DON Degrading Bacteria

RESEARCH LETTER
Thirteen novel deoxynivalenol-degrading bacteria are classified

within two genera with distinct degradation mechanisms
Ikuo Sato, Michihiro Ito, Masumi Ishizaka, Yoko Ikunaga, Yukari Sato, Shigenobu Yoshida,
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Motoo Koitabashi & Seiya Tsushima
National Institute for Agro-Environmental Sciences, Tsukuba, Japan
Correspondence: Seiya Tsushima, National Abstract

Institute for Agro-Environmental Sciences,
3-1-3 Kannondai, Tsukuba, Ibaraki 305-8604, The mycotoxin deoxynivalenol (DON), a secondary metabolite produced by
Japan. Tel./fax: +81 29 838 8351; species of the plant pathogen Fusarium, causes serious problems in cereal crop
e-mail: seya@affrc.go.jp production because of its toxicity towards humans and livestock. A biological
approach for the degradation of DON using a DON-degrading bacterium
Received 14 October 2011; revised 4 (DDB) appears to be promising, although information about DDBs is limited.
November 2011; accepted 14 November
We isolated 13 aerobic DDBs from a variety of environmental samples, includ-
2011. Final version published online 15
December 2011.
ing field soils and wheat leaves. Of these 13 strains, nine belonged to the
Gram-positive genus Nocardioides and other four to the Gram-negative genus
DOI: 10.1111/j.1574-6968.2011.02461.x Devosia. The degradation phenotypes of the two Gram types were clearly differ-
ent; all washed cells of the 13 strains degraded 100 lg mL 1 DON to below
Editor: J. Colin Murrell the detection limit (0.5 lg mL 1), but the conditions inducing the DON-
degrading activities differed between the two Gram types. The HPLC profiles
Keywords of the DON metabolites were also distinct between the two genera, although
Fusarium mycotoxin; trichothecenes; all strains produced 3-epi-deoxynivalenol. The Gram-positive strains showed
mycotoxin degradation; Nocardioides;
DON assimilation in media containing DON as a carbon source, whereas the
Devosia.
Gram-negatives did not. Our results suggest that aerobic DDBs are distributed
MICROBIOLOGY LETTERS
within at least two phylogenetically restricted genera, suggesting independent

evolution of the DON-degradation mechanisms.
DON have resulted in the establishment of legal limits

Introduction
ranging from 0.3 to 2.0 μg g−1 in several countries (Food
Several Fusarium species, mainly Fusarium graminearum, & Agriculture Organization, 2004).
infect many crops such as wheat and barley, and cause Although FHB is suppressed by fungicides and by the
Fusarium Head Blight (FHB) (Yoshizawa & Jin, 1995; use of resistant varieties, these measures do not reliably
Goswami & Kistler, 2004; Goswami et al., 2006; Yoshida & reduce DON levels to below legal limits. A biological
Nakajima, 2010). FHB induces not only reduction of crop method specific to the degradation of DON using microor-
yield but also accumulation of mycotoxins and results in ganisms could be a promising approach (Zhou et al., 2008;
huge economic losses (Windels, 2000). Deoxynivalenol He et al., 2010; Karlovsky, 2011). To date, several micro-
(3a,7a,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one; bial strains that degrade DON have been reported and their
DON; Fig. 1) is one of the most troublesome mycotoxins degradation products have been identified (Zhou et al.,
produced by FHB pathogens in crops. The main toxic 2008; He et al., 2010). It has been shown that DON reduc-
effect of DON at the cellular level in both humans and tion of the 12,13 epoxide group or its oxidation of the
livestock is the inhibition of protein synthesis by binding hydroxyl group on carbon 3 by the microbial strains cause
to the ribosome, and DON ingestion leads to weight loss, the decreased toxicity (Shima et al., 1997; Ericksen et al.,
feed refusal and vomiting (Ehrlich & Daigle, 1987; Middle- 2004; Karlovsky, 2011). The anaerobic bacterium Eubac-
brook and Leatherman, 1989a, b; Rotter et al., 1996; terium sp. strain BBSH797 was isolated from bovine
Pestka, 2010). The toxicity and frequent occurrence of rumen fluid and was reported to transform DON into
ª 2011 Federation of European Microbiological Societies FEMS Microbiol Lett 327 (2012) 110–117
Published by Blackwell Publishing Ltd. All rights reserved
Aerobic deoxynivalenol-degrading bacteria 111
* niacin, 5 mg Ca-pantothenate, 5 mg p-aminobenzoate and

