APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2006, p. 6070–6078
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 9
An Oxidoreductase Is Involved in Cercosporin Degradation
by the Bacterium Xanthomonas campestris pv. zinniae
Tanya V. Taylor,1Thomas K. Mitchell,1and Margaret E. Daub2*
Departments of Plant Pathology1and Botany,2North Carolina State University, Raleigh, North Carolina 27695
Received 28 February 2006/Accepted 5 July 2006
The polyketide toxin cercosporin plays a key role in pathogenesis by fungal species of the genus Cercospora.
The bacterium Xanthomonas campestris pv. zinniae is able to rapidly degrade this toxin. Growth of X. campestris
pv. zinniae strains in cercosporin-containing medium leads to the breakdown of cercosporin and to the
formation of xanosporic acid, a nontoxic breakdown product. Five non-cercosporin-degrading mutants of a
strain that rapidly degrades cercosporin (XCZ-3) were generated by ethyl methanesulfonate mutagenesis and
were then transformed with a genomic library from the wild-type strain. All five mutants were complemented
with the same genomic clone, which encoded a putative transcriptional regulator and an oxidoreductase.
Simultaneous expression of these two genes was necessary to complement the mutant phenotype. Sequence
analysis of the mutants showed that all five mutants had point mutations in the oxidoreductase gene and no
mutations in the regulator. Quantitative reverse transcription-PCR (RT-PCR) showed that the expression of
both of these genes in the wild-type strain is upregulated after exposure to cercosporin. Both the oxidoreduc-
tase and transcriptional regulator genes were transformed into three non-cercosporin-degrading bacteria to
determine if they are sufficient for cercosporin degradation. Quantitative RT-PCR analysis confirmed that the
oxidoreductase was expressed in all transconjugants. However, none of the transconjugants were able to
degrade cercosporin, suggesting that additional factors are required for cercosporin degradation. Further
study of cercosporin degradation in X. campestris pv. zinniae may allow for the engineering of Cercospora-
resistant plants by using a suite of genes.
Members of the fungal genus Cercospora are pathogenic to
plants, causing damaging leaf spot and blight diseases. The
genus collectively has a wide host range, with individual species
parasitizing specific hosts. Cercospora spp. can infect such hosts
as tobacco, corn, soybean, rice, sugar beet, and banana (5, 14,
17, 28, 40). Crop damage and losses can be very high.
Cercospora spp. are known for the production of the non-
host-specific toxin cercosporin. Cercosporin was first isolated
from dried mycelium of Cercospora kikuchii, a soybean patho-
gen, in 1957 by Kuyama and Tamura (23). It is deep red and
has the molecular formula C29H26O10(Fig. 1) (26, 46). Cer-
cosporin is produced by most members of the Cercospora ge-
nus and has been isolated from many Cercospora species as
well as from infected host plants (2, 4, 17, 18, 23, 28, 31, 43). It
is a polyketide secondary metabolite with toxicity to plants,
animals, bacteria, and fungi (14). Cercosporin is a photosensi-
tizer and uses light energy to produce the activated oxygen
species superoxide and singlet oxygen (11, 47). Although cer-
cosporin has been shown to produce both activated oxygen
species, singlet oxygen appears to cause the most damage,
leading to lipid peroxidation and the breakdown of cell mem-
branes (11–13, 15, 21, 24).
Production of the toxin cercosporin has been associated with
parasitism and the development of disease in host plants (14).
When cercosporin-deficient mutants of Cercospora kikuchii
and Cercospora nicotianae were inoculated onto their host
plants, the resulting lesions were fewer and smaller (8, 41).
Mutants of Cercospora zeae-maydis disrupted in the CZK3
mitogen-activated protein kinase kinase kinase and deficient in
both cercosporin production and conidiation caused only lim-
ited small chlorotic lesions on corn rather than the typical large
necrotic lesions (37). In addition, in the sugar beet, the disease
severity of Cercospora leaf spot disease has been shown to be
directly related to light intensity (7). Furthermore, previous
studies by Steinkamp et al. showed that ultrastructural changes
in the sugar beet caused by infection with Cercospora beticola
are almost identical to those caused by treatment with cerco-
sporin (13, 38, 39).
Presently, the control of diseases caused by Cercospora spe-
cies relies on the use of fungicides and appropriate cultural
practices such as tillage and crop rotation (44, 45). The devel-
opment of resistant cultivars has been attempted, but high
levels of resistance to Cercospora diseases are uncommon. Al-
though limited disease resistance has been found in corn, sugar
beet, and soybean (9, 33, 36, 44, 45), alternate methods of
control would be desirable. One such approach would be to
target cercosporin by engineering plants to express genes en-
coding degradation enzymes.
A patent for a method to identify bacteria that have the
ability to degrade cercosporin was issued to Robeson et al.
(35). Mixed bacterial populations were harvested from the soil,
leaf surfaces, and leaf tissue of C. beticola-infected sugar beet
plants and inoculated onto cercosporin-containing medium.
After incubation in the dark, bacteria that are able to degrade
cercosporin were identified by a clear halo surrounding the
colony on the red plates.
