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Applied and Environmental Microbiology, December 2001, p. 5721-5728, Vol. 67, No. 12
Boyce Thompson Institute, Cornell University, Ithaca, New
York 14853-1801,1 and USDA-Agricultural
Research Service, Department of Botany and Plant Pathology, Purdue
University, West Lafayette, Indiana 47907-11552
Received 7 March 2001/Accepted 20 September 2001
Pathogenic strains of the soilborne fungus Periconia
circinata produce peritoxins with host-selective toxicity
against susceptible genotypes of sorghum. The peritoxins are
low-molecular-weight, hybrid molecules consisting of a peptide and a
chlorinated polyketide. Culture fluids from pathogenic, toxin-producing
(Tox+) and nonpathogenic, non-toxin-producing
(Tox Milo disease, a root and
crown rot caused by the soilborne fungus Periconia circinata
(Mangin) Sacc., was the first major threat to the cultivation of
sorghum (Sorghum bicolor [L.]) in North America
(14). The majority of sorghums introduced into the
south-central United States from Africa were representatives of the
milo race with several desirable agronomic characteristics, including
drought tolerance (8). These introductions were
susceptible to P. circinata and were nearly
devastated by milo disease during the 1920s and 1930s
(14). High-frequency, spontaneous mutations in the
semidominant allele at the Pc locus (21) led to
the development in the 1930s of resistant genotypes, which remain the
primary strategy for the control of milo disease today
(8).
Pathogenic strains of P. circinata produce
peritoxins, which are low-molecular-weight, hybrid molecules consisting
of a peptide and a chlorinated polyketide (2, 16).
Peritoxins exhibit host-selective toxicity at concentrations as low as
1 ng ml Non-toxin-producing (Tox One approach to studies of toxin biosynthesis is to analyze the
metabolite profiles of toxin-producing and non-toxin-producing strains.
A mutation in a single gene that inactivates a single enzyme in the
biosynthetic pathway could result in the accumulation of the
intermediate that is the substrate of the defective enzyme or in
complete loss of production of all metabolites in the pathway, if a
regulatory gene is inactivated. The latter result could also occur if
multiple enzymes (or enzymatic activities) are encoded by genes in a
gene cluster (11) that is not found in non-toxin-producing strains. Such deletions occur in the maize pathogens Cochliobolus heterostrophus and Cochliobolus carbonum, in which only
the highly virulent, toxin-producing races possess the genes required
for the biosynthesis of their respective host-selective toxins; weakly virulent pathogens are missing the entire biosynthetic pathway (1, 3, 4, 13, 25, 28).
The primary objective of this research was to determine whether
nonpathogenic strains of P. circinata synthesize
and accumulate known metabolic intermediates in the pathway committed
to the biosynthesis of peritoxins. Do Tox Culture and bioassays of P.
circinata.
Single conidiophore isolates of
P. circinata were obtained from infected roots of
field-grown sorghum collected in Texas, Kansas, and California (Table
1). The strains were grown and maintained
on potato dextrose agar (Difco, Detroit, Mich.) in the dark at 25°C
and stored on silica gel at 4°C (7) or as agar disks
(5-mm diameter) in a 65% glycerol solution at
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5721-5728.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Differential Synthesis of Peritoxins and Precursors by Pathogenic
Strains of the Fungus Periconia circinata
and
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
) strains were analyzed directly by gradient
high-performance liquid chromatography (HPLC) with photodiode array
detection and HPLC-mass spectrometry to detect intermediates and final
products of the biosynthetic pathway. This approach allowed us to
compare the metabolite profiles of Tox+ and
Tox strains. Peritoxins A and B and the biologically
inactive intermediates, N-3-(E-pentenyl)-glutaroyl-aspartate,
circinatin, and 7-chlorocircinatin, were detected only in culture
fluids of the Tox+ strains. The latter two compounds were
produced consistently by Tox+ strains regardless of the
amount of peritoxins produced under various culture conditions. In
summary, none of the known peritoxin-related metabolites were detected
in Tox strains, which suggests that these strains may
lack one or more functional genes required for peritoxin biosynthesis.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
1 against sorghum genotypes that are
susceptible to the pathogen (16, 26). Treatment of sorghum
seedlings with culture filtrates or purified preparations of toxin
reproduces the biochemical and visible symptoms of the disease in a
genotype-specific manner (6, 7, 9, 23, 27). Strains that
lack the ability to produce peritoxins are nonpathogenic (7, 19,
20). Thus, the pathogenic ability of P. circinata is strictly dependent upon its ability to produce
peritoxin, which in turn is the determinant of the disease phenotype
(8).