* * * 5 mg thioctic acid per litre), and the indicated amounts of
* % 16
1
10 1 DON as a carbon source. Nutrient agar (NA; Difco, Grand
9 11
13
2 3 1*
1 Island, NY), 100-fold-diluted NA and R2A (Merck KGaA,
6
8
12 Darmstadt, Germany) agar plates were used for the iso-
1 7 15 4 * lation of DDBs. Three-fold-diluted R2A (1/3R2A) agar
5
%* plates were used for the precultures and colony counting
1* %* *

1* 14 of strains SS1, SS2, SS3, SS4, LS1, LS2, NKK1, NKJ1,
YUL1, YMN1, PFS1 and WSN05-2, while three-fold-
Fig. 1. Structure of deoxynivalenol (3a,7a,15-trihydroxy-12,13-epoxytri-
diluted Luria–Bertani (1/3LB; Difco) agar plates were used
chothec-9-en-8-one).
for strains SS5 and RS1.
de-epoxydized DON (Fuchs et al., 2002). In addition, Yu

Enrichment and isolation of bacteria from
et al. (2010) isolated 10 anaerobic bacteria from chicken
environmental samples
intestines, and each of these bacteria converted DON to
de-epoxy DON. Regarding aerobic microorganisms, one For the isolation of DDBs, samples were collected from
fungus and two bacteria have been isolated thus far. Shima the environment including wheat field soil, paddy field
et al. (1997) isolated the Gram-negative bacterial strain soil, uncultivated soil (at a shrine), and wheat leaves and
E3-39 from a soil sample, which was shown to metabolize wheat spikelets at the National Institute for Agro-Envi-
DON aerobically into 3-keto-4-deoxynivalenol. The fungus ronmental Sciences, Tsukuba, Ibaraki, Japan. Approxi-
Aspergillus tubingensis NJA-1 has been demonstrated to mately 0.1 g of the screening samples was suspended in
degrade DON, and an unidentified metabolite, which was 1 mL MM containing 100 lg mL 1 DON as sole carbon
postulated to be a hydrolysed product of DON, was found source, and the cultures were incubated with shaking at
in the culture medium (He et al., 2008). Ikunaga et al. 120 r.p.m. and 28 °C for 7 days. Then, 10 lL of these
(2011) isolated the DON-degrading and DON-assimilating cultures was added to 1 mL of the same media and sub-
bacterium Nocardioides sp. WSN05-2 and identified a jected to 7 days of incubation under the same conditions.
novel metabolite, 3-epi-deoxynivalenol (3-epi-DON). As This procedure was repeated two or more times. The
compared with reports on the isolation and degradation concentrations of DON in the culture media were moni-
mechanisms of anaerobic DON-degrading bacteria tored by HPLC as described below. Culture samples with
(DDBs), those of aerobic DDBs have been fewer. Here, we decreasing DON concentrations were selected, serially
provide for the first time a comprehensive phylogenetic diluted in sterile distilled water and plated on R2A agar,
and phenotypic analysis of aerobic DDBs. NA or 100-fold-diluted NA plates. The resulting plates
were incubated at 28 °C for 7 days. Randomly selected
bacterial colonies, approximately 107–108 cells, were sus-
Materials and methods
pended in 50 lL MM with 100 lg mL 1 DON, incubated
at 28 °C for 5 days and analysed for DON-degrading
Chemicals and media
ability using HPLC. DDBs were selected and stored in
DON was prepared as described by Clifford et al. (2003) 10% glycerol at 80 °C until use. The strains (accession
with the following modifications: F. graminearum H3 numbers MAFF311601 to MAFF311613) have been
(MAFF101551) was used as the DON producer and Wako- deposited at the Microorganisms Section (MAFF) of the
gel C-200 (Wako, Tokyo, Japan) was used for the purifica- NIAS Genebank, National Institute of Agrobiological Sci-
