J.Pesticide Sci.25, 357-364 (2000)

Original Article

Isolation of Pentoxazone-Transforming Microorganisms from Soil:

Their Characteristics and Metabolites

Kouji SATSUMA, Osamu HAYASHI, Kiyoshi SATO, Kazutoshi OHYAMA,

Shin-ichi MAKI, Motoo HASHIMURA and Yasuhiro KATO

Chemistry Division, The Institute of Environmental Toxicology, Uchimoriya-machi 4321, Mitsukaido, Ibaraki 303-0043, Japan

Kaken Pharmaceutical Co., Ltd., 28-8, Honkomagome 2-Chome, Bunkyo-ku, Tokyo 113-8650, Japan

(Received June 9, 1999; Accepted June 15, 2000)

Five microbial strains which have pentoxazone [3-(4-chloro-5-cyclopentyloxy-2-fluorophenyl)-5-isopropylidene-l, 3-oxazolidine-2, 4-dione]-transforming abilities were isolated from Ushi-ku paddy field soil.These strains, designated as 1B, 2B, 7B, 21B and 9A, have been tentatively identified by their morphological and physiological characteristics: Pseudomonas fluorescens, Bradyrhizobium

japonicum, two pathovars of Xanthomonas oryzae and Bacillus sp., respectively.These isolates favored or tolerated the lower oxygen concentration than air though they belong to aerobes.These isolates partly modified pentoxazone molecule to afford five different metabolites.Strains lB and 2B produced A-0505 [N-(4-chloro-5-cyclopentyloxy-2-fluorophenyl)-2-oxo-3-methylbutanamide]. Strain(s) 7B and 2l B produced, respectively, A-0480 (4-chloro-5-cyclopentyloxy-2-fluoroaniline) and A-1374 [N-(4-chloro-5-cyclopentyloxy-2-fluorophenyl)-2-hydroxy-3-methylbutanamide] after their transient accumulation of A-0505.Strain 9A produced two monohydroxylated compounds on cyclopentyl moiety: hydroxylated-pentoxazone and hydroxylated-A-0505.Strains 7B, 21B and 9A were able to transform A-0505 when it was provided as the initial substrate.This indicates that A-0505 could be a metabolic intermediate of these strains.

Key words: pentoxazone, soil microorganisms, isolated strains, pour plate, spread plate, cometabolism.

INTRODUCTION

Microbes in soil/water play an important role in degradation of xenobiotics released into the terrestrial and aquatic environment.Many microbial strains which individually mineralize xenobiotics as sole carbon or energy sources have been isolated.Population of such intensive degraders, however, is considered to be very scarce in natural soil.A selective and enrichment

procedure is thus necessary for isolation of these microbes.1-4) Mineralization has been observed in many cases of a soil without any possible history of

previous pesticide input.5-8) Pre-existing enzymes of indigenous microbes are likely to contribute to the degra-dation of recalcitrant xenobiotics such as synthetic organic pesticides.Our previous studies demonstrated that pentoxazone was mineralized in soils9) and rapidly transformed in diluted liquid medium inoculated with

paddy field soils as microbial sources.10) The ability to degrade pentoxazone is considered to be widely distribut-ed among ubiquitous soil microorganisms.In order to understand the system of the biodegradation of pentox-azone in detail, the soil microorganisms which metabol-ize pentoxazone were screened.The isolates were tax-onomically characterized and examined for their metabolites to clarify the catalytic feature.

MATERIALS AND METHODS

1.Chemicals Pentoxazone, labeled with and without 14C in the

phenyl ring were prepared as described in our previous paper.9) Reference standards of putative metabolites (A-0505, A-1374, A-1168, A-0480 and A-1814 [3-[4-chloro-5-(3-hydroxycyclopentyloxy)-2-fluorophenyl] -5-isopropylidene-l, 3-oxazolidine-2, 4-dione]) were synthe-sized by Sagami Chemical Research Center.Radiolabeled A-0505 (1222.2 MBq/mmol) was prepared by hydrolysis of [14C] pentoxazone (1132.2 MBq/mmol) in borate buffer solution (pH 9).11) Nutrient broth

* To whom correspondence should be addressed.1 Microbial Metabolism of Herbicide Pentoxazone (part 3),

See Ref.9 and 10.