) strains of the fungus
are distributed throughout the temperate regions of the world, but
toxin-producing (Tox+) strains are known to occur
only in regions of the United States coincident with the occurrence of
milo disease (7). Peritoxin-producing ability apparently
increased the pathological niche of P. circinata by enabling it to parasitize a single genotype of sorghum, which is the
only species known to be a host. The possibility that milo disease
arose suddenly as the result of a mutation in a previously benign,
saprophytic population of P. circinata was
dismissed by Odvody et al. (19) because the disease
developed over a wide geographic area of the United States within a
very short period. Furthermore, the fungus has not mutated to races
that are pathogenic to other genotypes or groups of sorghum during the
past 70 years (8).
strains synthesize peritoxins in such low quantities relative to
Tox+ strains that toxin concentrations are not
high enough to elicit disease symptoms? Do Tox
strains synthesize only the inactive metabolites in the peritoxin biosynthetic pathway or no pathway intermediates at all? In order to
address these questions, a secondary objective was to evaluate culture
conditions to determine those most likely to support the production of
peritoxins and their precursors in any strain capable of making such
compounds. Our results provide the basis for subsequent molecular
approaches to characterize the genes for peritoxin biosynthesis in
P. circinata and to determine whether they are
absent or mutated in nonpathogenic, nontoxigenic strains.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C.
TABLE 1.
Isolates of P. circinata used in this study
strains have been deposited in the American
Type Culture Collection (ATCC 32725, ATCC 32726, ATCC 32727, and ATCC
32728). We determined the pathogenicity of the strains as described
previously (6, 7, 19) by growing seedlings of susceptible
or resistant sorghum genotypes in a nutrient solution containing
conidia (3 × 102 to 5 × 102 ml1) of the fungal
isolate or by planting sorghum seeds in soil or vermiculite infested
with a mixture of conidia and mycelium.
Culture fluids containing metabolites produced by the
Tox+ and Tox strains were
tested for their genotype-specific phytotoxicity against intact
seedlings or excised leaves and by root growth inhibition and
electrolyte leakage bioassays as described previously (6, 7, 8,
9, 16, 19). In the bioassays and the pathogenicity tests,
near-isogenic cultivars `Colby' (Pc Pc) and `Resistant
Colby' (pc pc) were used as the toxin-sensitive,
susceptible genotype and toxin-insensitive, resistant genotype,
respectively (21).
For analyses of metabolites produced in culture, P. circinata strains were grown by one of two methods. The
majority of the assays were conducted with culture fluids harvested
from strains grown in 16-oz, narrow-mouthed, glass prescription bottles
(Brockway Glass Co., Inc., Brockway, Pa.) containing 100 ml of liquid
modified Fries' medium supplemented with 0.1% yeast extract (MFY)
(7, 20). Five disks (5-mm diameter) from the growing edge
of 5- to 10-day-old cultures grown on potato dextrose agar plates were cut with a cork borer and transferred to each prescription bottle, which was incubated upright without agitation in the dark at 25°C for
approximately 24 h with the bottle cap loosely tightened. After
24 h, each bottle was incubated on its flattened side undisturbed for at least 20 days. Fungal growth and toxin production were most
consistent when the majority of the agar was removed from each disk of
inoculum by cutting it away with a scalpel so that the disks floated on
the surface of the liquid medium.
An alternative culture method for in vitro toxin production was
examined. Disposable polystyrene tissue culture flasks with a 0.2-µm
vented cap (Falcon no. 3107; Becton Dickinson, Franklin Lakes, N.J.)
were used as culture vessels instead of prescription bottles. Three
disks of inoculum (5-mm diameter) were transferred to 14.4 ml of liquid
MFY and incubated as described above for at least 12 days.
HPLC-DAD analyses of metabolites.
Culture fluids of
P. circinata were assayed over time by
high-performance liquid chromatography-diode array detection
(HPLC-DAD) for the purpose of determining the kinetics of
production of the known peritoxins and precursors. We developed a
method to analyze small volumes of culture fluids directly from growing
cultures without extensive purification steps. These studies allowed us to determine the time of maximal peritoxin production in each type of
culture vessel in order to most efficiently characterize the
differences among multiple Tox+ and
Tox isolates.