tion of DON. Preparation of 3-epi-DON was as described ences, Tsukuba, Ibaraki, Japan.
by Ikunaga et al. (2011). Mineral salt medium (MM) of
Kirimura et al. (1999) was employed with slight modifica-
Taxonomic characterization of the isolated
tion: the medium contained (L−1) 1.6 g Na2HPO4, 1 g
bacteria
KH2PO4, 0.5 g MgSO4·7H2O, 0.5 g NaNO3, 0.5 g
(NH4)2SO4, 0.025 g CaCl2·2H2O, 2 mL trace metal solu- The DDBs were characterized by Gram staining and the
tion (1.5 g FeCl2·4H2O, 0.190 g CoCl2·6H2O, 0.1 g 16S rRNA genes were analysed as described by Ikunaga
MnCl2·4H2O, 0.07 g ZnCl2, 0.062 g H3BO3, 0.036 g et al. (2011). This yielded approximately 1200 bp of use-
Na2MoO4·2H2O, 0.024 g NiCl2·6H2O and 0.017 g ful 16S rRNA gene sequence. Sequences similar to the
CuCl2·2H2O per litre), 1 mL vitamin solution (2 mg 16S rRNA gene of isolated strains were identified using
biotin, 2 mg folic acid, 5 mg thiamine–HCl, 5 mg ribofla- BLAST searches (National Center for Biotechnology Infor-
vin, 10 mg pyridoxine–HCl, 50 mg cyanocobalamin, 5 mg mation, http://www.ncbi.nlm.nih.gov). The 16S rRNA
FEMS Microbiol Lett 327 (2012) 110–117 ª 2011 Federation of European Microbiological Societies
112 I. Sato et al.
gene sequences determined in this study have been depos- Japan). Significant differences between the means were
ited in the DNA Data Bank of Japan (DDBJ) under acces- determined by using a t-test and Tukey test variance anal-
sion numbers AB627753 to AB627765. ysis (P < 0.05).
HPLC analysis Results
Culture samples were filtered through 0.45-lm membranes
Isolation of DDBs and taxonomic
(Advantec, Tokyo, Japan) and 10 lL was directly injected
characterization
on to an HPLC system. The HPLC system (Waters, Mil-
ford, MA) consisted of a 600E pump, a 2487 dual absor- A total of 169 environmental samples (61 soil, 78 wheat
bance detector, a Waters Symmetry C18 column (3.9 mm leaf and 30 wheat spikelet samples) were used for enrich-
ID 9 150 mm; Waters) and EMPOWER 2 software. The ment culture. Thirteen samples were found to have
mobile phase contained methanol and water (15:85, v/v) decreased DON in the media, two of which were leaf
at a flow rate of 1.0 mL min 1. A wavelength of 220 nm samples and the other 11 of which were soil samples.
was used. The column was heated to 40 °C. Authentic Thirteen DDBs were isolated from every enrichment cul-
DON and 3-epi-DON were detected at retention times of ture using the R2A agar or 100-fold-diluted NA plates.
6.5 and 4.5 min, respectively. Gram staining revealed that nine strains were Gram-
positive and four were Gram-negative. The bacterial 16S
rRNA genes were analysed and the results are summa-
Characterization of DON-degrading activity in
rized in Table 1. Phylogenetic analysis was performed by
washed cells
constructing neighbour-joining trees. As shown in Fig. 2a,
The DDBs were cultured on the agar plates at 28 °C for the Gram-positive strains (SS1, SS2, SS3, SS4, LS1, LS2,
5 days. These bacteria (OD600 nm of 0.2) were then preincu- YMN1, YUL1, PFS1) were closely related to the genus
bated in DON mineral medium (DMM, MM containing Nocardioides in the family Nocardioidaceae, forming four
20 lg mL 1 DON), 1/3R2A and 1/3LB liquid media at clusters. Levels of 16S rRNA gene sequence similarity ran-
28 °C for 24 h. After incubation, bacterial cells were recov- ged from 92% to 100%. The Gram-negative strains (SS5,
ered by centrifugation at 6000 g for 10 min, washed twice RS1, NKK1, NKJ1) were closely related to the genus Dev-
with 50 mM potassium phosphate buffer (pH 7.0), and osia in the family Hyphomicrobiaceae, forming two clus-