In previous work in our laboratory, we followed up on the
findings of Robeson et al. and screened bacteria for the ability
to degrade cercosporin. Out of 244 isolates that were screened,
* Corresponding author. Mailing address: Department of Botany,
2214 Gardner Hall, North Carolina State University, Raleigh, NC
27695-7616. Phone: (919) 513-3807. Fax: (919) 515-3436. E-mail:
43 isolates that were able to degrade cercosporin were identi-
fied based on the production of a clear halo when bacteria
were cultured on cercosporin-containing medium (30). Of the
isolates tested, Xanthomonas species had the largest propor-
tions of cercosporin degraders, and among those, all 32
isolates of Xanthomonas campestris pv. zinniae that were
tested were particularly active. We subsequently showed
that cercosporin degradation leads to the production of a
green breakdown product identified as xanosporic acid (29)
(Fig. 1). Xanosporic acid was shown to be nontoxic to a
cercosporin-sensitive mutant of C. nicotianae (30). The lac-
tonized derivative of xanosporic acid, xanosporolactone, was
used in a plant infiltration assay of tobacco and was also
shown to be nontoxic (30).
The goal of the work reported here is to isolate and char-
acterize the gene(s) responsible for cercosporin degradation by
a strain of X. campestris pv. zinniae that rapidly degrades cer-
cosporin (XCZ-3). We show that the ability of XCZ-3 to de-
grade cercosporin requires a region of DNA containing genes
encoding an oxidoreductase and a transcriptional regulator.
MATERIALS AND METHODS
Bacterial cultures. Mutants of strains XCZ-3 (30) and XCZ-3 were main-
tained on Luria-Bertani (LB) medium (10 g of tryptone, 10 g of NaCl, and 5 g of
yeast extract per liter [pH 7.2]) containing rifampin at 100 ?g/ml. Xanthomonas
axonopodis (pseudonym X. campestris) pv. pruni (34, 42) strains XAP-76, XAP-
50, XAP-75, XAP-1, XAP-39, XAP-49, XAP-34, XAP-31, and XAP-79 were
obtained from David Ritchie (North Carolina State University) and maintained
on LB medium without antibiotics. Isolates of Xanthomonas axonopodis pv.
vesicatoria (34, 42) (XAV-79 and XAV-126), also obtained from David Ritchie,
were maintained on LB medium containing 200 ?g/ml copper sulfate. Pseudo-
monas syringae pv. tomato DC3000 and Pseudomonas syringae pv. pisi 209-21-3R
were obtained from Peter Lindgren (North Carolina State University) and main-
tained on LB medium containing rifampin (as described above). All of the
bacterial isolates were maintained at 28°C in the dark.
Cercosporin stocks and chemicals. Cercosporin was isolated and purified from
mycelial cultures of Cercospora nicotianae as previously described (11). Dried
cercosporin crystals were dissolved in acetone for use in media. All other chem-
icals were obtained from Sigma-Aldrich Co. (St. Louis, MO) unless noted other-
wise. All restriction enzymes were obtained from Promega Corporation (Madi-
Chemical mutagenesis of Xanthomonas campestris pv. zinniae and screening
for mutants unable to degrade cercosporin. Three of the non-cercosporin-de-
grading mutants (120-2, MutA, and MutB) were created through ethyl methane-
sulfonate (EMS) mutagenesis according to the protocol described previously by
Thorne et al. (40). The other two non-cercosporin-degrading mutants (MutC and
MutD) were obtained by first transforming the wild type with the multicopy
vector pLAFR6 (obtained from Brian Staskawicz, University of California,
Berkeley, CA) containing both the transcriptional regulator and the oxidoreduc-
tase. EMS mutagenesis was then performed to yield the mutants. Screening for
non-cercosporin-degrading mutants (Fig. 2) was carried out as previously de-
scribed (30). For MutC and MutD, vector pLAFR6 was maintained through
antibiotic selection until the mutants were discovered and then removed through
plasmid curing. Mutant colonies were grown on LB medium containing rifampin
at 100 ?g/ml to ensure that the mutants retained the RifR marker.
Box-PCR. The whole-cell method of Box-PCR was performed according to a
protocol established previously by Louws et al. (27) in order to confirm that the
non-cercosporin-degrading mutants were derived from isolate XCZ-3. The fol-
lowing cycle conditions were used: 95°C for 2 min followed by 35 cycles of 94°C
for 3 s, 92°C for 30 s, 50°C for 1 min, and 65°C for 8 min. Ten microliters of each
PCR product was separated on a 1.5% agarose gel at 36 V for approximately 15 h
at 4°C. The banding patterns on the ethidium bromide-stained gel were viewed
under UV light.
Xanthomonas campestris pv. zinniae genomic library construction. Genomic
DNA was purified from XCZ-3 as described previously (3). A genomic library
was created based on the protocol described previously by Thorne et al. (40). The
genomic DNA was digested with Sau3A, and fragments of approximately 21 kb
were selected. The fragments were cloned into the BamHI restriction site of the
expression vector pLAFR6, which carries tetracycline resistance as the selectable
marker. Plasmids were then transformed into Epicurian Coli JM109 competent
cells (Stratagene, La Jolla, CA). Transformants were selected for tetracycline
resistance. A total of 2,016 clonal colonies were selected in order to form the
library. Colonies were grown in 96-well plates and stored at ?80°C.
Complementation assays. Conjugation of the non-cercosporin-degrading mu-
tants with the library clones was carried out using a triparental mating procedure
with a helper cell line containing the pRK2013 plasmid (16). The library clones
were cultured in LB liquid medium containing tetracycline at 20 ?g/ml and
incubated at 37°C. The helper cell line was cultured in LB liquid medium at 37°C.