1 over 60 min with the column temperature
held at 30°C. Authentic peritoxins A and B (PtxA and PtxB,
respectively), circinatin, 7-chlorocircinatin (7-Cl-C), and
N-3-(E-pentenyl)-glutaroyl-aspartate (PGA) were
previously isolated in the laboratory of V. Macko (2, 15,
16; V. Macko and D. Arigoni, unpublished data) and used as
standards for this study. Since none of the authentic compounds exhibited distinctive or useful absorbance spectra in the range of 200 to 600 nm, the eluate was monitored by absorbance at 205 nm, near the
maximum absorbance for all of the compounds. Peaks were identified by
coinjection of culture fluids with authentic compounds or by separate
analyses of authentic compounds and samples, followed by electronic
stacking of computer-generated chromatograms for comparison of elution
times. The reproducibility of integration of multiple peak areas and
heights was within 3 and 1%, respectively, of the average when a
single compound was injected at three separate times on the same day.
These values were within 8 and 5%, respectively, of the average when a
sample of mixed standards was injected at two different times,
approximately 1 year apart.
For each isolate grown in tissue culture flasks, independent cultures
were grown and analyzed in triplicate for each time point, or equal
aliquots of culture fluid from each replicate were combined and
analyzed as one averaged sample. For prescription bottle cultures,
triplicate samples from single cultures grown for multiple time points
were analyzed. Space limitations imposed by the large size of the
prescription bottles usually precluded culturing independent replicates
of all time points or strains. In the analyses reported here, our goal
was to detect the peritoxins and precursors in crude culture fluid
samples and characterize the chromatographic profiles of such samples
without routine quantification of individual components.
HPLC-mass spectrometry (MS) analyses of metabolites.
Culture
fluids of the pathogenic strain S+4-1 and the nonpathogenic
strain CSC 9-1 were harvested from prescription bottle cultures at 25 days postinoculation. They were concentrated approximately 1.5- to
2-fold, and 5- to 10-µl samples were analyzed on a Micromass Quatro I
mass spectrometer, utilizing low-resolution positive electrospray
ionization with a probe voltage of 3.5 kV, a cone voltage of 25 V, and
a pepper pot voltage of 400 V. The nebulizer gas was run at 20 liters
of nitrogen h1, the drying gas was run at 350 liters of nitrogen h1, and the source
temperature was 100°C. Data acquisition and processing were
controlled by a Micromass MassLynx NT data system. Analytical conditions were the same as those described above for HPLC-DAD analyses, except that a 1- by 150-mm reversed-phase
C18 column (100-Å particle size; Vydac
218TP5115) was used at a flow rate of 50 µl/min. Limits of detection
for the Quatro I were not reported.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Toxin bioassays. In all bioassays performed in this study (Table 1), as well as in experiments with over 50 isolates conducted during the past 25 years (6, 7, 8, 9, 15, 16, 19, 26, 27; data not shown), only Tox+ strains were pathogenic. No exceptions have been observed to the conclusion that pathogenicity of P. circinata, i.e., the ability to cause root rot and associated disease symptoms beyond small, restricted cortical lesions on the root, absolutely requires the ability to produce peritoxins.
Differential production of peritoxins and precursors by pathogenic
isolates. (i) HPLC-DAD analyses.
Pathogenic strains of
P. circinata produced two known toxins, PtxA and
PtxB, which eluted at 12.1 and 19.1 min, respectively (Fig.
1 and 2).
The biologically inactive precursors PGA, circinatin, and 7-Cl-C eluted
at 18.9, 23.7, and 26.3 min, respectively. The limits of detection for
PtxA, PtxB, PGA, circinatin, and 7-Cl-C were measured as 0.1, 0.1, 0.2, 0.05, and 0.25 µg, respectively. The chromatographic profile of
strain S+4-1 (Fig. 1 and 2) is the recognizable, standard profile that
is typical of pathogenic, toxin-producing strains. We used this same
strain in our previous work (2, 5, 15, 16, 26, 27) for the
analysis, isolation, identification, and structural characterization of
the metabolites produced by P. circinata.
