for DtxR function.
DtxR functions as an iron-dependent global regulator of me-
tabolism in C. diphtheriae, and characterization of the family
of genes in the DtxR regulon and their products is currently
in progress. Production of DT and siderophore is coordinately
regulated during the transition from iron-replete to iron-defi-
cient growth conditions [135]. Production of both DT and sid-
erophore is derepressed in C. diphtheriae C7(b)hm723, which
is deficient in DtxR function, and complementation with the
wild type dtxR1 allele restores repressibility of both DT and
siderophore production under high-iron growth conditions
[112]. Cloning of chromosomal loci from C. diphtheriae C7(2)
and screening for promoters that are functional in E. coli and
repressible by DtxR under high-iron growth conditions led
to the identification of five different iron-regulated promoter/
operators designated IRP1 through IRP5 [120, 122, 136]. A 38-
kDa lipoprotein that functions as a putative ferric siderophore
receptor is encoded downstream from IRP1 [136], a 15-kDa
protein homologous to several bacterial regulatory proteins in
the AraC family is encoded downstream from IRP3 [122], and
JID 2000;181 (Suppl 1)
open-reading frames encoding a 15-kDa protein and a putative
67–amino acid polypeptide with unknown functions are en-
coded downstream from IRP4 and IRP5, respectively [122].
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DtxR also regulates the hmuO gene of C. diphtheriae that en-
codes heme oxygenase [106, 108]. In addition, at least 14 pro-
teins have been identified that are preferentially expressed by
C. diphtheriae under low-iron conditions, but whether DtxR
regulates them has not been established [137].
DtxR is now recognized as the prototype for a family of
homologous iron-dependent regulatory proteins (generically
called IdeR) found in a number of bacteria outside the genus
Corynebacterium, including Brevibacterium lactofermentum
[138], Mycobacterium smegmatis and Mycobacterium tubercu-
losis [136, 139, 140], Streptomyces lividans and Streptomyces
pilosus [141], Staphylococcus epidermidis and Staphylococcus
aureus [142], and Treponema pallidum [143]. The amino acid
sequences of these proteins are highly conserved (50%–60%
overall identity with DtxR), although domains 1 and 2 exhibit
much higher conservation, and domain 3 shows much greater
variability. Strikingly, the amino acids that have been implicated
as important for function in the anion-cation binding site 1
and in metal-binding site 2 are identical among all of these
DtxR homologs, except for a deduced H79D substitution in
the IdeR protein encoded by B. lactofermentum 138].
Molecular Epidemiology of tox and dtxR Genes
in C. diphtheriae
Cloning and sequencing of the tox gene in the early 1980s
and of the dtxR gene in the early 1990s made it possible to
study directly the evolution of these genes among strains of C.
diphtheriae isolated at different times and in different locations.
Concomitantly, the diphtheria epidemic that began in 1990 in
the Russian Federation, which is the largest outbreak of diph-
theria in the developed world since the 1960s, presented a
unique opportunity to determine whether specific alleles of tox
or dtxR might be useful as molecular markers for specific clones
of C. diphtheriae that circulate or evolve during the course of
a large epidemic. Related molecular methods were used to de-
termine why some nontoxinogenic isolates of C. diphtheriae
carry tox gene sequences but do not produce DT, as discussed
in a previous section of this review.
The polymerase chain reaction (PCR), using specific forward
and reverse primers within the tox gene corresponding to a
segment of the A domain of DT, was investigated as a rapid
method to determine whether clinical isolates of C. diphtheriae
were toxinogenic [144–146]. The accuracy of the test compared
favorably with the Elek test for detecting production of DT
and is a useful addition to the diagnostic armamentarium. Re-
cently, optimized standard conditions were developed to detect
toxinogenic C. diphtheriae directly from clinical specimens by
use of PCR with two sets of primers specific for the A and B
domains of DT [147]. The estimated sensitivity levels for use
JID 2000;181 (Suppl 1) Diphtheria Toxin and the tox Gene
of these two primer sets were 50 and 500 cfu per PCR mixture.
Although PCR-based tests could give misleading results con-
cerning toxinogenicity of strains that have tox gene sequences
but do not produce biologically active DT, such strains are rare
among clinical isolates of C. diphtheriae.
Traditional methods for differentiating bacterial strains, such
as biotyping, antibiograms, phage typing, and analysis of re-
striction fragment length polymorphisms, have gained only
limited acceptance as tools for molecular epidemiologic studies
of C. diphtheriae because they lacked sufficient power to dis-
criminate among strains or they were too cumbersome to be-
come widely used (or both). Recently, ribotyping, multilocus
enzyme electrophoresis, and pulsed-field gel electrophoresis
have been applied with considerable success for molecular epi-
demiologic comparison of C. diphtheriae isolates from the epi-
demic in Russia and from reference culture collections [148,
149]. These studies demonstrated that a distinct clonal group
of isolates (called the ET 8 complex) emerged in 1990 in Russia
and became progressively more prevalent as the epidemic
developed.