The non-cercosporin-degrading mutants were cultured in LB liquid medium
containing 100 ?g/ml rifampin at 28°C in the dark. For conjugation, 133 ?l of
each culture was mixed together and plated onto a 0.45-?m nitrocellulose mem-
brane on LB solid medium using a 96-well replicator. The bacteria were allowed
to dry on the membrane for 15 min and were then grown for 48 h at 28°C in the
dark. Colonies were then transferred onto LB solid medium containing 100
?g/ml rifampin and 20 ?g/ml tetracycline. For screening, a minimum of three
colonies per conjugation were collected and transferred onto nutrient agar solid
medium containing 50 ?M cercosporin. As a control, the colonies were also
grown on nutrient agar solid medium containing 0.5% acetone (used to solubilize
cercosporin). A density of 12 colonies per plate was used for the screening
process. Two controls, the cercosporin-degrading isolate XCZ-3 and the non-
cercosporin-degrading isolate XAP-76, were also inoculated onto each plate. The
colonies were incubated in the dark at 28°C for 12 days and screened for a clear
halo surrounding the colony. Colonies were also screened on M9 minimal me-
dium to identify auxotrophs.
FIG. 1. Chemical structure of cercosporin and the breakdown
product (xanosporic acid) produced by X. campestris pv. zinniae isolate
FIG. 2. Non-cercosporin-degrading XCZ-3 mutants. Mutants show
a lack of a clear halo when grown on cercosporin-containing medium.
The positive control (XCZ-3 [wild type]) appears at the top of the
plate, and the negative control (XAP-76) appears at the bottom of the
VOL. 72, 2006 CERCOSPORIN DEGRADATION BY X. CAMPESTRIS PV. ZINNIAE6071
Subcloning of complementing library clone. Subcloning was carried out
through the use of restriction enzyme digests with EcoRI and HindIII. The
digested clone was separated on a 1.0% agarose gel, and the individual bands
were excised and purified using a QIAGEN (Valencia, CA) gel purification kit.
Subclones were ligated into the expression vector pLAFR6 (linearized with
EcoRI) and transformed into DH5? chemically competent cells (Invitrogen,
Carlsbad, CA) according to the manufacturer’s protocol. Complementation as-
says were performed with each subclone as described above.
Sequencing of the subclone. The single complementing subclone (a 7.5-kb
fragment obtained from the EcoRI digest) was sheared using a Hydroshear.
Sheared fragments were separated on a 1.0% agarose gel, and DNA fragments
between 1 and 2 kb were excised and purified using a QIAGEN (Valencia, CA)
gel purification kit. The End-Repair kit (Epicenter, Madison, WI) was used
to blunt end the fragments. Fragments were ligated to pBluescript II SK(?)
(Stratagene, La Jolla, CA), and the ligation was transformed into DH10B elec-
trocompetent cells (Invitrogen, Carlsbad, CA). Colonies were chosen using blue/
white screening and ampicillin resistance and then cultured overnight. Plasmids
were purified from each colony and were amplified by PCR using the standard
primers T3 and T7. The PCR products were sequenced at the North Carolina
State University Genome Research Laboratory.
Sequencing of mutant genes. The transcriptional regulator and oxidoreductase
from each non-cercosporin-degrading mutant were sequenced to identify muta-
tions. Sequences were amplified by high-fidelity PCR using a combination of
both Taq DNA polymerase (Promega, Madison, WI) and Pfu Ultra high-fidelity
DNA polymerase (Stratagene, La Jolla, CA). PCR products were separated on
a 1.0% agarose gel, and the desired bands were excised. The Promega Wizard SV
gel and PCR Clean-Up kit were used to purify the DNA from the agarose gel.
The bands were then cloned into the pGEM-T Easy vector system (Promega,
Madison, WI) according to the manufacturer’s protocol. The genes were se-
quenced using pGEM-T Easy sequencing primers T7 and SP6.
Gene complementation. The oxidoreductase gene was PCR amplified from
XCZ-3 using primers F8kb1 (5?-TCCTGCTCGATGTCAATGC-3?) and R8kb1
(5?-GAAGTTCTAGGGGCGACCAG-3?). The PCR product was cloned into
the pGEM-T Easy vector, excised by restriction digestion with EcoRI, and
cloned into pLAFR6. A second construct containing both the transcriptional
regulator and the oxidoreductase gene was made in the same fashion using
primers newsense2 (5?-AGCACCATTGACGCAAGCACC-3?) and R8kb1 (de-
scribed above). A third construct containing 330 bp at the 3? end of the tran-
scriptional regulator and all of the oxidoreductase was made by amplification
with primers F915 (5?-TGATCGGGCCATTACTGCTGTT-3?) and R8kb1 (de-
scribed above). All three pLAFR6 constructs were tested for their ability to
complement the non-cercosporin-degrading mutants.