Consequently, regardless of the compression or expansion of the HPLC
profiles that may result from chromatographic variables (such as column
dimensions, gradient elution parameters, etc.), we know the chemical
identity of many of the peaks in the chromatogram because the basic
pattern is consistent. All three Tox+ strains
analyzed in this study (Table 1) exhibited remarkably similar
metabolite profiles even though S+4-1 was isolated from a different
geographic location than were the other two.
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strains analyzed by
HPLC-DAD (Fig. 1 and 2). These results were also observed for replicate
cultures of the Tox isolate CSC 9-1 grown in
time course experiments in tissue culture flasks (data not shown). The
same results were observed when CSC 9-1 and all other
Tox isolates (Table 1) were grown for 20 to 25 days in prescription bottles or for 15 days in tissue culture flasks
(Fig. 2). Since PGA and PtxB had retention times (18.9 and 19.1 min,
respectively) similar to those of unknown compounds produced by
Tox strains (Fig. 1 and 2), it was sometimes
difficult to rule out the presence of these compounds in culture fluids
of Tox strains analyzed by HPLC-DAD.
Comparisons of full-scale chromatographic profiles of metabolites of
Tox+ and Tox strains
showed that Tox strains generally produced
greater amounts of unknown metabolites eluting between 8 and 12 min
than did Tox+ strains (Fig. 1B and 2). Many of
the same compounds (based on elution time) were produced by
Tox+ strains but in substantially smaller
quantities. The similarities and differences in the overall metabolite
profiles of the Tox+ and
Tox strains suggest that chemical profiling
could be used to determine the toxin-producing phenotype of
P. circinata strains.
(ii) HPLC-MS analyses.
HPLC-MS (Fig.
3) confirmed that culture fluids of the
pathogenic strain S+4-1 contained ions for all of the known
peritoxin-related compounds. These metabolites, listed in order of
their retention times on the column used for HPLC-MS analyses, are PtxA
(19.4 min), PtxB (26.5 min), PGA (27.1 min), circinatin (30.8 min), and
7-Cl-C (33.2 min). The peritoxin-related compounds were identified by
the presence of their molecular ions [M+H]+ at
the expected retention times and by characteristic isotopic peaks,
which are indicative of the number of chlorine molecules in each
compound (18). For example, the
[M+H]+ of PtxA was 575.2, and the isotopic
peaks detected in the region of this molecular ion were characteristic
for the presence of three chlorine atoms. This pattern of isotopic
peaks and the size of the molecular ion identified the compound eluting
at 19.4 min as PtxA. PtxB and 7-Cl-C exhibited isotopic clusters
characteristic of chlorinated compounds with three or one chlorine
molecule(s), respectively. The isotopic peaks for PGA and circinatin
were typical of nonchlorinated compounds. Molecular ions for each of
the five peritoxin-related metabolites were not detected by select ion monitoring during chromatography of the culture fluids of the nonpathogenic isolate CSC 9-1. Thus, we were unable to detect any
peritoxin-related compounds in strain CSC 9-1 using the HPLC-MS analytical conditions reported here. In combination with the HPLC-DAD data from all of the Tox strains, as well as
highly sensitive bioassays capable of detecting as little as 1 ng of
peritoxins ml1, the results suggested that
nonpathogenic strains of P. circinata do not
produce peritoxins or their known precursors.
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Conditions for production of peritoxins and precursors.
Production of peritoxins and precursors by the pathogenic,
Tox+ isolate S+4-1, cultured in prescription
bottles, was assayed every 3 to 5 days, starting at 4 days
postinoculation and ending at 46 days postinoculation (Fig.
4). The earliest accumulation of all
peritoxin-related metabolites in prescription bottles was at 8 days
postinoculation. Major peaks in 4-day-old cultures that were observed
prior to the 3.25-min elution time (data not shown) and at 3.9-, 4.5-, 8.9-, and 13.1-min elution times were due to unknown components of the
medium. These compounds were detected at day 0 and were still evident,
but at significantly reduced levels, through 8 days postinoculation.
Production of PtxA and PtxB was comparable at days 20 (32 mg of PtxA
liter1, 12 mg of PtxB
liter1) and 25 (28 mg of PtxA
liter1, 15 mg of PtxB
liter1) postinoculation. Maximal production of
all peritoxin-related metabolites combined was detected at 25 days
postinoculation, at which time we measured the production of the
peritoxin precursors PGA, circinatin, and 7-Cl-C at quantities of
approximately 17, 30, and 136 mg liter1,
respectively. PGA and PtxB were generally more difficult to resolve and
detect at all time points than were PtxA and the other precursors. By
41 days postinoculation, peaks for PtxA and PtxB were not clearly
resolved, whereas circinatin and 7-Cl-C were still easily detectable at
46 days postinoculation.