These studies were extended by using PCR to analyze hetero-
geneity of the tox and dtxR genes among 72 isolates of C.
diphtheriae from Russia and Ukraine that were collected start-
ing before and extending throughout the early part of the pres-
ent epidemic [150]. Primers were designed so that the amplicons
from four sets spanned dtxR, the amplicons from eight sets
spanned tox, and the amplicons were of appropriate size for
analysis by single-strand conformation polymorphism (SSCP)
patterns. All four regions in dtxR and three regions in tox
showed significant variations in the sizes or numbers (or both)
of amplicons, and dtxR and tox could be classified into 12 and
6 different types, respectively. Most epidemic isolates from Rus-
sia were of dtxR types 2 and 8 (all biotype gravis), most epi-
demic isolates from Ukraine were of dtxR type 5 (all biotype
gravis), and isolates of both types were of tox types 3 and 4.
Biotype mitis isolates were of dtxR type 1. Thus, PCR-SSCP
analysis allowed clear association of isolates of distinct geo-
graphic and temporal origins with particular dtxR and tox
types.
Direct sequencing of the dtxR and tox alleles was done on
the 72 isolates from Russia and Ukraine subjected previously
to SSCP analysis [151]. One silent point mutation was detected
in the region of tox encoding the A domain of DT, and three
silent mutations were detected in the region of tox encoding
the B domain of DT, but the amino acid sequences of all the
DT proteins encoded by these tox alleles were identical. Greater
variability was detected in dtxR. A total of 35 point mutations
was detected, 9 of which resulted in amino acid substitutions
in the carboxyl-terminal half of DtxR and 26 of which were
silent. The 9 mutations that resulted in amino acid substitutions
were distributed among 7 SSCP types and resulted in 5 different
amino acid sequences. These findings demonstrated that varia-
S163
tion in SSCP types correlated well with differences in nucleotide
sequences of the dtxR and tox alleles.
In conclusion, SSCP analysis is a useful screening method
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for molecular epidemiologic studies of the dtxR and tox genes,
but the functional consequences of the differences in deduced
amino acid sequences in DtxR among clinical isolates of C.
diphtheriae remain to be established.
Acknowledgments
I thank the present and former postdoctoral fellows, students, and
technicians in my laboratory who contributed so greatly to our research
on C. diphtheriae, corynephages, diphtheria toxin, and the diphtheria
toxin repressor. I also thank my collaborators, Wim G. J. Hol (Uni-
versity of Washington, Seattle) and Issar Smith (Public Health Research
Institute, New York City), and the scientists in their laboratories who
participated in our collaborative studies for their outstanding contri-
butions to studies of the diphtheria toxin repressor and its mycobac-
terial homologs. Finally, I acknowledge with gratitude the mentorship
and long-term friendship of Lane Barksdale (deceased), who introduced
me to C. diphtheriae during my graduate studies at New York Uni-
versity Medical Center (New York City).
References
1. Pappenheimer AM Jr. Diphtheria: studies on the biology of an infectious
disease. Harvey Lect 1980; 76:45–73.
2. Barksdale L. Corynebacterium diphtheriae and its relatives. Bacteriol Rev
1970; 4:378–422.
3. Pappenheimer AM Jr. The pathogenesis of diphtheria. In: Howie JW, O’Hea
AJ, eds. Mechanisms of microbial pathogenicity. Fifth Symposium of
the Society for General Microbiology. Cambridge: Cambridge University
Press, 1955:40–56.
4. Pappenheimer AM Jr. The story of a toxic protein, 1888–1992. Protein Sci
1993; 2:292–8.
5. Wood WB Jr. From miasmas to molecules. New York: Columbia University
Press, 1961.
6. Freeman VJ. Studies on the virulence of bacteriophage-infected strains of
Corynebacterium diphtheriae. J Bacteriol 1951; 61:675–88.
7. Groman NB. The relation of bacteriophage to the change of Corynebac-
terium diphtheriae from avirulence to virulence. Science 1953; 117:297–9.
8. Groman NB. Evidence for the induced nature of the change from non-
toxigenicity to toxigenicity in Corynebacterium diphtheriae as a result of
exposure to specific bacteriophage. J Bacteriol 1953; 66:184–91.
9. Barksdale WL, Pappenheimer AM Jr. Phage-host relationships in nontoxi-
genic and toxigenic diphtheria bacilli. J Bacteriol 1954; 56:220–32.
10. Barksdale L, Arden SB. Persisting bacteriophage infections, lysogeny, and
phage conversions. Annu Rev Microbiol 1974; 28:265–99.
11. Groman NB. Conversion by corynephages and its role in the natural history
of diphtheria. J Hyg (Lond) 1984; 93:405–17.
12. Holmes RK, Barksdale L. Genetic analysis of tox1 and tox2 bacteriophages
of Corynebacterium diphtheriae. J Virol 1969; 3:586–98.
13. Uchida T, Gill DM, Pappenheimer AM Jr. Mutation in the structural gene
for diphtheria toxin carried by temperate phage. Nat New Biol 1971;
233:8–11.
14. Matsuda M, Barksdale L. Phage-directed synthesis of diphtherial toxin in
non-toxinogenic Corynebacterium diphtheriae. Nature 1966; 210:911–3.