resuspended in the same buffer containing 100 lg mL 1 ters, and their 16S rRNA gene sequence similarities
DON to achieve an OD600 nm of 0.8 (equivalent to 1.3 mg ranged from 95% to 100%.
dry weight mL 1). Samples were incubated at 25 °C, col-
lected at various time points, filtered and subjected to
Characterization of DON-degradation
HPLC. Initial DON degradation rates were measured
phenotypes
within the period of linear DON degradation. Three buf-
fers, noninoculated cells containing DON, inoculated cells The initial DON degradation rates using the washed
without DON and inoculated autoclaved cells (121 °C, cells of the strains preincubated with DMM, 1/3LB and
20 min) containing DON, were also analysed as controls. 1/3R2A were examined (Table 1). All of the strains pre-
incubated with DMM showed DON-degrading activities,
and degraded 100 lg mL 1 of DON to below the detec-
Cell growth experiment
tion limit (0.5 lg mL 1) after the 24 h of incubation.
Cells were inoculated into MM with or without 100 Among the strains, SS5 and RS1 showed high rates of
lg mL 1 DON and incubated at 120 r.p.m. and 28 °C DON degradation, which were more than three times
after precultivating on the agar plates at 28 °C for 5 days. those of the other strains. Although strains NKK1 and
Culture media were collected every other day, appropri- NKJ1 were closely related to strains SS5 and RS1, the
ately diluted and spread onto 1/3LB agar plates for strains degradation rates were lower. Strains SS5, RS1 and NKJ1
SS5 and RS1, and onto 1/3R2A agar plates for the other expressed DON-degrading activities regardless of the
strains. These agar plates were incubated at 28 °C for preincubation media used. Preincubation with 1/3LB
7 days, and the numbers of colonies were counted. enhanced the DON-degrading activities of strains SS5 and
RS1, but repressed that of NKK1. These results provided
insight into the diversity of DON-degradation phenotypes
Statistical methods
within closely related strains. Meanwhile, all of the Gram-
Statistical analysis of the data was carried out using JMP positive strains exhibited high DON-degrading activities
software (version 5.01J; SAS Institute Japan, Tokyo, by preincubation with DMM, although they exhibited
(a)
(b)
Fig. 2. Phylogenetic trees based on partial 16S rRNA gene sequences of strains belonging to Gram-positive (a) and Gram-negative (b) bacteria
and related organisms. Bars indicate 1% sequence divergence. GenBank accession numbers are shown in parentheses. The accession numbers of
DDBs isolated in this study are as follow: AB627753, SS1; AB627754, SS2; AB627755, SS3; AB627756, SS4; AB627757, SS5; AB627758, RS1;
AB627759, LS1; AB627760, LS2; AB627761, NKK1; AB627762, NKJ1; AB627763, YUL1; AB627764, YMN1; AB627765, PFS1.
very low activities by preincubation with 1/3R2A or 1/3LB. Figure 3a and b show the time course of DON degra-
That the buffer with autoclaved cells did not decrease the dation, and HPLC elution profiles of DON and its
concentration of DON and that the buffer filtrates during metabolites in washed cells of representative strains LS1,
DON degradation also did not (data not shown) indicate SS5 and these autoclaved strains. The profiles of the two
that the decrease of DON is attributed to the enzymatic strains showed at least three peaks in addition to the
reactions catalysed in the living cells. DON peak (6.5 min); one peak corresponded to the peak
114 I. Sato et al.
Table 1. Characterization of aerobic DDBs
Initial DON-degradation rate of strains preincubated