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isolates. These experiments were also of
value in examining the possibility of decreasing culture vessel size
and shortening culture time without reducing peritoxin production in
Tox+ isolates. Culture fluids of the pathogenic,
Tox+ isolate S+4-1 and the nonpathogenic,
Tox isolate CSC 9-1 were harvested at 4, 6, 8, 12, 16, and 20 days postinoculation, and samples were analyzed by
gradient HPLC (data not shown). PtxA, circinatin, and 7-Cl-C were
detected in the Tox+ isolate at the earliest
sampling time of 4 days postinoculation. Maximal production of all
peritoxin-related metabolites occurred in this isolate by 12 days
postinoculation, although amounts detected at 16 and 20 days
postinoculation were generally comparable. No peritoxin-related
metabolites were ever detected in the Tox
isolate CSC 9-1 grown under these conditions.
Stability of toxin production in P.
circinata.
Pringle and Sheffer (20)
reported that toxin production in P. circinata
was a stable phenotype. However, we detected on numerous occasions and
in all Tox+ isolates analyzed (Table 1)
substantial reductions in the production of PtxA and PtxB after storage
of stock cultures on silica gel at 4°C or in glycerol at 70°C for
more than approximately 3 years. This was the case for cultures grown
and stored at both the Boyce Thompson Institute in Ithaca, N.Y., and
the USDA-Agricultural Research Service laboratory in West Lafayette,
Ind. Pathogenic, toxin-producing strains that became toxin deficient
after storage also exhibited reduced virulence in planta (data not
shown). However, in such peritoxin-deficient isolates, production of
the inactive precursors circinatin and 7-Cl-C was stable, easily
detectable, and, in some cases, greatly increased compared with levels
in pathogenic Tox+ strains. To restore normal
levels of peritoxin production and virulence, the strains were
inoculated onto susceptible sorghum plants, allowed to grow in planta
for several weeks, and then recovered as monoconidial cultures.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Differential production of peritoxin-related metabolites by
pathogenic strains of P. circinata.
A primary goal of this research was to determine whether
Tox strains of P. circinata synthesize any peritoxin-related metabolites. Using HPLC-DAD analyses (with detection limits ranging from 0.05 to
0.25 µg depending on the metabolite), we could not detect the known
peritoxins or their inactive precursors in six
Tox strains. Similarly, none of the
peritoxin-related metabolites were detected in culture fluids from a
single Tox strain analyzed by HPLC-MS.
Additionally, peritoxin activity was not detectable in
Tox isolates in bioassays that can detect as
little as 1 ng of peritoxin ml1
(8). These results suggest that the lack of symptom
induction in plants treated with Tox strains or
their culture fluids is due to an inability to synthesize peritoxins.
strains. This possibility
would not be surprising given the trend that is emerging from the
studies of other fungal plant pathogens (1, 10, 22, 25,
28) that, like P. circinata, produce secondary metabolites that influence host specificity. In these other
plant-pathogenic fungi, nonpathogenic strains neither produce toxin nor
carry the genes required for toxin biosynthesis. The fact that many
genes that determine host specificity are absent in nonpathogenic races
raises questions regarding the evolution of pathogenic races of fungi
that produce host-specific toxins. Horizontal gene transfer has been
proposed as the means by which genes for host-specific toxin
biosynthesis are acquired (1, 10, 28, 29).
Peritoxin biosynthesis in P. circinata. The peritoxins and precursors consist of two amino acids, a cyclic D-lysine and a D-aspartic acid, attached to a C10 polyketide unit, which is multiply chlorinated in the active toxins (2, 8, 15, 16, 26). Experiments monitoring incorporation of 1-[13C]acetate and 1,2-[13C2]acetate provided evidence for the polyketide origin of the C10 moiety in circinatin and PtxA and PtxB (2; D. Arigoni, personal communication). The observed incorporation pattern suggested that circinatin is a precursor of the chlorinated compounds. This hypothesis was supported by the isolation of the monochloro derivative of circinatin, 7-Cl-C, from culture fluids of P. circinata (Fig. 3) (5; Macko and Arigoni, unpublished).