15. Matsuda M, Barksdale L. System for the investigation of the bacteriophage-
directed synthesis of diphtherial toxin. J Bacteriol 1967; 93:722–30.
S164 Holmes
16. Gill DM, Uchida T, Singer RA. Expression of diphtheria toxin genes carried
by integrated and nonintegrated phage beta. Virology 1972; 50:664–8.
17. Holmes RK. Characterization and genetic mapping of nontoxinogenic
(tox) mutants of corynebacteriophage beta. J Virol 1976; 19:195–207.
18. Laird W, Groman N. Orientation of the tox gene in the prophage of
corynebacteriophage beta. J Virol 1976; 19:228–31.
19. Laird W, Groman N. Prophage map of converting corynebacteriophage
beta. J Virol 1976; 19:208–19.
20. Rappuoli R, Ratti G. Physical map of the chromosomal region of Cory-
nebacterium diphtheriae containing corynephage attachment sites attB1
and attB2. J Bacteriol 1984; 158:325–30.
21. Ratti G, Covacci A, Rappuoli R. A tRNA(2Arg) gene of Corynebacterium
diphtheriae is the chromosomal integration site for toxinogenic bacte-
riophages [letter]. Mol Microbiol 1997; 25:1179–81.
22. Buck GA, Groman NB. Physical mapping of beta-converting and gamma-
nonconverting corynebacteriophage genomes. J Bacteriol 1981; 148:
131–42.
23. Costa JJ, Michel JL, Rappuoli R, Murphy JR. Restriction map of cory-
nebacteriophages beta c and beta vir and physical localization of the
diphtheria tox operon. J Bacteriol 1981; 148:124–30.
24. Michel JL, Rappuoli R, Murphy JR, Pappenheimer AM Jr. Restriction
endonuclease map of the nontoxigenic corynephage gamma c and its
relationship to the toxigenic corynephage beta c. J Virol 1982; 42:510–8.
25. Greenfield L, Bjorn MJ, Horn G, et al. Nucleotide sequence of the structural
gene for diphtheria toxin carried by corynebacteriophage beta. Proc Natl
Acad Sci USA 1983; 80:6853–7.
26. Kaczorek M, Delpeyroux F, Chenciner N, et al. Nucleotide sequence and
expression of the diphtheria tox228 gene in Escherichia coli. Science
1983; 221:855–8.
27. Ratti G, Rappuoli R, Giannini G. The complete nucleotide sequence of
the gene coding for diphtheria toxin in the corynephage omega (tox1)
genome. Nucleic Acids Res 1983; 11:6589–95.
28. Giannini G, Rappuoli R, Ratti G. The amino-acid sequence of two non-
toxic mutants of diphtheria toxin: CRM45 and CRM197. Nucleic Acids
Res 1984; 12:4063–9.
29. Groman N, Cianciotto N, Bjorn M, Rabin M. Detection and expression
of DNA homologous to the tox gene in nontoxinogenic isolates of Co-
rynebacterium diphtheriae. Infect Immun 1983; 42:48–56.
30. Rappuoli R, Ratti G, Perugini M, Murphy JR. Detection and physical
map of a omega tox1-related defective prophage in Corynebacterium
diphtheriae Belfanti 1030(2)tox2. J Virol 1985; 54:194–8.
31. Holmes RK, Barksdale L. Comparative studies with tox plus and tox minus
corynebacteriophages. J Virol 1970; 5:783–94.
32. Buck GA, Cross RE, Wong TP, Loera J, Groman N. DNA relationships
among some tox-bearing corynebacteriophages. Infect Immun 1985; 49:
679–84.
33. Leong D, Murphy JR. Characterization of the diphtheria tox transcript in
Corynebacterium diphtheriae and Escherichia coli. J Bacteriol 1985; 163:
1114–9.
34. Cianciotto NP, Groman NB. Characterization of bacteriophages from tox-
containing, non-toxigenic isolates of Corynebacterium diphtheriae. Mi-
crob Pathog 1997; 22:343–51.
35. Rappuoli R, Perugini M, Ratti G. DNA element of Corynebacterium diph-
theriae with properties of an insertion sequence and usefulness for epi-
demiological studies. J Bacteriol 1987; 169:308–12.
36. Pappenheimer AM Jr. Diphtheria toxin. Annu Rev Biochem 1977; 46:69–94.
37. Collier RJ. Diphtheria toxin: mode of action and structure. Bacteriol Rev
1975; 39:54–85.
38. Strauss N, Hendee ED. The effect of diphtheria toxin on the metabolism
of HeLa cells. J Exp Med 1959; 109:144–63.
39. Collier RJ, Pappenheimer AM Jr. Studies on the mode of action of diph-
theria toxin. II. Effect of toxin on amino acid incorporation in cell-free
systems. J Exp Med 1964; 120:1019–39.
JID 2000;181 (Suppl 1)
40. Collier RJ. Effect of diphtheria toxin on protein synthesis: inactivation of
one of the transfer factors. J Mol Biol 1967; 25:83–98.
41. Goor RS, Pappenheimer AM Jr. Studies on the mode of action of diphtheria
toxin. III. Site of toxin action in cell-free extracts. J Exp Med 1967; 126:
Downloaded from https://academic.oup.com/jid/article-abstract/181/Supplement_1/S156/839052 by Universitas Indonesia user on 27 December 2018
899–912.