358 日本 農薬 学会 誌 第25巻 第4号 平 成12年11月

medium was purchased from Difco Laboratories and agar (bacteriological) was purchased from Wako Pure Chemical Co, Ltd. All other chemicals used were of reagent grade. Pure water was prepared by a Milli-QTM Pure Water Preparation System (Japan Milli-pore Co, Ltd. ).

2. Media The nutrient broth and the 100-fold diluted nutrient

broth were prepared for liquid media as described in our

previous paper. 10) For the solid medium, agar was added to the broth (nutrient agar or diluted nutrient agar) at the concentration of 1. 2% followed by autoclav-ing at 121C. When necessary, labeled or unlabeled

pentoxazone was added at the concentration of 0. 5 jig/ml after autoclaving and cooling.

3. Isolation of Microorganisms The soil sample was collected from the experimental

paddy field at Research Station of the Japan Association for Advancement Phyto-Regulators (Ushiku-City, Ibaraki-Pref. ). This soil was passed through a 5-mm sieve and stored at 5C in the dark prior to use. The soil was taken into a 200-ml glass vessel and was incubated under upland condition at 25C for about two weeks. A

portion of the soil equivalent to 2. 00 g on dry weight basis (2. 82 g of wet weight) was taken into a test tube, added with 7. 18 ml of sterile pure water and shaken vigorously for 30 min. The mixture was then centrifuged at 3500 rpm for 10 min. The supernatant (100 p1) was then transferred into 10 ml of the diluted nutrient broth in the test tube with a stainless steal cap. The medium was serially diluted 100-times with sterile

pure water after 12 days of cultivation at 25C with gentle shaking. Each diluted medium (0. 5 ml) was then spread on the diluted nutrient agar plate (spread plate tech-nique) or mixed with still unsolidified (about 45C) diluted nutrient agar (pour plate technique). Both agar

plates were containing unlabeled pentoxazone. These inoculated agar plates were cultured at 25C for 9 days. Each single colony was then picked up randomly and reincubated in the diluted nutrient broth containing

[14C]pentoxazone of 0. 5 u g/ml. Then the microbial strains which degrade pentoxazone were selected by comparing degradation profiles in inoculated liquid media with non-inoculated control. The selected de-

graders were maintained by subculture at two weeks intervals with unlabeled pentoxazone.

4. Identification of Isolates The isolated strains were characterized using "Bergey's

Manual of Systematic Bacteriology. "12) Most examina-tions for characterization were performed according to conventional method. 13> Characterization was also con-ducted by a Biolog identification system (Biolog Inc.,

Hayward, CA). With respect to strain 2B and 9A, further identification was conducted by NCIMB Japan Co., Ltd.

5. Inoculation and Incubation The diluted nutrient broth containing [14C] pentox-

azone or [14C] A-0505 was used to determine the metabolites of each isolate. The isolates grown on the diluted nutrient broth containing unlabeled pentoxazone for 7 days were transferred (10 p1) into the broth contain-ing 14C-labeled compounds. Each inoculated broth was then incubated at 25C with gentle shaking in the dark. At appropriate intervals, the incubated broth was taken out and determined radioactive metabolites.

6. Analytical Method Each incubated sample described above was loaded

onto a C18 cartridge column. The column was succes-sively eluted with 20-ml portion of ethyl acetate and methanol. Both eluates were combined and concen-trated by rotary-evaporator. The concentrated organic eluate was subjected to TLC for separation and

quantification of [14C] 4pentoxazone and its radioactive metabolites. A pre-coated silica gel 60F254 on alumi-num plate (Merck) was used. Each sample was devel-oped by two different solvent systems (one-dimensional development). Radioactive components were detected by a radio-TLC-analyzer system (RAYTEST). Distri-bution of the radioactivity on TLC chromatogram was determined by the integration of the area of each peak.

