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Application of Matrix-Assisted Laser-Desorption/Ionization Time-Of Vol. 37, No. 4, 305–311 (2012) MALDI-TOF MS application in APEOn-degrading bacteriaJ. Pestic. from Sci. the 37environment(4), 305–311 (2012)305 DOI: 10.1584/jpestics.D12-023 Original Article Application of matrix-assisted laser-desorption/ionization time-of-ight mass spectrometry for the identication of alkylphenol polyethoxylate-degrading bacteria in the environment Yudai H, Manabu W, Kaduki N, Akifumi H, and Hiroto T* Department of Environmental Bioscience, Meijo University, 1–501 Shiogamaguchi, Tenpaku-ku, Nagoya, Aichi 468–8502, Japan (Received March 19, 2012; Accepted August 2, 2012) In order to clarify the usefulness of the S10-GERMS (S10-spc-alpha operon gene-encoded ribosomal protein mass spec- trum) method for the discrimination of microbial isolates from the environment, the isolates with octylphenol polyethoxylate (OPEOn)-degrading capability from several soils in Japan were classied into 4 biodegradation patterns based on dierent nal metabolic toxicants. Some isolates were identied as genera Chelatococcus and Mesorhizobium which have never been reported as OPEOn-degrading bacteria based on 16S rRNA gene sequences. e ndings from this study demonstrate that the S10-GERMS method successfully discriminates the isolates at the strain level in the genus Pseudomonas. Moreover, this method is better than 16S rRNA gene sequence similarity because it precisely demonstrated that OPEOn-degrading bacteria in the genera Chelatococcus and Mesorhizobium might be new species. e S10-GERMS method is suggested as a useful tool for the discrimi- nation and monitoring of man-made chemical-degrading bacteria isolated from the environment. © Pesticide Science Society of Japan Keywords: alkylphenol polyethoxylate-degrading bacteria, octylphenol polyethoxylate, biodegradation, S10-GERMS method, MALDI-TOF MS. Electronic supplementary materials e online version of this article contains supplementary materials (Supplemental Figure S1, Supplemental Tables S1 and S2), which is available at http://www.jstage.jst.go.jp/browse/jpestics/. knowledge of APEO biodegradation has revealed that there Introduction n are two biodegradation mechanisms: 1) exo-type shortening of Since pesticides that are discharged into the non-target environ- the EO chain accompanied by either oxidation of the EO moi- ment through surface water and/or groundwater transform into ety (oxidative biodegradation)4–8); or 2) nonoxidative hydroxy their biodegraded products (metabolic toxicants), and may exert shift.9,10) Moreover, environmental elements, such as Mg2+, certain chemical selective pressure on wildlife, including ani- Ca2+, and Fe3+, signicantly inuence the nal degradation me- mals, plants, and microorganisms the establishment of their life- tabolites.3,11) cycle impact assessment in the environment is becoming more In a previous study, we demonstrated that bacteria that can important in an environmentally conscious material cycle soci- degrade OPEOn to estrogenic and antiandrogenic metabolites 12,13) ety. Nonionic surfactant octylphenol polyethoxylates (OPEOn), are ubiquitous in paddy elds in Japan. Furthermore, the which are one of the alkylphenol polyethoxylates (APEOn), are study of the dynamic relationship between the microbial diver- easily degraded to octylphenols (OP), octylphenol monoethox- sity and biodegradation capacity of OPEOn using enrichment ylate (OPEO1), octylphenol diethoxylate (OPEO2), and the cor- cultures of various sediments from Iwata River in Japan revealed responding octylphenol carboxylates (OPEC1,2) in the environ- that OPEOn-degrading bacteria are widely distributed at the 14) ment. In particular, accumulated OP, OPEO1, and OPEO2, with sites of dierent types of human activity along Iwata River. their relatively high hydrophobicity in the environment, act as is implies that the quantity and quality of the resultant eco- estrogen agonists and androgen antagonists.1–3) Accumulated toxicity depended on the metabolites in the environment. ere- fore, it is important to identify degrading bacteria rapidly for * To whom correspondence should be addressed. the evaluation and monitoring of the degrading activity of man- E-mail: [email protected] made chemicals. Published online November 14, 2012 Currently, matrix assisted laser desorption/ionization time- © Pesticide Science Society of Japan of-ight mass spectrometry (MALDI-TOF MS) is used for the 306 Y. Hotta et al. Journal of Pesticide Science identication of microorganisms, especially, in routine clini- commercial name Triton X-100 (TX-100), were purchased cal microbiological diagnostics because of its accuracy, speed, from Wako (Japan) and Aldrich Chemical Co. (USA). A liquid and cost eectiveness. However, a complete and representative basal salt medium with 0.1% (w/v) of TX-100 as a sole carbon database is an essential requirement for the accurate identica- source, called the TX-A medium, was used for the biodegrada- tion of isolates by MALDI-TOF MS method which is based on tion test and isolation