42. Honjo T, Nishizuka Y, Hayaishi O. Adenosine diphosphoribosylation of
aminoacyl transferase II by diphtheria toxin. Cold Spring Harb Symp
Quant Biol 1969; 34:603–68.
43. Drazin R, Kandel J, Collier RJ. Structure and activity of diphtheria toxin.
II. Attack by trypsin at a specific site within the intact toxin molecule.
J Biol Chem 1971; 246:1504–10.
44. Gill DM, Pappenheimer AM Jr. Structure-activity relationships in diph-
theria toxin. J Biol Chem 1971; 246:1492–5.
45. Collier RJ, Kandel J. Structure and activity of diphtheria toxin. I. Thiol-
dependent dissociation of a fraction of toxin into enzymically active and
inactive fragments. J Biol Chem 1971; 246:1496–503.
46. Gill DM, Dinius LL. Observations on the structure of diphtheria toxin. J
Biol Chem 1971; 246:1485–91.
47. Yamaizumi M, Mekada E, Uchida T, Okada Y. One molecule of diphtheria
toxin fragment A introduced into a cell can kill the cell. Cell 1978; 15:
245–50.
48. Oppenheimer NJ, Bodley JW. Diphtheria toxin. Site and configuration of
ADP-ribosylation of diphthamide in elongation factor 2. J Biol Chem
1981; 256:8579–81.
49. Van Ness BG, Howard JB, Bodley JW. ADP-ribosylation of elongation
factor 2 by diphtheria toxin. NMR spectra and proposed structures of
ribosyl-diphthamide and its hydrolysis products. J Biol Chem 1980; 255:
10710–6.
50. Bodley JW, Dunlop PC, VanNess BG. Diphthamide in elongation factor
2: ADP-ribosylation, purification, and properties. Methods Enzymol
1984; 106:378–87.
51. Chung DW, Collier RJ. The mechanism of ADP-ribosylation of elongation
factor 2 catalyzed by fragment A from diphtheria toxin. Biochim Biophys
Acta 1977; 483:248–57.
52. Moehring JM, Moehring TJ. Characterization of the diphtheria toxin-
resistance system in Chinese hamster ovary cells. Somatic Cell Genet
1979; 5:453–68.
53. Moehring JM, Moehring TJ, Danley DE. Posttranslational modification
of elongation factor 2 in diphtheria-toxin–resistant mutants of CHO-K1
cells. Proc Natl Acad Sci USA 1980; 77:1010–4.
54. Chen JY, Bodley JW, Livingston DM. Diphtheria toxin–resistant mutants
of Saccharomyces cerevisiae. Mol Cell Biol 1985; 5:3357–60.
55. Foley BT, Moehring JM, Moehring TJ. Mutations in the elongation factor
2 gene which confer resistance to diphtheria toxin and Pseudomonas
exotoxin A. Genetic and biochemical analyses. J Biol Chem 1995; 270:
23218–25.
56. Dorland RB, Middlebrook JL, Leppla SH. Receptor-mediated internali-
zation and degradation of diphtheria toxin by monkey kidney cells. J
Biol Chem 1979; 254:11337–42.
57. Middlebrook JL, Dorland RB, Leppla SH. Association of diphtheria toxin
with Vero cells. Demonstration of a receptor. J Biol Chem 1978; 253:
7325–30.
58. Middlebrook JL, Dorland RB. Response of cultured mammalian cells to
the exotoxins of Pseudomonas aeruginosa and Corynebacterium diph-
theriae: differential cytotoxicity. Can J Microbiol 1977; 23:183–9.
59. Naglich JG, Rolf JM, Eidels L. Expression of functional diphtheria toxin
receptors on highly toxin-sensitive mouse cells that specifically bind ra-
dioiodinated toxin. Proc Natl Acad Sci USA 1992; 89:2170–4.
60. Naglich JG, Metherall JE, Russell DW, Eidels L. Expression cloning of a
diphtheria toxin receptor: identity with a heparin-binding EGF-like
growth factor precursor. Cell 1992; 69:1051–61.
61. Mekada E, Senoh H, Iwamoto R, Okada Y, Uchida T. Purification of
diphtheria toxin receptor from Vero cells. J Biol Chem 1991; 266:
20457–62.
JID 2000;181 (Suppl 1) Diphtheria Toxin and the tox Gene
62. Hooper KP, Eidels L. Localization of a critical diphtheria toxin-binding
domain to the C-terminus of the mature heparin-binding EGF-like
growth factor region of the diphtheria toxin receptor. Biochem Biophys
Res Commun 1995; 206:710–7.
63. Mitamura T, Higashiyama S, Taniguchi N, Klagsbrun M, Mekada E.
Diphtheria toxin binds to the epidermal growth factor (EGF)–like do-
main of human heparin-binding EGF-like growth factor/diphtheria toxin
receptor and inhibits specifically its mitogenic activity. J Biol Chem
1995; 270:1015–9.