7. Identification of the Metabolites Two metabolites produced by strain 9A (9AM 1 and

9AM2) were characterized by LC/MS and LC-MS/MS analyses. Mass spectrometer (Thermoquest TSQ-700) was connected to the outlet of HPLC system used in our

previous study9) through an electrospray ionization (ESI) interface. The operating condition of the mass spectrometer were as follows: capillary temperature

(250C), spray voltage (4. 5 kV) and multiplier voltage (1000 V). The product ion spectra were available by collision induced dissociation (CID, 1. 2 mTorr argon, -25 eV) with multiplier voltage at 1200 V. The other

radioactive metabolites were identified by TLC co-chromatography as described in our previous paper. 9)

RESULTS

1. Morphological Observations of the Isolates Five microbial strains with pentoxazone-transforming

ability were isolated. The isolates except 9A were Gram-negative, non-sporeforming rods. The isolates 1 B and 2B were motile by several (l B) or polar (2B) flagel-lum. The flagellum of the isolates 7B and 21B could not be observed. The motility of 21 B, however, was recog-nized by a hanging drop method. The isolate 2l B

Journal of Pesticide Science 25 (4) November 2000 359

shows unique characteristics compared with other bacte-rial strains isolated: small cell size and generation time twice longer than those of others. The isolate 9A was Gram-positive, endospore-forming

long rods, moderately motile by peritrichous flagella.When the strain grew in broth media their cells settled on the bottom of the tube and tended to become fragmented into rods.On the agar plate, cells occured singly or in chains of considerable length, so that mycelium-like

growth was observed. These morphological and some physiological charac-

teristics examined by conventional method are summar-ized in Table 1, as basic information of the isolates.

2.Identification of the Isolates Strains 1B, 7B and 2l B were tentatively identified by

comparing their utilization patterns of various carbon sources with the Biolog strain database: Pseudomonas

fluorescens, and two pathovars of Xanthomonas oryzae, respectively.The similarity indices of three strains were 0.747, 0.614 and 0.825, respectively.Strains 2B and 9A were examined on the base of the substrate availability including various saccharide and organic acids at NCIMB Japan Co., Ltd.The strains were finally classified into genus Bradyrhizobium and Bacillus, respectively.Since only one species of Bradyrhizobium is presently recognized, strain 2B was tentatively identified as Bradyrhizobium japonicum.Strain 9A

possesses common characteristics of B.cereus and B.thuringiensis.Further examination will be needed to clarify which species the isolated Bacillus belongs to.

3.Identification of Unknown Products Fig.1 illustrates the mass spectrum and product ion

(PI) spectrum from quasi-molecular ion (QM-) of 9AM 1.The QM- at m/z 368 was 16 amu greater than that of pentoxazone (m/z 352).The both spectra were similar to those of A-1814, 3-[4-chloro-5-(3-hydroxycyclopentyloxy)-2-fluorophenyl] -5-isopropyl-idene-l, 3-oxazolidine-2, 4-dione as shown in Fig.2.Based on these results, it is concluded that 9AM 1 has a hydroxyl group on the cyclopentyl moiety of pentox-azone.This metabolite was referred to as "hydroxylated -pentoxazone".

The mass spectra and PI spectra of 9AM2 and A-0505 are shown in Fig.3 and 4, respectively.As discussed below, the QM- at m/z 342 of 9AM2 was 16 amu greater than that of A-0505 (m/z 326) and ultimately 9AM2 was characterized as hydroxy-cyclopentyl derivative of A-0505.The fragment ions possessing hydroxy-cyclopentyl moiety (m/z 322 and 252) in Fig.3 were 16 amu greater than those containing cyclopentyl moiety (m/z 306 and 236) of A-0505.Additionally, the PI spectra of both 9AM2 and A-0505 have the common fragment ions (m/z 238 and 167) produced by the loss of hydroxy-cyclopentyl (9AM2) and cyclopentyl (A-0505) moiety.Therefore it is concluded that metabolite 9AM2 has a hydroxyl group on the cyclopentyl moiety similar to 9AM 1.This metabolite was referred to as "hydroxylated -A-0505."

The position of hydroxylation on cyclopentyl moiety of 9AM 1 and 9AM2 is also supported by following experimental facts ; Firstly, with respect to the corn-

Table 1 Morphological and physiological characteristics of pentoxazone

degrading microorganisms isolated from Ushiku paddy field soil.

w : weak reaction, x : unknown.