Quantitative RT-PCR of oxidoreductase and regulator in wild-type XCZ-3
cells. XCZ-3 cells were grown in LB liquid medium or LB medium containing 50
?M cercosporin or an equal volume of acetone (0.5%). RNA was isolated at 24 h
using the Promega SV Total RNA isolation kit. A total of 1 ?g RNA for each
sample was used to make cDNA using an Applied Biosystems (Foster City, CA)
TaqMan Reverse Transcription Reagents kit. Primers for the oxidoreductase and
transcriptional regulator genes were designed using Primer Express (Applied
Biosystems, Foster City, CA) software and acquired from Sigma Genosys (The
Woodlands, TX). For the oxidoreductase, primers F1 (5?-TGCGCTACGAAC
GGATCA-3?) and R1 (5?-GGCACGAGCTTGGGAATGT-3?) were used. For
the transcriptional regulator, primers F2 (5?-TCTTCAATCGCTTGGTCGA
T-3?) and R2 (5?-ATCGCTTCAAGCTTGTCCAG-3?) were used. As an inter-
nal control, the internal transcribed sequence that resides between the 16S and
23S rRNAs was amplified using primers F-ITS (5?-GTTCCCGGGCCTTGTAC
ACAC-3?) and R-ITS (5?-GGTTCTTTTCACCTTTCCCTC-3?) (20). Quantita-
tive reverse transcription-PCR (RT-PCR) was carried out using 2? SYBR
Green Master Mix (Applied Biosystems, Foster City, CA) with the following
conditions: 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 60°C for 1 min,
and 72°C for 20 s. The sample plate was read after every cycle. Analysis of the
quantitative RT-PCR results was carried out according to a method described
previously by Livak and Schmittgen (25).
Southern analysis. A labeled probe was created using digoxigenin (DIG)
(Roche, Indianapolis, IN) and primers F8kb1 (5?-TCCTGCTCGATGTCAATG
C-3?) and R8kb1 (5?-GAAGTTCTAGGGGCGACCAG-3?) according to the
manufacturer’s protocol. Genomic DNA was isolated (3) for Southern analysis
from XCZ-3, XAP-76, XAV-79, XAV-126, P. syringae pv. tomato DC3000, P.
syringae pv. pisi 209-21-3R, XAP-50, XAP-75, XAP-1, XAP-39, XAP-49, XAP-
34, XAP-31, and XAP-79. A total of 5 ?g of genomic DNA was digested with 10
?l EcoRI from Promega (Madison, WI) in a total volume of 100 ?l according to
the manufacturer’s protocol. The digestion was carried out at 37°C for 18.5 h.
The EcoRI enzyme was heat inactivated for 15 min at 65°C. The DNA was
cleaned using the Promega Wizard DNA Clean-Up kit, and the entire digest was
separated on a 0.7% agarose gel for Southern analysis. Treatment and transfer
of the Southern gel were carried out according to a method described previously
by Ausubel et al. (3), with the exception that the gel was soaked in 0.25 N HCl
for 8 min instead of 30 min. After transfer, the membrane was exposed to
hybridization buffer for 4 h at 65°C and then incubated overnight at 65°C with
hybridization buffer containing the DIG-labeled probe. The Southern blot was
developed according to the manufacturer’s protocol for DIG-labeled probes
(Roche, Indianapolis, IN).
Transformation of the regulator and oxidoreductase genes into non-cer-
cosporin-degrading bacteria. The original complementing 23-kb library clone
contained in the pLAFR6 vector (p6-23kb) was conjugated into non-cercosporin-
degrading bacteria (XAV-79, XAV-126, and P. syringae pv. tomato DC3000)
according to the triparental mating protocol described above. Transconjugant
bacteria, as well as the corresponding wild-type strains, were grown in LB liquid
medium containing 50 ?M cercosporin or 0.5% acetone. RNA was isolated at
24 h and used to make cDNA as previously described. Quantitative RT-PCR was
carried out using the F1 and R1 primers mentioned above in order to assess the
expression of the oxidoreductase gene. Primers F-ITS and R-ITS were used to
amplify the internal transcribed spacer region as an internal control.
Time course of cercosporin degradation by transconjugants. Cercosporin deg-
radation was assessed by growing the transconjugants and the corresponding
wild-type strains in 50 ml LB liquid medium containing 60 ?M cercosporin.
Five-milliliter aliquots were taken at 0, 24, and 48 h and stored at ?20°C until
ready for use. Each sample was extracted twice with 2 ml chloroform (30),
followed by partitioning of the cercosporin into the aqueous phase using 1 N
NaOH. Mixtures were vortexed and centrifuged, and the supernatant was col-
lected. The supernatant was treated with 1 N HCl (to partition cercosporin into
the organic phase) and extracted with an additional 1 ml of chloroform. The
chloroform extractions for the individual samples were combined to yield a total
of 5 ml for each culture. Cercosporin degradation was quantified by measuring
the absorbance at 472 nm using a Beckman DU 650 spectrophotometer. The
experiment was carried out twice.
Nucleotide sequence accession number. The nucleotide sequence spanning the
oxidoreductase and regulator genes has been deposited in GenBank under ac-
cession number DQ087176.