64. Iwamoto R, Higashiyama S, Mitamura T, Taniguchi N, Klagsbrun M,
Mekada E. Heparin-binding EGF-like growth factor, which acts as the
diphtheria toxin receptor, forms a complex with membrane protein
DRAP27/CD9, which up-regulates functional receptors and diphtheria
toxin sensitivity. EMBO J 1994; 13:2322–30.
65. Mitamura T, Iwamoto R, Umata T, et al. The 27-kD diphtheria toxin
receptor–associated protein (DRAP27) from Vero cells is the monkey
homologue of human CD9 antigen: expression of DRAP27 elevates the
number of diphtheria toxin receptors on toxin-sensitive cells. J Cell Biol
1992; 118:1389–99.
66. Hooper KP, Eidels L. Glutamic acid 141 of the diphtheria toxin receptor
(HB-EGF precursor) is critical for toxin binding and toxin sensitivity.
Biochem Biophys Res Commun 1996; 220:675–80.
67. Mitamura T, Umata T, Nakano F, et al. Structure-function analysis of the
diphtheria toxin receptor toxin binding site by site-directed mutagenesis.
J Biol Chem 1997; 272:27084–90.
68. Morris RE, Gerstein AS, Bonventre PF, Saelinger CB. Receptor-mediated
entry of diphtheria toxin into monkey kidney (Vero) cells: electron mi-
croscopic evaluation. Infect Immun 1985; 50:721–7.
69. Lemichez E, Bomsel M, Devilliers G, et al. Membrane translocation of
diphtheria toxin fragment A exploits early to late endosome trafficking
machinery. Mol Microbiol 1997; 23:445–57.
70. Draper RK, Simon MI. The entry of diphtheria toxin into the mammalian
cell cytoplasm: evidence for lysosomal involvement. J Cell Biol 1980; 87:
849–54.
71. Sandvig K, Olsnes S. Diphtheria toxin entry into cells is facilitated by low
pH. J Cell Biol 1980; 87:828–32.
72. Hu VW, Holmes RK. Evidence for direct insertion of fragments A and B
of diphtheria toxin into model membranes. J Biol Chem 1984; 259:
12226–33.
73. Montecucco C, Tomasi M, Schiavo G, Rappuoli R. Hydrophobic photo-
labelling of pertussis toxin subunits interacting with lipids. FEBS Lett
1986; 194:301–4.
74. Moskaug JO, Sandvig K, Olsnes S. Low pH-induced release of diphtheria
toxin A–fragment in Vero cells. Biochemical evidence for transfer to the
cytosol. J Biol Chem 1988; 263:2518–25.
75. Kagan BL, Finkelstein A, Colombini M. Diphtheria toxin fragment forms
large pores in phospholipid bilayer membranes. Proc Natl Acad Sci USA
1981; 78:4950–4.
76. Papini E, Rappuoli R, Murgia M, Montecucco C. Cell penetration of
diphtheria toxin. Reduction of the interchain disulfide bridge is the rate-
limiting step of translocation in the cytosol. J Biol Chem 1993; 268:
1567–74.
77. Lanzrein M, Falnes PO, Sand O, Olsnes S. Structure-function relationship
of the ion channel formed by diphtheria toxin in Vero cell membranes.
J Membr Biol 1997; 156:141–8.
78. Olsnes S, van Deurs B, Sandvig K. Protein toxins acting on intracellular
targets: cellular uptake and translocation to the cytosol. Med Microbiol
Immunol 1993; 182:51–61.
79. Lord JM, Roberts LM. Toxin entry: retrograde transport through the
secretory pathway. J Cell Biol 1998; 140:733–6.
80. Merion M, Schlesinger P, Brooks RM, Moehring JM, Moehring TJ, Sly
WS. Defective acidification of endosomes in Chinese hamster ovary cell
mutants cross-resistant: to toxins and viruses. Proc Natl Acad Sci USA
1983; 80:5315–9.
S165
81. Choe S, Bennett MJ, Fujii G, et al. The crystal structure of diphtheria
toxin. Nature 1992; 357:216–22.
82. Bennett MJ, Choe S, Eisenberg D. Refined structure of dimeric diphtheria
toxin at 2.0 Å resolution. Protein Sci 1994; 3:1444–63.
Downloaded from https://academic.oup.com/jid/article-abstract/181/Supplement_1/S156/839052 by Universitas Indonesia user on 27 December 2018
83. Bennett MJ, Eisenberg D. Refined structure of monomeric diphtheria toxin
at 2.3 Å resolution. Protein Sci 1994; 3:1464–75.
84. Weiss MS, Blanke SR, Collier RJ, Eisenberg D. Structure of the isolated
catalytic domain of diphtheria toxin. Biochemistry 1995; 34:773–81.
85. Bell CE, Eisenberg D. Crystal structure of diphtheria toxin bound to ni-
cotinamide adenine dinucleotide. Biochemistry 1996; 35:1137–49.
86. Bell CE, Eisenberg D. Crystal structure of nucleotide-free diphtheria toxin.
Biochemistry 1997; 36:481–8.
87. Domenighini M, Magagnoli C, Pizza M, Rappuoli R. Common features
of the NAD-binding and catalytic site of ADP-ribosylating toxins. Mol
Microbiol 1994; 14:41–50.