with indicated media (lg h 1 mg dry cells 1)*
Source and Closely related species
Strain year of isolation (% 16S rRNA gene sequence similarity) DMM† 1/3LB 1/3R2A
‡
WSN05-2 Soil, 2005 Nocardioides sp. AN3 (100) 18 ± 1.1CDEa
< 1.0b
< 1.0b
SS1 Soil, 2006 Nocardioides sp. WSN05-2 (99) 22 ± 1.7Ca < 1.0c 4.7 ± 0.46b
SS2 Soil, 2006 Nocardioides mesophilus MSL-22 (97) 5.3 ± 0.30Fa < 1.0b < 1.0b
SS3 Soil, 2006 Nocardioides sp. WSN05-2 (100) 19 ± 0.58CDa < 1.0b < 1.0b
SS4 Soil, 2006 Nocardioides ginsengisegetis Gsoil 485 (100) 15 ± 1.0CDEFa < 1.0c 2.0 ± 0.21b
SS5 Soil, 2006 Devosia sp. 4_C16_46 (96) 70 ± 8.1Ba 150 ± 14b 15 ± 1.2c
RS1 Soil, 2006 Devosia sp. 4_C16_46 (96) 150 ± 10Ab 270 ± 20a 140 ± 8.0b
LS1 Wheat leaf, 2006 Nocardioides ginsengisegetis Gsoil 485 (100) 15 ± 0.50CDEFa < 1.0c 2.4 ± 0.24b
LS2 Wheat leaf, 2006 Nocardioides ginsengisegetis Gsoil 485 (100) 14 ± 0.40CDEFa < 1.0c 1.9 ± 0.15b
NKK1 Soil, 2008 Devosia ginsengisoli Gsoil 326 (99) 7.3 ± 0.47EFa < 1.0c 13 ± 1.0b
NKJ1 Soil, 2009 Devosia ginsengisoli Gsoil 326 (99) 8.6 ± 0.85DEFb 12 ± 1.2a 12 ± 1.0a
YUL1 Soil, 2009 Nocardioides sp. WSN05-2 (99) 22 ± 2.0Ca < 1.0c 5.2 ± 0.23b
YMN1 Soil, 2009 Nocardioides sp. WSN05-2 (100) 17 ± 1.2CDEa < 1.0b < 1.0b
PFS1 Soil, 2010 Nocardioides sp. WSN05-2 (98) 21 ± 2.1Ca < 1.0c 4.1 ± 0.32b
*The experiment was conducted twice, and similar results were obtained. One representative result is shown. Data are mean ± SD of triplicates.
The same capital letters within the DMM column are not significantly different (P < 0.05). The same lower case letters within each same line are
not significantly different (P < 0.05).
†
Cells autoclaved after preincubation in DMM were also analysed and all the cells exhibited no DON degradation (< 1.0 lg h 1 mg dry cells 1).
‡
WSN05-2 was previously isolated by Ikunaga et al. (2011).
in the authentic standards of 3-epi-DON (4.5 min), indi- carbon source. The degradation products shown in Fig. 3
cating that both strains produced 3-epi-DON. The HPLC were detected during the growth of the Norcardioides
elution profiles also revealed unidentified peaks at 3.0 strains in MM with DON (Fig. 4), suggesting that the
and 6.9 min in the RS1 sample, and at 1.6 and 4.8 min strains assimilate DON through the repeated release and
in the LS1 sample. These peaks were not detected when intake of DON and its metabolites.
DDBs were autoclaved or were incubated without DON
(Fig. 3c), indicating that these peaks were the products
Discussion
derived from DON. Similar results were obtained from
the other Gram-positive and Gram-negative strains (data Only two bacterial aerobic DDBs (strains WSN05-2 and
not shown). These results indicated that the Gram-nega- E3-39) had been reported previously, with WSN05-2
tive and Gram-positive strains produce different DON belonging to the genus Nocardioides and E3-39 being of
metabolites other than 3-epi-DON. the Agrobacterium–Rhizobium group (Shima et al., 1997).
The time-dependent change in cell growth and the However, the present 16S rRNA gene sequence analyses
DON-degradation capabilities of the strains inoculated in revealed that E3-39 is most closely related to Devosia sp.
MM media containing 100 lg mL 1 DON (Fig. 4) were 4_C16_46. Thus, all aerobic DDBs reported to date are
examined. Growth of each Gram-positive strain (WSN05-2, closely related to the genera Nocardioides and Devosia
LS1, SS1, SS2) on DON was enhanced compared with only. By contrast, all previously reported anaerobic DDBs
that without DON and was accompanied by a decrease of were Gram-positive and encompassed a variety of genera
DON level. After at least 6 days of incubation, the con- (Eubacteria, Anaerofilum, Collinsella, Bacillus) and the
centrations of DON in the media with these strains were order Clostridiales (Yu et al., 2010). These results high-
below the detection limit. Growth promotion and DON light the clear phylogenetic differences between aerobic
degradation of the other Gram-positive strains after and anaerobic DDBs.
6 days of incubation were observed (data not shown). In We also characterized the DON-degradation pheno-
contrast, the Gram-negative bacterium RS1 showed nei- types of the aerobic strains in this study, identifying three
ther growth promotion nor declining DON concentra- key differences between the Gram-positive and Gram-