Isolation of Xanthomonas campestris pv. zinniae mutants un-
able to degrade cercosporin. Wild-type XCZ-3, when grown on
cercosporin, produces a clear halo surrounding the colony
(Fig. 2). By contrast, non-cercosporin-degrading strains do
not produce a halo and are purple due to the uptake of cer-
cosporin. XCZ-3 cultures were mutagenized with EMS and
screened by transferring individual colonies onto cercosporin-
containing medium. The presence or absence of halo forma-
tion was determined at 12 days. Two initial screens resulted in
the isolation of three non-cercosporin-degrading XCZ-3 mu-
tants. These mutants were named 120-2, MutA, and MutB
(Fig. 2). When tested on M9 minimal medium, none of the
mutants were auxotrophic. All three of the mutants also re-
tained the rifampin resistance marker. Mutants were screened
by Box-PCR to confirm their identities. The banding patters of
all three mutants matched that of the wild-type strain (data not
Identification of complementing genes. Complementation of
the non-cercosporin-degrading mutants with the genomic li-
brary was carried out using the triparental mating procedure as
outlined in Materials and Methods. The transconjugant colo-
nies were screened for cercosporin degradation on cercosporin-
containing medium. Out of 2,016 library clones that were
screened, a single clone of 23 kb that restored cercosporin
degradation to all three mutants was found. The 23-kb clone
was subcloned, and a 7.5-kb subclone that complemented all
the mutants was identified. This subclone was sequenced using
6072 TAYLOR ET AL.APPL. ENVIRON. MICROBIOL.
the shotgun method, the sequences were assembled, and the
resulting contig assemblies were compared against the NCBI
database using the blastx function. Six genes were identified.
These genes had sequence similarities to a pectate lyase (E
value ? 1e?104), a soluble lytic murein transglycosylase (E
value ? e?169), a TonB-dependent receptor (E value ? e?179),
an oxidoreductase (E value ? 2e?82), a transcriptional regula-
tor (E value ? 2e?37), and a choline dehydrogenase (E value ?
2e?96) (Fig. 3).
Initial efforts were focused on the transcriptional regulator
and oxidoreductase genes. Subcloning indicated that digestion
of the complementing 23-kb library clone with HindIII elimi-
nated complementation ability, and the complementing 7.5-kb
subclone has two HindIII digestion sites, one each in the tran-
scriptional regulator and oxidoreductase genes. In addition,
the oxidoreductase was a likely candidate based on the pro-
posed reaction for the formation of xanosporic acid from cer-
cosporin, which involves an oxygen insertion followed by the
spontaneous rearrangement of the molecule (29). The region
spanning the transcriptional regulator and oxidoreductase was
amplified by PCR and tested for the ability to complement
mutants 120-2, MutA, and MutB; these sequences restored the
cercosporin-degrading phenotype to all three mutants.
The nucleotide sequence spanning the oxidoreductase and reg-
ulator is shown in Fig. 4. The genes are transcribed in the same
direction and are located only 61 bp apart. A blastx search
(NCBI) showed that the translated form of the oxidoreductase
gene is most similar to an oxidoreductase from Xanthomonas
axonopodis pv. citri strain 306 (GenBank accession no.
AAM36534; E value ? 2e?114) (10), a flavoprotein monooxygen-
ase from Pseudomonas syringae pv. syringae strain B728a (acces-
sion no. YP_233205; E value ? 9e?105) (19), and a monooxygen-
ase from Pseudomonas syringae pv. tomato strain DC3000
(accession no. NP_790149; E value ? e?85) (6). An alignment
between the two xanthomonad oxidoreductase sequences, carried
out using the programs Vector NTI 8 (Informax, Inc., Frederick,
MD) and Clustal W 1.82 (EMBL-EBI, Heidelberg, Germany),
showed that the two genes are 63% similar and 51.9% identical at
the amino acid level. Despite the similarity, the XCZ-3 oxi-
doreductase gene does not have any defining domains or motifs
known to be associated with monooxygenases according to an
ExPASy PROSITE motif scan.
The translated XCZ-3 transcriptional regulator shows the
highest similarity to a transcriptional regulator for cryptic he-
molysin from X. axonopodis pv. citri strain 306 (GenBank ac-
cession no. AAM36535; E value ? e?37) (10). An alignment of
these two genes using Vector NTI and Clustal W showed
70.1% similarity and 58.6% identity at the amino acid level.
Interestingly, the homologous transcriptional regulator from
X. axonopodis pv. citri strain 306 lies directly upstream of the
homologous oxidoreductase. As in XCZ-3, these two genes are
transcribed in the same direction and are only 30 bp apart. An
alignment of the region containing these two genes from
XCZ-3 and X. axonopodis pv. citri strain 306 shows 55.2%
similarity at the nucleotide level.
The second most similar match to the transcriptional regu-
lator is a MarR transcriptional regulatory protein from
Gloeobacter violaceus strain PCC 7421 (GenBank accession no.
NP_926807) that shows 53% similarity, with an E value of
5e?12(32). An ExPASy PROSITE motif scan revealed that the
XCZ-3 transcriptional regulator contains a MarR-type helix-
turn-helix domain (Fig. 4). The helix-turn-helix domain is often
associated with DNA binding in transcriptional regulators (1).
The gene marR of Escherichia coli, which is involved in the
negative regulation of multiple antibiotic resistance, is the
most common representative of the MarR family of helix-turn-
helix transcriptional regulators.
Search for additional genes. Two additional mutants,
termed MutC and MutD, were obtained by EMS mutagenesis
of XCZ-3 expressing the multicopy vector pLAFR6 containing
both the transcriptional regulator and the oxidoreductase in an
effort to identify mutations in genes other than the transcrip-
tional regulator and the oxidoreductase. Both MutC and MutD
showed the same non-cercosporin-degrading phenotype as the
other mutants when grown on cercosporin-containing medium
(Fig. 2). Although the mutations in MutC and MutD were
generated while carrying multiple copies of the wild-type tran-
scriptional regulator and oxidoreductase genes, they were still
complemented by the region containing the wild-type tran-
scriptional regulator and oxidoreductase genes, indicating that
the new mutations occurred in the same region already iden-
Sequencing of mutants. The region spanning the transcrip-
tional regulator and the oxidoreductase genes was sequenced
in each of the five non-cercosporin-degrading XCZ-3 mutants.