360 日本 農薬 学会 誌 第25巻 第4号 平 成12年11月

pounds which contain both cycloalkanes (cyclopentane or cyclohexane) ring and benzene ring, it is known that the former ring is preferentially hydroxylated. For example, cyclopentane ring of pencycuron (a fungicide)14) and cyclohexane ring of phencyclidine (an anesthetic)15) are reported to be hydroxylated by mi-crobial metabolism. Secondly, on microbial metabo-lism of vinclozolin, 16) a fungicide which contains both alkyl side chain and benzene ring, no hydroxylated metabolite was detected.

4. Metabolites of Pentoxazone and A-0505 Fig. 5 shows the biodegradation profile of pentox-

azone into various metabolites. Biodegradation of

pentoxazone was initiated by the oxazolidine ring open-

ing and resulted in the formation of A-0505 as the

principal metabolite. Strains 1 B and 2B could not fur-ther degrade A-0505. Strains 7B and 2l B converted from A-0505 into A-0480 and A-1374, respectively.

Strain 9A uniquely hydroxylated pentoxazone on cyclopentyl moiety and also produced monohydroxylat-ed A-0505. Strain 9A produced small amount of several other unknown metabolites (not shown in the figure). Hydrated-pentoxazone, which was formed in buffer solu-tion at neutral or higher pH range, 11) was detected from each medium except 9A-inoculated medium. Strains 1B, 2B, 7B and 2l B could not further degrade this com-

pound. Fig. 6 shows the transformation of A-0505 by the

isolates. All isolates except strain l B were able to trans-

Fig. 1 Mass spectrum (A) and product ion spectrum (B) of 9AM 1 (hydroxylated-pentoxazone) with negative ion ESI-LC/MS and MS/MS.

Fig. 2 Mass spectrum (A) and product ion spectrum (B) of synthetic 3-[4-chloro-5-(3-hydroxycyclopentyloxy)-2-fluorophenyl]-5-isopropylidene-l, 3-oxazolidine-2, 4-dione with negative ion ESI-LC/MS and MS/MS.

Journal of Pesticide Science 25 (4) November 2000 361

form A-0505.Strain 2B produced a small amount (16% at maximum) of A-1374.Strains 7B and 21B produced large amounts of A-0480 and A- l 374, respectively.In addition, strain 2l B produced a small amount of A-0480.Strain 9A produced hydroxylated-A-0505 and a few unidentified metabolites (not shown in the figure).Although dissipation of A-0505 by strain 1 B was not evidently recognized, trace amount of A-1374 was detect-ed.

DISCUSSION

1.Characteristics of Isolates Five microbial strains which belong to four genera

have been isolated in this work.Kuwatsuka7) and MacRae18) summarized that Pseudomonas, Xanth-omonas and Bacillus have been frequently isolated from natural environment as pesticide degraders.Particular-

ly, Pseudomonas is well-known as a genus which degrades various xenobiotics.Pseudomonas fluores-cens (strain l B) is a representative aerobe frequently found in natural microcosms as pesticide degraders.Glovleva et al.16> reported that fungicide vinclozolin which has 1, 3-oxazolidine-2, 4-dione moiety similar to

pentoxazone was decomposed to 2-[[(3, 5-dichloro-phenyl)carbamoyl] oxy] -2-methyl-3-butenoic acid and 3, 5-dichlorophenyl-2-hydroxy-2-methylbut-3-enanilide by Pseudomonas fluorescens.These two degradation

products correspond to hydrated-pentoxazone and A-0505 in the present study, respectively.Thus species Pseudomonas fluorescens may commonly posses trans-formation ability for 1, 3-oxazolidine-2, 4-dione ring struc-ture. Genus Bacillus (strain 9A) is also known to possess a

broad spectrum of availability for various xenobiotics.18)

Fig.3 Mass spectrum (A) and product ion spectrum (B) of 9AM2 (hydroxylated-A-0505) with negative ion ESI-LC/MS and MS/ MS.

Fig.4 Mass spectrum (A) and product ion spectrum (B) of A-0505 with negative ion ESI-LC/MS and MS/MS.