We suggest the following biosynthetic relationships among the known peritoxin-related metabolites: PGA is proposed as an early precursor from which circinatin, 7-Cl-C, PtxB, and PtxA are synthesized in that order. Precursors of PGA, as well as other intermediates involved in the conversion of PGA to circinatin or 7-Cl-C to PtxB, likely exist. Such precursors and intermediates may be labile, degraded, or rapidly converted or may accumulate in extremely low quantities that are undetectable by the analytical methods that we have employed. Knowledge of the structures and synthesis of the peritoxins and precursors allows us to predict the kinds of genes that are required for biosynthesis of these compounds. Because the known peritoxin-related metabolites consist of two modified nonprotein amino acids linked to a C10 polyketide moiety, we predict that a peptide synthetase (17) and a polyketide synthase (12) are key enzymes required for biosynthesis. Both enzymes are multifunctional, multidomain enzymes that catalyze the synthesis of complex nonribosomally synthesized peptides or polyketides, respectively. Since the C10 polyketide moiety of 7-Cl-C and the peritoxins is chlorinated, a chloroperoxidase or some other type of novel halogenase (24) is predicted to be required as well.Optimal culture conditions for production of peritoxin-related metabolites. In vitro production of peritoxins by P. circinata is dependent, at least in part, on physical conditions that are not well understood. For example, peritoxin production is not detected in aerated shake cultures and is suppressed in standing cultures grown in Erlenmeyer flasks, while production of the precursor circinatin is significantly enhanced under these growth conditions (15). In contrast, peritoxin accumulation is relatively abundant in stale standing cultures grown in glass Roux or prescription bottles (20, 26). In the studies reported here, a secondary goal was to scale down the size of culture vessels while maintaining optimal peritoxin production by Tox+ isolates. We designed culture conditions to maintain the surface-area-to-volume ratio provided in the prescription bottles.
Toxin production by pathogenic strains of P. circinata was consistently good when the fungus was grown in prescription bottles for at least 20 to 25 days (16, 20). The production of peritoxins and precursors in tissue culture flasks was usually comparable to, and sometimes better than, production in prescription bottles, with generally less variability among replicates. Tissue culture flasks have several advantages over prescription bottles for peritoxin production: they are disposable and smaller and can be stacked, contamination problems are less likely, and the time to maximal peritoxin production is reduced by about 1 week. Even when peritoxin production in Tox+ isolates was reduced as a result of changes in strains during storage or unfavorable culture conditions, circinatin and 7-Cl-C were easily detected. Thus, we hypothesize that circinatin production is consistently and specifically associated with Tox+ genotypes and that circinatin production can be used as a reliable phenotypic marker to distinguish Tox+ and Tox strains or
mutants, as long as at least small amounts of PtxA are detectable.
The development of efficient and reproducible methods for culturing and
analyzing P. circinata for in vitro production of the peritoxins and precursors will increase the number of strains that
can be screened and enable the identification of mutants with targeted
disruptions in peritoxin biosynthetic genes. Ultimately, such efforts
are expected to lead to the cloning of genes responsible for peritoxin biosynthesis.
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ACKNOWLEDGMENTS |
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We thank Heather McLane and Mark McClenning for technical assistance and D. Arigoni and his colleagues at the ETH in Zürich, Switzerland, for their long-standing and valuable collaboration.
This work was supported in part by a grant to A.C.L.C. from the Park Foundation, Ithaca, N.Y. MS analyses were conducted by the Mass Spectrometry Laboratory, School of Chemical Sciences, University of Illinois, supported in part by a grant from the National Institute of General Medical Sciences (GM 27029). The Quatro mass spectrometer was purchased in part with a grant from the Division of Research Resources, National Institutes of Health (RR 07141).
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FOOTNOTES |
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* Corresponding author. Mailing address: Boyce Thompson Institute, Cornell University, Tower Rd., Ithaca, NY 14853-1801. Phone: (607) 254-1355. Fax: (607) 254-2958. E-mail: acc7{at}cornell.edu.
Present address: Department of Medicine, University of Rochester,
Rochester, NY 14607.
Present address: Oridigm Corporation, Seattle, WA 98103-8012.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
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Discussion
References
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Applied and Environmental Microbiology, December 2001, p. 5721-5728, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5721-5728.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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