Sequence analysis identified single point mutations in the
oxidoreductase gene in all five mutants but no mutations in the
transcriptional regulator (Fig. 4). Each mutation in the oxi-
doreductase gene resulted in an amino acid change. The pre-
FIG. 3. Complementing fragment derived from sequencing the
complementing 7.5-kb subclone. Contig 9 alone complemented all five
VOL. 72, 2006 CERCOSPORIN DEGRADATION BY X. CAMPESTRIS PV. ZINNIAE 6073
dicted wild-type oxidoreductase is 400 amino acids long, and
homology suggests that the active site resides between amino
acids 150 and 340. The mutations in MutA, MutB, and MutD
fall within this region. In 120-2, the start codon was changed to
a threonine, eliminating the protein start site. In MutA, a
tryptophan residue was changed to a stop codon, resulting in a
truncated protein. The mutations found in MutB, MutC, and
MutD change three different glycines to aspartic acid. When
these three mutations were analyzed by NNPREDICT, the
secondary structure was predicted to be affected only in MutB.
However, this substitution replaces a single hydrogen with a
bulky and acidic side group (CH2COO), which is likely to affect
the tertiary structure and function of the protein in all three of
Importance of regulator sequences in mutant complemen-
tation. As all mutants were defective in the oxidoreductase
only, we tested the sequences required to complement the
mutants. The entire oxidoreductase gene with the upstream
intergenic regions plus 113 bases of the regulator (nucleotides
391 to 1948) (Fig. 4) were amplified and cloned into the
pLAFR6 vector. This construct was unable to complement any
of the non-cercosporin-degrading mutants. By contrast, a con-
struct containing an additional 217 bases of the regulator (nu-
cleotides 174 to 1948) (Fig. 4) was able to complement all five
mutants. Although this construct does not include the full-
length gene for the transcriptional regulator, it does include
the potential active site (Fig. 4).
Presence of cercosporin upregulates gene expression. Quan-
titative RT-PCR was used to determine whether gene expres-
sion of either the wild-type oxidoreductase or the wild-type
transcriptional regulator was regulated by the presence of cer-
cosporin. Results were compared to data for expression in cells
grown in LB medium and in LB medium with 0.5% acetone,
used to solubilize cercosporin. Expression of the two genes in
the acetone control was increased by approximately three- to
fourfold over the untreated control (Fig. 5). When treated with
cercosporin, expression of the transcriptional regulator and
oxidoreductase were strongly upregulated, 17-fold and 136-
fold, respectively, compared to the untreated control.
Southern analysis of oxidoreductase homologues in cer-
cosporin-degrading and non-cercosporin-degrading species.
A series of cercosporin-degrading bacteria (XCZ-3, XAP-50,
XAP-75, XAP-1, XAP-39, XAP-49, XAP-34, XAP-31, and
XAP-79) and non-cercosporin-degrading bacteria (XAP-76,
XAV-79, XAV-126, P. syringae pv. tomato DC3000, and P.
syringae pv. pisi 209-21-3R) was assayed by Southern analysis
for the presence of oxidoreductase homologues using a digoxi-
genin-labeled probe amplified from wild-type XCZ-3 oxi-
FIG. 4. Sequence containing the putative transcriptional regulator and oxidoreductase genes of XCZ-3 (GenBank accession no. DQ087176).
The dashed line represents the transcriptional regulator, and the solid line represents the oxidoreductase. The dark triangles pointing to specific
nucleotides in boldface type show the location of each of the mutations. The shaded area shows the sequence that is homologous to MarR genes
and that potentially contains a helix-turn-helix motif. The underlined codons in boldface type are possible alternative start sites that are in frame
with the transcriptional regulator. Sites of primers used for amplification are also shown. Nucleotide and amino acid changes resulting from the
mutations are as follows: 120-2, T3C and start codon3threonine; MutA, G3A and tryptophan3stop; MutB, MutC, and MutD, all G3A and
glycine3aspartic acid. Changes in secondary structure are predicted only for MutB (NNPREDICT analysis).
6074 TAYLOR ET AL.APPL. ENVIRON. MICROBIOL.
doreductase (Fig. 6). All of the cercosporin-degrading isolates
were found to contain a sequence that hybridized to the oxi-
doreductase probe. With the exception of P. syringae pv. to-
mato DC3000, all of the non-cercosporin-degrading isolates
lacked a detectable oxidoreductase homologue. Although P.
syringae pv. tomato DC300 was unable to degrade cercosporin,
Southern analysis detected a band that hybridized to the
XCZ-3 oxidoreductase probe. A subsequent NCBI BLAST
search identified a similar sequence (GenBank accession no.
NP_790149; E value ? e?85) (6).
The transcriptional regulator and oxidoreductase are not
sufficient for cercosporin degradation in non-cercosporin-de-
grading species. To determine if the presence of the transcrip-
tional regulator and oxidoreductase genes alone is sufficient for
cercosporin degradation, non-cercosporin-degrading bacteria
were conjugated with the 23-kb complementing library clone
(in pLAFR6) containing the wild-type transcriptional regula-
tor and oxidoreductase genes. Quantitative RT-PCR was used
to assess oxidoreductase gene expression in each of the
transconjugants upon exposure to cercosporin. Results showed
that the oxidoreductase was expressed in all of the transcon-
jugants and appeared to be regulated similarly to XCZ-3, with
increased expression upon treatment with cercosporin (Fig. 7).