88. Domenighini M, Rappuoli R. Three conserved consensus sequences identify
the NAD-binding site of ADP-ribosylating enzymes, expressed by eu-
karyotes, bacteria and T-even bacteriophages. Mol Microbiol 1996; 21:
667–74.
89. Mueller JH, Miller PA. Production of diphtheria toxin of high potency
(100 Lf) on a reproducible medium. J Immunol 1941; 40:21–32.
90. Mueller JH. Nutrition of the diphtheria bacillus. Bacteriol Rev 1940; 4:
97–134.
91. Mueller JH. The influence of iron on the production of diphtheria toxin.
J Immunol 1941; 42:343–51.
92. Pappenheimer AM Jr, Johnson S. Studies on diphtheria toxin production.
I. The effect of iron and copper. Br J Exp Pathol 1936; 17:335–41.
93. Murphy JR, Pappenheimer AM Jr, de Borms ST. Synthesis of diphtheria
tox-gene products in Escherichia coli extracts. Proc Natl Acad Sci USA
1974; 71:11–5.
94. Kanei C, Uchida T, Yoneda M. Isolation from Corynebacterium diphtheriae
C7(beta) of bacterial mutants that produce toxin in medium with excess
iron. Infect Immun 1977; 18:203–9.
95. Welkos SL, Holmes RK. Regulation of toxinogenesis in Corynebacterium
diphtheriae. II. Genetic mapping of a tox regulatory mutation in bac-
teriophage beta. J Virol 1981; 37:946–54.
96. Welkos SL, Holmes RK. Regulation of toxinogenesis in Corynebacterium
diphtheriae. I. Mutations in bacteriophage beta that alter the effects of
iron on toxin production. J Virol 1981; 37:936–45.
97. Murphy JR, Skiver J, McBride G. Isolation and partial characterization
of a corynebacteriophage beta, tox operator constitutive–like mutant
lysogen of Corynebacterium diphtheriae. J Virol 1976; 18:235–44.
98. Murphy JR, Michel JL, Teng M. Evidence that the regulation of diphtheria
toxin production is directed at the level of transcription. J Bacteriol
1978; 135:511–6.
99. Holmes RK. Genetic aspects of toxinogenesis in bacteria. In: Schlessinger
D, ed. Microbiology—1975. Washington, DC: American Society for Mi-
crobiology, 1975:296–301.
100. Murphy JR, Bacha P. Regulation of diphtheria toxin production. In: Schles-
singer D, ed. Microbiology—1979. Washington, DC: American Society
for Microbiology, 1979:181–6.
101. Russell LM, Holmes RK. Initial characterization of the ferric iron transport
system of Corynebacterium diphtheriae. J Bacteriol 1983; 155:1439–42.
102. Cryz SJ Jr, Russell LM, Holmes RK. Regulation of toxinogenesis in Co-
rynebacterium diphtheriae: mutations in the bacterial genome that alter
the effects of iron on toxin production. J Bacteriol 1983; 154:245–52.
103. Russell LM, Cryz SJ Jr, Holmes RK. Genetic and biochemical evidence
for a siderophore-dependent iron transport system in Corynebacterium
diphtheriae. Infect Immun 1984; 45:143–9.
104. Budzikiewicz H, Bossenkamp A, Taraz K, Pandey A, Meyer JM. Cory-
nebactin, a cyclic catecholate siderophore from Corynebacterium glutami-
cum ATCC 14067 (Brevibacterium sp. DSM 20411). Z Naturforsch [C]
1997; 52:551–4.
105. Russell LM, Holmes RK. Highly toxinogenic but avirulent Park-Williams
S166 Holmes
8 strain of Corynebacterium diphtheriae does not produce siderophore.
Infect Immun 1985; 47:575–8.
106. Schmitt MP. Utilization of host iron sources by Corynebacterium diph- 125.
theriae: identification of a gene whose product is homologous to eu-
karyotic heme oxygenases and is required for acquisition of iron from
heme and hemoglobin. J Bacteriol 1997; 179:838–45. 126.
107. Wilks A, Schmitt MP. Expression and characterization of a heme oxygenase
(Hmu O) from Corynebacterium diphtheriae. Iron acquisition requires
oxidative cleavage of the heme macrocycle. J Biol Chem 1998; 273: 127.
837–41.
108. Schmitt MP. Transcription of the Corynebacterium diphtheriae hmuO gene
is regulated by iron and heme. Infect Immun 1997; 65:4634–41.
109. Boyd J, Murphy JR. Analysis of the diphtheria tox promoter by site-directed 128.
mutagenesis. J Bacteriol 1988; 170:5949–52.
110. Fourel G, Phalipon A, Kaczorek M. Evidence for direct regulation of
diphtheria toxin gene transcription by an Fe21-dependent DNA-binding
repressor, DtoxR, in Corynebacterium diphtheriae. Infect Immun 1989; 129.
57:3221–5.
111. Boyd J, Oza MN, Murphy JR. Molecular cloning and DNA sequence
analysis of a diphtheria tox iron-dependent regulatory element (dtxR)
from Corynebacterium diphtheriae. Proc Natl Acad Sci USA 1990; 87: 130.