tions during the 8 days of incubation. Similar results negative strains. First, there is an obvious difference in the
were obtained for the other Gram-negative strains (data DON-assimilating abilities, as only the Gram-positive
not shown). These results indicated that only the Gram- strains utilized DON as the carbon source. To our
positive strains are capable of assimilating DON as the knowledge, DON-assimilating bacteria are limited to the
Fig. 3. DON-degradation by washed cells of strains LS1 and RS1. (a) Time course of DON-degradation by the washed cells of LS1 (upper panel)
and RS1 (lower panel) incubated in 50 mM potassium phosphate buffer with (●) or without (○) 100 lg mL 1 DON after preincubation in DMM.
Data points at each incubation time marked with an asterisk are significantly different according to t-test (P < 0.05). (b) HPLC profiles of DON and
DON metabolites in buffer containing DON with LS1 (upper panel) and with RS1 (middle panel) or without either (lower panel) at various
incubation times. Incubation times are indicated in the upper right of each HPLC profile. DON (grey bar) and 3-epi-DON (dashed grey bar) are
identified by the retention times of authentic standards. Unidentified major peaks detected in the samples of washed cells are indicated by arrows.
A representative result from three independent experiments is shown. (c) HPLC profiles of the washed cells incubated in buffer without DON.
Gram-positive bacteria that we isolated. On the other The Gram-positive strains needed preincubating in DON-
hand, it is interesting that the Gram-negative strains containing media for maximal expression of degradation
exhibited no DON-assimilating abilities even though they activities, suggesting that they possess some regulatory
were isolated using the enrichment culture with DON as system for the expression of DON-degrading enzyme or
the carbon source. This result might imply that the micro- DON-uptake machineries. Together with the finding that
bial consortia, composed of both Gram-negative DDBs Gram-positive strains can assimilate DON, we postulate
and other microorganisms, performed cooperative catabo- that the Gram-positive strains are native DON-degraders
lism of DON in the enrichment culture media. We whose DON-assimilating abilities play a key role in their
assume that the Gram-negative DDBs were responsible for survival in nature. By contrast, the Gram-negative strains
the initial DON-degradation steps while the other micro- might be casual degraders, given that they did not assimi-
organisms degraded the DON-derived products. Such late DON or need preincubating in DON-containing
cooperative catabolism has been reported for the micro- media for expression of DON-degrading activities. Note
bial degradation of chloronitrobenzenes and atrazine that it was not on mineral media containing DON as car-
(Park et al., 2002; Smith et al., 2005). Further analysis of bon source but on complete media such as diluted NA
the enrichment culture media in this study could lead to and R2A agar plates that we isolated DDBs. Use of the
the isolation of novel microorganisms that promoted complete media in our study resulted in the successful
metabolism in the latter half of the DON-degradation isolation of the casual DON-degraders.
pathway. The third difference is the DON metabolites produced.
The second difference is the ability to express DON- HPLC analysis revealed that the two Gram types pro-
degradation activities under preincubation conditions. duced different DON metabolites, suggesting differences
116 I. Sato et al.
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DON Degrading Bacteria

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DON Degrading Bacteria

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RESEARCH LETTER

Thirteen novel deoxynivalenol-degrading bacteria are classified

Correspondence: Seiya Tsushima, National Abstract

within at least two phylogenetically restricted genera, suggesting independent

DON have resulted in the establishment of legal limits

* niacin, 5 mg Ca-pantothenate, 5 mg p-aminobenzoate and

de-epoxydized DON (Fuchs et al., 2002). In addition, Yu

HPLC analysis Results

Table 1. Characterization of aerobic DDBs

Initial DON-degradation rate of strains preincubated

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