362 日本農 薬学会 誌 第25巻 第4号 平成12年ll月

In this study, strain 9A characteristically transformed

pentoxazone not only to hydroxylation products but also to small amounts of various metabolites (data not shown). This unique metabolic profile may contribute to the total degradation of pentoxazone by providing diverse substrates for the other soil microorganisms.

Strain 2B, Bradyrhizobium japonicum, is known as a root nodule bacterium. Although Bradyrhizobium is normally present as swollen form (bacteroid), the strain transformed pentoxazone in a free living state in this

study. Two strains of Xanthomonas oryzae, 7B and 21B, are pathovars causing bacterial leaf blight on rice

plant. These three strains (2B, 7B and 21B) probably exist in ryzosphere as symbiotic and pathogenic microbes. Such microbes greatly involved with plant

growth could interestingly accelerate the degradation of herbicide pentoxazone. We obtained these five isolates from agar plates prepar-

ed by pour plate technique. The oxygen concentration in the agar layer which embeds colonies would be rather

Fig. 5 Dissipation of pentoxazone and its metabolites formed by microorganisms isolated from paddy field soil.

●: pentoxazone, ○: A-0505, ◇: hydrated-pentoxazone, ■: A-0480, □: A-1374, ◆: hydroxylated-pentoxazone, ×: hydr0xylated-

A-0505.

Non-inoculated control 1B 216

2e 7B 9A

Fig. 6 Dissipation of A-0505 and its metabolites formed by microorganisms isolated from paddy field soil.

○: A-0505, ■: A-0480, □: A-1374, ×: hydroxylated-A-0505.

Non-inoculated control 1B 21B

2B 7B 9A

Journal of Pesticide Science 25 (4) November 2000 363

lower than that of ambient atmosphere. The isolated

pentoxazone degraders are thus considered to favor (2B and 21B) or tolerate (1B, 7B and 9A) lower oxygen concentrations by their great variation of respiratory enzymes. 19) These results suggest that microaerophiles could play an important role in initial transformation of

pentoxazone in aquatic environment.

2. Metabolites of Isolates Not only such various microbial strains contribute to

the transformation of pentoxazone, but each stain has

produced unique metabolites. Fig. 7 shows the chemi-cal structure of the respective metabolites and the scheme of pentoxazone transformation by the isolates. Each metabolite produced by the isolates well reflects the contents of those detected in liquid media inoculated with soil slurries. 10) Thus the metabolites would not be

peculiar for indigenous soil microbes. Especially A-0505 is abiotically produced through hydrolysis, so that availability of A-0505 is important for total degradation of pentoxazone. Strains 7B, 2l B and 9A can transform A-0505 (Fig. 6) and the metabolites were identical with those produced when pentoxazone was applied. This indicates that these strains are able to utilize A-0505 as a metabolic intermediate. The hydroxylated compounds produced by strain 9A

were not detected in liquid media inoculated with soil slurries. 10) When strain 9A was individually cultured in liquid medium with pentoxazone, hydroxylated-A-0505 increased following transient accumulation of

hydroxylated-pentoxazone. It seems reasonable to assume that hydroxylated-A-0505 could be produced from hydroxylated-pentoxazone via the opening of ox-azolidine ring followed by decarboxylation. Since strain 9A can also hydroxylate A-0505 as shown in Fig. 6, hydroxylated-A-0505 could be partly produced direct-ly from A-0505.

3. Feature of Microbial Metabolism of Pentoxazone In general, it is considered that there are few microbial

species which mineralize a pesticide individually in soils without previous treatment of pesticides. 20-22) Selective and enrichment procedure is in most cases necessary to isolate such extreme degraders. 1-4) On the other hand, many papers have demonstrated that the most novel synthetic pesticides including pentoxazone could be mineralized in soils without being exposed to the

pesticides. 5-9) These facts suggest that partial transfor-mation of the pesticides performed by ubiquitous mi-crobial community would be functional steps. Janke et al. 23) summarized in their review that "microbial associa-tions based on initial co-metabolic steps" is important for total degradation of xenobiotics in natural environment. Imai et al. 24) isolated soil microorganisms which degrade herbicide molinate through cometabolism. They esti-mated the ratio of degraders against total microbial

population in soil as about 30%. We obtained five microbial strains from twenty-five microbial colonies in the present study. This indicates that 20% of culturable microbial strains can decompose pentoxazone. The

Fig. 7 The scheme of pentoxazone transformation by microbial strains isolated from paddy field soil.