5968–72.
112. Schmitt MP, Holmes RK. Iron-dependent regulation of diphtheria toxin
and siderophore expression by the cloned Corynebacterium diphtheriae
repressor gene dtxR in C. diphtheriae C7 strains. Infect Immun 1991; 131.
59:1899–904.
113. Handke K. Regulation of ferric iron transport in Escherichia coli K12:
isolation of a constitutive mutant. Mol Gen Genet 1981; 182:288–92.
114. Schmitt MP, Holmes RK. Characterization of a defective diphtheria toxin
132.
repressor (dtxR) allele and analysis of dtxR transcription in wild-type
and mutant strains of Corynebacterium diphtheriae. Infect Immun
1991; 59:3903–8.
133.
115. Boyd JM, Hall KC, Murphy JR. DNA sequences and characterization of
dtxR alleles from Corynebacterium diphtheriae PW8(2), 1030(2), and
C7hm723(2). J Bacteriol 1992; 174:1268–72.
134.
116. Schmitt MP, Twiddy EM, Holmes RK. Purification and characterization
of the diphtheria toxin repressor. Proc Natl Acad Sci USA 1992; 89:
7576–80.
135.
117. Tao X, Boyd J, Murphy JR. Specific binding of the diphtheria tox regulatory
element DtxR to the tox operator requires divalent heavy metal ions
and a 9-base pair interrupted palindromic sequence. Proc Natl Acad Sci
136.
USA 1992; 89:5897–901.
118. Tao X, Murphy JR. Binding of the metalloregulatory protein DtxR to the
diphtheria tox operator requires a divalent heavy metal ion and protects
the palindromic sequence from DNase I digestion. J Biol Chem 1992;
267:21761–4.
119. Schmitt MP, Holmes RK. Analysis of diphtheria toxin repressor–operator 137.
interactions and characterization of a mutant repressor with decreased
binding activity for divalent metals. Mol Microbiol 1993; 9:173–81.
120. Schmitt MP, Holmes RK. Cloning, sequence, and footprint analysis of two
promoter/operators from Corynebacterium diphtheriae that are regulated 138.
by the diphtheria toxin repressor (DtxR) and iron. J Bacteriol 1994; 176:
1141–9.
121. Tao X, Murphy JR. Determination of the minimal essential nucleotide
sequence for diphtheria tox repressor binding by in vitro affinity selec- 139.
tion. Proc Natl Acad Sci USA 1994; 91:9646–50.
122. Lee JH, Wang T, Ault K, Liu J, Schmitt MP, Holmes RK. Identification
and characterization of three new promoter/operators from Corynebac- 140.
terium diphtheriae that are regulated by the diphtheria toxin repressor
(DtxR) and iron. Infect Immun 1997; 65:4273–80. 141.
123. Krafft AE, Tai SP, Coker C, Holmes RK. Transcription analysis and nu-
cleotide sequence of tox promoter/operator mutants of corynebacterio-
phage beta. Microb Pathog 1992; 13:85–92.
124. Wang Z, Schmitt MP, Holmes RK. Characterization of mutations that 142.
JID 2000;181 (Suppl 1)
inactivate the diphtheria toxin repressor gene (dtxR). Infect Immun
1994; 62:1600–8.
Tao X, Zeng HY, Murphy JR. Transition metal ion activation of DNA
binding by the diphtheria tox repressor requires the formation of stable
Downloaded from https://academic.oup.com/jid/article-abstract/181/Supplement_1/S156/839052 by Universitas Indonesia user on 27 December 2018
homodimers. Proc Natl Acad Sci USA 1995; 92:6803–7.
Qiu X, Verlinde CL, Zhang S, Schmitt MP, Holmes RK, Hol WG. Three-
dimensional structure of the diphtheria toxin repressor in complex with
divalent cation co-repressors. Structure 1995; 3:87–100.
Schiering N, Tao X, Zeng H, Murphy JR, Petsko GA, Ringe D. Structures
of the apo- and the metal ion–activated forms of the diphtheria tox
repressor from Corynebacterium diphtheriae. Proc Natl Acad Sci USA
1995; 92:9843–50.
Ding X, Zeng H, Schiering N, Ringe D, Murphy JR. Identification of the
primary metal ion–activation sites of the diphtheria tox repressor by x-
ray crystallography and site-directed mutational analysis. Nat Struct Biol
1996; 3:382–7.
Qiu X, Pohl E, Holmes RK, Hol WG. High-resolution structure of the
diphtheria toxin repressor complexed with cobalt and manganese reveals
an SH3-like third domain and suggests a possible role of phosphate as
co-corepressor. Biochemistry 1996; 35:12292–302.
Pohl E, Qui X, Must LM, Holmes RK, Hol WG. Comparison of high-
resolution structures of the diphtheria toxin repressor in complex with
cobalt and zinc at the cation-anion binding site. Protein Sci 1997; 6:
1114–8.
Pohl E, Holmes RK, Hol WG. Motion of the DNA-binding domain with
respect to the core of the diphtheria toxin repressor (DtxR) revealed in
the crystal structures of apo- and holo-DtxR. J Biol Chem 1998; 273:
22420–7.