P. syringae pv. tomato DC3000 transconjugants, in particular,
showed high levels of expression, equal to or greater than that
in wild-type strain XCZ-3.
Although the oxidoreductase was expressed in the transcon-
jugants, cercosporin degradation assays in both solid and liquid
media failed to detect cercosporin degradation by the oxi-
doreductase-expressing transconjugants. A time course assay
FIG. 5. Quantitative RT-PCR analysis of expression of the transcriptional regulator and oxidoreductase genes in XCZ-3 following exposure of
the bacterium to cercosporin (CE). Numbers above the bars are increases in expression (n-fold) compared to the untreated control, which was
normalized to 1. The acetone control (Acetone Ctrl) contained 0.5% acetone, which was used to solubilize cercosporin.
FIG. 6. Southern hybridization of total DNA isolated from cercosporin-degrading (XCZ-3, XAP-50, XAP-75, XAP-1, XAP-39, XAP-49,
XAP-34, XAP-31, and XAP-79) and non-cercosporin-degrading (XAP-76, XAV-79, XAV-126, P. syringae pv. tomato DC3000, and P. syringae pv.
pisi 209-21-3R) isolates using a PCR-amplified digoxigenin-labeled oxidoreductase gene as a probe. Wild-type strain XCZ-3 was used in lane 1 as
a positive control. Sequences homologous to the oxidoreductase are present in all cercosporin-degrading isolates and in the non-cercosporin-
degrading isolate P. syringae pv. tomato DC3000.
VOL. 72, 2006CERCOSPORIN DEGRADATION BY X. CAMPESTRIS PV. ZINNIAE 6075
of cercosporin degradation in liquid medium is shown in Fig. 8.
Some variability is evident in readings at 0 h due to the inability
to completely extract cercosporin from the bacteria. Levels of
cercosporin in LB medium alone remained constant over the
48 h of the experiment. Incubation of cercosporin with the
wild-type control, XCZ-3, and the complemented mutant,
MutA ? p6-23kb, resulted in an almost complete loss of cer-
cosporin by 48 h. By contrast, incubation of medium containing
cercosporin with MutA and the non-cercosporin-degrading
isolates XAV-79, XAV-126, and P. syringae pv. tomato
DC3000 resulted in only a small decrease in the apparent
cercosporin concentration, likely due to the uptake of cer-
cosporin into the cells and our inability to completely extract
the cercosporin from the cells. The transconjugants were un-
able to degrade cercosporin, showing no more loss of cer-
cosporin than their wild-type counterparts. To ensure that
other genes on the 23-kb clone were not interfering with deg-
radation, the pLAFR6 vector containing only the transcrip-
tional regulator and the oxidoreductase genes was conjugated
into the non-cercosporin-degrading bacteria. Cercosporin deg-
radation by these transconjugants was still not achieved (data
Many species in the genus Cercospora produce cercosporin
and rely on its toxicity to cause disease in their host plants.
Research efforts to avoid or limit the exposure of a host plant
to the toxin may be of great benefit in disease prevention. We
have chosen to focus on toxin degradation as a potential
method to achieve toxin and disease resistance. Previous re-
ports of toxin degradation by transgenic plants have shown that
this method can be successful in yielding disease resistance.
For example, transformation of sugarcane with a gene encod-
ing detoxification of the toxin albicidin provided protection
against infection by Xanthomonas albilineans, the causal agent
of leaf scald on susceptible sugarcane (48). Also, the Tri101
gene from Fusarium graminearum, which is responsible for
self-protection of this fungus from their trichothecene toxins,
provided protection against the trichothecene 4,15-diacetoxy-
scirpenol in transgenic tobacco (22).
In previous work in our laboratory, we identified bacteria
FIG. 7. Quantitative RT-PCR analysis of expression of oxidoreduc-
tase in transconjugants of the non-cercosporin-degrading bacteria
XAV-79, XAV-126, and P. syringae pv. tomato (PST) DC3000 com-
pared to the cercosporin (CE)-degrading strain XCZ-3. The figure
shows estimated total oxidoreductase transcripts in nontransformed
cells (wild type) compared to transconjugants (Transconj) grown with
and without cercosporin. Untreated indicates the 0.5% acetone control
(used to solubilize cercosporin). All transconjugants expressed oxido-
reductase and showed increased expression in response to cercosporin.
FIG. 8. Degradation of cercosporin in liquid culture by cer-
cosporin-degrading and non-cercosporin-degrading isolates and oxi-
doreductase-expressing transconjugants. Isolates tested were wild-type
strain XCZ-3, XCZ-3 non-cercosporin-degrading mutant MutA, wild-
type non-cercosporin-degrading isolates XAP-76, XAV-79, XAV-126,
and P. syringae pv. tomato (PST) DC3000, and the oxidoreductase-
expressing transconjugants (?p6-23kb) of XAV-79, XAV-126, P. sy-
ringae pv. tomato DC3000, and MutA. Cercosporin degradation was
assayed by measuring the absorbance at 472 nm after extraction of
cercosporin from culture filtrates at 0, 24, and 48 h as described in
Materials and Methods. Cercosporin in LB alone (Untreated) was
included as a control. Small decreases in apparent cercosporin con-
centrations with the non-cercosporin-degrading bacteria are due to the
uptake of cercosporin by bacteria in the medium. Only XCZ-3 and the
complemented MutA mutant were able to degrade cercosporin. Re-
sults shown are from one of two experiments performed. OD472, op-
tical density at 472 nm.