C: chemical; 1B, 2B, 7B, 21B and 9A: isolated microbial strains. Letters in parenthesis represent weak reactions.

364 日本農 薬学 会誌 第25巻 第4号 平成12年11月

main metabolites such as A-0505, A-1374, A-0480 and

hydrated-pentoxazone produced by the isolates were

similar to those produced by soil microbial community

in liquid media.10 In conclusion, the isolates are pos-

sible participants of microbial association which contrib-

ute the initial co-metabolic step of pentoxazone biode-

gradation.

ACKNOWLEDGMENTS

We are grateful to Dr.Kenji Hirai of Sagami Chemical Research Center for providing us with the reference compounds and helpful suggestions.We also wish to express our special thanks to Mrs.Kiyomi Imai of Sagami Chemical Research Center for synthesis of the reference compounds used in series of our investigation.

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4) L.L.Smith-Grenier & A.Adkins: Can.J.Microbiol.42, 221 (1996)

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23, 1 (1998) 8) Abstr.; 9th Int.Congr.Pestic.Chem.(IUPAC), London,

6A-005, 6A-009, 6A-O11, 6A-014 and 6A-015, 1998 9) K.Satsuma, 0.Hayashi, K.Sato, M.Hashimura & Y.Kato:

J.Pesticide Sci.25, 201 (2000) 10) K.Satsuma, 0.Hayashi, K.Sato, M.Hashimura & Y.Kato:

J.Pesticide Sci.25, 89 (2000) 11) K.Satsuma, 0.Hayashi, K.Sato, M.Hashimura & Y.Kato:

J.Pesticide Sci.In preparation.12) N.R.Krieg & J.G.Holt: "Bergey's Manual of Systematic

Bacteriology, Vol.1, 8th ed., " The Williams & Wilkins Co., London, p.141, 1984

13) T.Hasegawa (ed.): "Biseibutsu no Bunrui to Doutei 2nd ed., " Gakkai Shuppan Center, Tokyo, 1990 (in Japanese)

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要 約

ペ ン トキサゾ ン分解性 土壌微生物 の単離=そ の特 徴

と代謝産物*

薩摩孝次, 林 靖, 佐藤 清, 大山和俊

牧 伸一, 橋村元雄, 加藤保博

除草剤ペン トキサゾンを代謝分解す る微生物 を牛久水田

土壌よ り希釈平板を用いて単離 した.得 られた5菌 株の特

徴 を調べ, またこれらの単離菌によるペ ン トキサゾンの代

謝物を同定 した.こ れら5株 の単離菌(lB, 2B, 7B, 21B,

および9A)は, その形態的および生理的特徴から, それぞ

れ, Pseudomonas fluorescens, Bradyrhizobium/aponicum,

2系 統のXanthomonas oryzae, およびBacillus sp.と分類あ

るいは同定 された.こ れら5株 の分解菌は好気性菌 として

分類され るものの, 塗抹法ではな く, すべて混釈法 を用い

て作成 した平板から単離された.こ のことからこれらの単

離菌は大気に比べ低い酸素濃度 を好むか, あるいは耐える

菌株であると考えられた.こ れ らの単離菌は, 100倍 希釈肉

エキス培地中で速やかにペン トキサゾンを分解 した.代 謝

産物はそれぞれの菌株により異なっていた.主 な代謝物は

lBお よび2B株 ではA-0505, 7B株 ではA-0480, 21B株 では

A-1374, 9A株 ではシクロペンチル環がモノ水酸化 を受け

た2種 の化合物であった.A-0505を 出発物質 として与える

試験により, 7B, 21Bお よび9A株 においてはA-0505を 中

間代謝物 とすることが確認された.

*除 草 剤ペ ン トキサ ゾ ンの微 生物 分解(第3報).