White A, Ding X, vanderSpek JC, Murphy JR, Ringe D. Structure of the
metal-ion–activated diphtheria toxin repressor/tox operator complex.
Nature 1998; 394:502–6.
Tao X, Murphy JR. Cysteine-102 is positioned in the metal binding acti-
vation site of the Corynebacterium diphtheriae regulatory element DtxR.
Proc Natl Acad Sci USA 1993; 90:8524–8.
Goranson-Siekierke J, Pohl E, Hol WGJ, Holmes RK. Anion-coordinating
residues at binding site 1 are essential for the biological activity of the
diphtheria toxin repressor. Infect Immun 1999; 67:1806–11.
Tai SP, Krafft AE, Nootheti P, Holmes RK. Coordinate regulation of
siderophore and diphtheria toxin production by iron in Corynebacterium
diphtheriae. Microb Pathog 1990; 9:267–73.
Schmitt MP, Predich M, Doukhan L, Smith I, Holmes RK. Characteri-
zation of an iron-dependent regulatory protein (IdeR) of Mycobacterium
tuberculosis as a functional homolog of the diphtheria toxin repressor
(DtxR) from Corynebacterium diphtheriae [published erratum appears in
Infect Immun 1996; 64:681]. Infect Immun 1995; 63:4284–9.
Tai SS, Zhu YY. Cloning of a Corynebacterium diphtheriae iron-repressible
gene that shares sequence homology with the AhpC subunit of alkyl
hydroperoxide reductase of Salmonella typhimurium. J Bacteriol 1995;
177:3512–7.
Oguiza JA, Tao X, Marcos AT, Martin JF, Murphy JR. Molecular cloning,
DNA sequence analysis, and characterization of the Corynebacterium
diphtheriae dtxR homolog from Brevibacterium lactofermentum. J Bac-
teriol 1995; 177:465–7.
Dussurget O, Rodriguez M, Smith I. An ideR mutant of Mycobacterium
smegmatis has derepressed siderophore production and an altered oxi-
dative-stress response. Mol Microbiol 1996; 22:535–44.
Doukhan L, Predich M, Nair G, et al. Genomic organization of the my-
cobacterial sigma gene cluster. Gene 1995; 165:67–70.
Gunter-Seeboth K, Schupp T. Cloning and sequence analysis of the Co-
rynebacterium diphtheriae dtxR homologue from Streptomyces lividans
and S. pilosus encoding a putative iron repressor protein. Gene 1995;
166:117–9.
Hill PJ, Cockayne A, Landers P, Morrissey JA, Sims CM, Williams P.
JID 2000;181 (Suppl 1) Diphtheria Toxin and the tox Gene
SirR, a novel iron-dependent repressor in Staphylococcus epidermidis.
Infect Immun 1998; 66:4123–9.
143. Hardham JM, Stamm LV, Porcella SF, et al. Identification and transcrip-
tional analysis of a Treponema pallidum operon encoding a putative ABC
transport system, an iron-activated repressor protein homolog, and a
glycolytic pathway enzyme homolog. Gene 1997; 197:47–64.
144. Pallen MJ. Rapid screening for toxigenic Corynebacterium diphtheriae by
the polymerase chain reaction. J Clin Pathol 1991; 44:1025–6.
145. Pallen MJ, Hay AJ, Puckey LH, Efstratiou A. Polymerase chain reaction
for screening clinical isolates of corynebacteria for the production of
diphtheria toxin. J Clin Pathol 1994; 47:353–6.
146. Mikhailovich VM, Melnikov VG, Mazurova IK, et al. Application of PCR
for detection of toxigenic Corynebacterium diphtheriae strains isolated
during the Russian diphtheria epidemic, 1990 through 1994. J Clin Mi-
crobiol 1995; 33:3061–3.
S167
147. Nakao H, Popovic T. Development of a direct PCR assay for detection of
the diphtheria toxin gene. J Clin Microbiol 1997; 35:1651–5.
148. De Zoysa A, Efstratiou A, George RC, et al. Molecular epidemiology of
Corynebacterium diphtheriae from northwestern Russia and surrounding
Downloaded from https://academic.oup.com/jid/article-abstract/181/Supplement_1/S156/839052 by Universitas Indonesia user on 27 December 2018
countries studied by using ribotyping and pulsed-field gel electrophoresis.
J Clin Microbiol 1995; 33:1080–3.
149. Popovic T, Kombarova SY, Reeves MW, et al. Molecular epidemiology of
diphtheria in Russia, 1985–1994. J Infect Dis 1996; 174:1064–72.
150. Nakao H, Pruckler JM, Mazurova IK, et al. Heterogeneity of diphtheria
toxin gene, tox, and its regulatory element, dtxR, in Corynebacterium
diphtheriae strains causing epidemic diphtheria in Russia and Ukraine.
J Clin Microbiol 1996; 34:1711–6.
151. Nakao H, Mazurova IK, Glushkevich T, Popovic T. Analysis of hetero-
geneity of Corynebacterium diphtheriae toxin gene, tox, and its regulatory
element, dtxR, by direct sequencing. Res Microbiol 1997; 148:45–54.