of OPEOn-degrading bacteria. e TX-A a principle called bacterial ngerprinting. erefore, a database medium is described in a previous study.11) e TX-A medium in which bacterial species are mislabeled or with a low num- with 100 mM sucrose, called the TX-sucrose medium, was used ber of registered bacterial species can cause misidentication for bacteria that do not shorten the ethoxylate (EO) chain of by MALDI-TOF MS. In environmental microorganisms, the OPEOn. number of isolated and characterized prokaryotic species is still relatively low; thus, it is dicult to apply the MALDI-TOF MS 2. Isolation of OPEOn-utilizing bacteria method based on bacterial ngerprinting to the identication OPEOn-degrading bacteria were isolated from several soils in of environmental microorganisms. erefore, the S10-GERMS Japan. e isolated bacteria and sampling sites are shown in (S10-spc-alpha operon gene-encoded ribosomal protein mass Table 1. e isolation procedures of OPEOn-degrading bacteria spectrum) method using ribosomal proteins coded in the S10- are the same as those used for others except for the medium.11) spc-alpha operon as biomarkers was developed to achieve a rap- Briey, 0.5 g soil was added to 5 mL of a TX-A medium in a test id and simple bacterial discrimination and typing method and tube. Aer shaking at 30°C for 2 weeks, a 100-µL sample suspen- demonstrated the advantages of such a bioinformatics-based ap- sion solution was transferred to 5 mL of a newly prepared TX-A proach over bacterial ngerprinting.15–17) e objective of this medium. is procedure was performed three times. Finally, a study is to clarify the usefulness of the S10-GERMS method for 100-µL enriched sample solution was spread on the basal salt the discrimination of bacteria isolated from the environment. medium agar plate with 1% (w/v) TX-100. Aer incubation at For that purpose, therefore, various OPEOn-degrading bacteria 30°C for 1 week, single colonies were selected and subjected to were also isolated. further studies. is study demonstrated that the S10-GERMS method is clearly more suitable than 16S rRNA gene sequence similarity 3. Bacterial biodegradation test for the rapid discrimination of OPEOn-degrading bacteria iso- e isolated bacteria were incubated in 5 mL TX-A or TX-su- lated from the environment that were grouped on the basis of crose media. Aer shaking at 30°C for 14 days, the culture me- nal metabolic toxicants due to their degrading activity. dium was extracted with an equal volume of ethyl acetate with 25 mg/L n-eicosane, and the ethylacetate layer was analyzed by Materials and Methods gas chromatography (GC) and MALDI-TOF MS. GC analysis 1. Culture media of biodegradation products was performed under the condi- 11) t-Octylphenol polyethoxylates (OPEOn), which have the tions described previously. MALDI-TOF MS measurements of Table 1. Bacterial isolates used in this study Strain Source of isolation Biodegradation pattern Assignment of bacteria Proteobacteria Homology (%) S5 Paddy eld, Fukuoka 3 Pseudomonas putida γ 98.8 FMP1 Paddy eld, Fukuoka 3 Pseudomonas putida γ 99.2 EC31 Upland eld, Aichi 4 Pseudomonas putida γ 98.8 BSN03 Ueda river, Aichi 1 Chelatococcus asaccharovorans α 97.1 BSN07 Ibi river, Mie 1 Chelatococcus asaccharovorans α 97.1 BSN11 Nagara river, Gifu 1 Sphingopyxis macrogoltabidus α 99.9 BSN20 Ueda river, Aichi 2 Sphingopyxis terrae α 99.9 BSN22 Upland eld, Gifu 1 Sphingopyxis soli α 98.5 BSN24 Yamayoke river, Mie 1 Chelatococcus asaccharovorans α 97.1 BSN48 Upland eld, Aichi 1 Sphingopyxis soli α 98.5 BSN51 Upland eld, Saitama 1 Sphingopyxis ginsengisoli α 99.4 BSN53 Upland eld, Saitama 2 Sphingopyxis soli α 99.6 BSN54 Upland eld, Saitama 2 Sphingopyxis macrogoltabidus α 99.2 BSN55 Iwata river, Mie 2 Sphingobium cloacae α 98.0 BSN58 Upland eld, Aichi 3 Mesorhizobium thiogangeticum α 98.5 BSN59 Iwata river, Mie 1 Chelatococcus asaccharovorans α 97.1 BSN60 Iwata river, Mie 1 Chelatococcus asaccharovorans α 97.1 Vol. 37, No. 4, 305–311 (2012) MALDI-TOF MS application in APEOn-degrading bacteria from the environment 307 Fig. 1. Biodegradation pattern of TX-100 by representative OPEOn-degrading bacteria. (a) GC analysis of biodegradation products by BSN07 (Pattern 1), (b) GC analysis of biodegradation products by BSN53 (Pattern 2), (c) GC analysis of bio- degradation products by FMP1 (Pattern 3), (d) MALDI-TOF MS analysis of biodegradation products by EC31 (Pattern 4). carboxylated products of OPEOn were performed using a Voy- were as follows: (1) 2 min at 98°C, (2) 30 cycles of 10 sec at 98°C, ager DE-PRO time-of-ight mass spectrometer (Applied Biosys- 30 sec at 50–55°C, and 6.5 min at 68°C. PCR and sequencing tems, USA). As the matrix for sample ionization, 5,10,15,20-tet- primers for the genera Mesorhizobium and Chelatococcus strains rakis (pentauorophenyl) porphyrin (F20TPP; MW 974.6; Sig- used in this study were designed on the basis of consensus nu- ma, USA) was used. About 2 mg F20TPP was dissolved in 1 mL cleotide sequences of S10 and spc operons from 17 genome-se- ethyl acetate to make the matrix solution.
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