6076TAYLOR ET AL.APPL. ENVIRON. MICROBIOL.
that are capable of degrading cercosporin. Of 244 isolates
tested, the most efficient degraders were pathovars of X.
campestris that are pathogenic on zinnia. X. campestris pv.
zinniae isolates were shown to rapidly degrade cercosporin to
produce the nontoxic breakdown product xanosporic acid (30).
The degradation reaction was hypothesized to be caused by an
oxygen insertion into one of the quinoid rings adjacent to the
ketone carbonyl (29). The execution of this step would require
enzymatic activity, but the subsequent rearrangement that re-
sults in xanosporic acid may be spontaneous. We hypothesized
that cercosporin degradation is carried out by a single enzyme,
perhaps by cytochrome P450.
The goal of this work was to isolate the gene(s) required for
cercosporin degradation by X. campestris pv. zinniae. We iden-
tified two genes, encoding a putative transcriptional regulator
and an oxidoreductase, required for cercosporin resistance.
Homology searches showed that the transcriptional regula-
tor homologue has similarity to a transcriptional regulator
for cryptic hemolysin and a MarR transcriptional regulator.
The oxidoreductase homologue has similarity to an oxi-
doreductase and two monooxygenases. Although the regu-
lator and oxidoreductase are most similar to a region
present in X. axonopodis pv. citri, the function of these
homologous genes has not yet been experimentally con-
firmed in X. axonopodis pv. citri.
Sequence analysis of the region spanning the transcriptional
regulator and the oxidoreductase in the mutants identified a
single point mutation present in the oxidoreductase of each
mutant and no mutations in the transcriptional regulator. Our
studies suggest, however, that the transcriptional regulator
may play an important role in the regulation of oxidoreductase.
A construct containing the wild-type oxidoreductase gene plus
the intergenic region and ca. 100 bases of the regulator did not
complement the non-cercosporin-degrading mutants, whereas
a construct containing both the transcriptional regulator and
the oxidoreductase genes did complement the mutants. It is
not clear if the requirement is for the regulator or for upstream
regulatory sequences. Transformation of the mutants with a
construct containing the oxidoreductase gene and upstream
sequences spanning 330 bases into the transcriptional regula-
tor gene proved to be as efficient in complementing the mu-
tants as did the construct containing the intact regulator. This
construct did contain the active site of the transcriptional reg-
ulator as well as sequences encoding five possible start sites
that may allow the terminal portion of the regulator to be
translated in frame with the original sequence, resulting in the
expression of the potentially necessary active site. It is inter-
esting that we were unable to clone the oxidoreductase gene
alone into high-expression vectors; recovered clones were slow
to grow and died after only 5 days (data not shown). When the
gene for the transcriptional regulator was included upstream of
the oxidoreductase in constructs in the high-expression vectors,
however, clones that grew normally were readily recovered.
These results suggest that the transcriptional regulator plays a
critical role in regulating the expression of oxidoreductase.
Finally, our quantitative RT-PCR studies showed that both the
regulator and the oxidoreductase genes were upregulated after
exposure to cercosporin, supporting our hypothesis that ex-
pression of both genes is necessary to yield cercosporin deg-
The region containing both the transcriptional regulator and
the oxidoreductase was transformed into other non-cercosporin-
degrading bacteria in an attempt to determine if these genes
were sufficient for degradation. Quantitative RT-PCR analysis
confirmed that the oxidoreductase was expressed and was up-
regulated in the presence of cercosporin. However, the
transconjugants were unable to degrade cercosporin, indicat-
ing that the genes are not sufficient for cercosporin degrada-
tion in bacteria. Although we attempted to identify additional
genes involved in cercosporin degradation, the two newer mu-
tants, MutC and MutD, also contained a point mutation in the
oxidoreductase gene. These results suggest that if additional
genes are involved in the degradation process, mutations in
these genes may not be recoverable, perhaps due to lethality.
Interestingly, Southern analysis showed that all bacterial iso-
lates tested that are able to degrade cercosporin contain a
sequence that hybridizes to the XCZ-3 oxidoreductase gene
that was used as a probe. With one exception, the bacterial
isolates tested that are unable to degrade cercosporin do not
contain a detectable oxidoreductase homologue. The excep-
tion is P. syringae pv. tomato DC3000, which does not have the
ability to degrade cercosporin but does contain a sequence
homologous to the oxidoreductase. These results are consis-
tent with our hypothesis that additional genes may be neces-
sary for cercosporin degradation. Further study is needed to
identify additional genes. Once found, expression of these
genes in plants may provide a novel strategy for the control of
Cercospora spp. and the diseases that they cause.
We thank David Ritchie and Peter Lindgren (North Carolina State
University, Raleigh, NC) for providing the bacterial strains and Brian
Staskawicz (University of California, Berkeley, CA) for use of the
pLAFR6 vector. We also thank Frank Louws and Heriberto Velez
(North Carolina State University, Raleigh, NC) for assistance with the
Box-PCR and oxidoreductase expression experiments, respectively.
This work was supported in part by Pioneer Hi-Bred International,
Inc., and an NIH Biotechnology Training Grant fellowship.
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