Isolation and Characterization of Bacillus Thuringiensis (Bacillaceae: Bacillales) Strains from an Urban Environment

Journal of Insect Biotechnology and Sericology 83, 71-76 (2014)

Isolation and characterization of Bacillus thuringiensis (Bacillaceae: Bacillales) strains from an urban environment

Yuuichi Yamamoto1†, Yoshinori Hatakeyama1†＊, Kazuyo Enomoto1, Tomoaki Shigano1, Hisayuki Oda2 and Hidetoshi Iwano1

1 Laboratory of Applied Entomology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan 2 Laboratory of Environmental Science, FCG Research Institute, Inc. 1-1-20-6F Aomi, Koto-ku, Tokyo, 135-0064, Japan (Received July 31, 2014; Accepted December 19, 2014)

Bacillus thuringiensis (Ishiwata, 1901; Berliner, 1915) produces parasporal inclusions prior to spore formation; these inclusions are insecticidal. Therefore, B. thuringiensis is used as a microbial pesticide. B. thuringiensis has been detected conventionally in the natural environment, such as in the soil and on plant surfaces as well as in farms and natural fields. To date, the detection rate of B. thuringiensis (the BT index) with standard methods is very low. In this study, we developed a highly efficient detection method to isolate B. thuringiensis from urban environments. In our experiments, The urban environment used was defined as the campus of the College of Biore- source Sciences at Nihon University. The urban samplings were composed of a mixed microbe layer including B. thuringiensis. The BT index was consistently >50% in this study. Our results suggested that the urban environment we examined is a reservoir of many varieties of B. thuringiensis. Key words: Bacillus thuringiensis, Serotype, Cry gene, Urban environment, Isolation, Characterization

INTRODUCTION MATERIALS AND METHODS Bacillus thuringiensis (Ishiwata, 1901; Berliner, 1915) 1. Detection of B. thuringiensis from an urban (Bacillaceae: Bacillales) is a gram-positive, motile, facul- environment tatively aerobic and ubiquitous bacterium. B. thuringiensis To establish an urban source of B. thuringiensis, we produces parasporal inclusions prior to spore formation, considered several factors including: efficiency, ease of and these inclusions usually have insecticidal properties. the search, and proximity to a large population; therefore, Thus, B. thuringiensis is used as a microbial pesticide to we investigated various sites located in and around the control insects in fields (Bravo et al., 2007). Some B. campus of the College of Bioresource Sciences at Nihon thuringiensis Cry proteins exhibit potently toxic insecti- University, our primary test site (Fig. 1). Furthermore, ex- cidal activity (Bravo et al., 2007). Each B. thuringiensis aminations were performed indoors and outdoors at 20 lo- strain produces a different insect toxin; therefore, many B. cations around the test site. An attempt at B. thuringiensis thuringiensis strains have been examined for their insecti- isolation was conducted for a total of 30 samples collect- cidal potentials already. ed from the urban environment including artifacts, such as Generally, B. thuringiensis strains are isolated from the a doorknob, or a floor. Accordingly, in our experiments, soil in farms and natural fields. The detection rate of B. the urban environment was defined as the campus of the thuringiensis (BT index) using standard methods is very College of Bioresource Sciences at Nihon University. low (Ohba et al., 2000). For example, the BT index has B. thuringiensis isolation was carried out according to the been reported to be 3.0% in soil (Hyakutake and Mizuki, following method. Each sample from the urban sites was 2008), 3.4% in plant surfaces (Mizuki et al., 1999), and plated on MYP selection medium agar plates (Kyokuto 4.4% in river water (Ichimatsu et al., 2000). These data Pharmaceutical Industrial Co. Ltd.) to identify B. thuringi- obtained using current, standard methods indicate a need ensis; although the MYP agar plate is an inspection nutri- for a more efficient detection method of B. thuringiensis. ent medium plate for detecting Bacillus cereus, it can be In this study, we developed a highly efficient detection used to isolate B. thuringiensis, because they are closely method to isolate B. thuringiensis in urban environments. related bacteria. The MYP agar plates were used by stamping them on the sampling location. At the water side, we used a cotton swab to dip the sample and applied it to the surface of the MYP agar plates (Fig. 2). The MYP agar plates recovered from various sites were incu- † These authors contributed equally to this work. bated at 37°C for 3-4 days. Colonies with morphological *To whom correspondence should be addressed. features of B. cereus were examined under a phase-con- Fax: +81-466-84-3520. Tel: +81-466-84-3520. trast microscope. Although B. thuringiensis and B. cereus Email: [email protected] were both detected on the MYP agar plates, those that 72 Yamamoto et al. formed parasporal inclusions were designated as B. primer described by Juárez-Pérez et al. (1997). Using this thuringiensis. Then, B. thuringiensis isolates were main- primer, can detected the band in the vicinity of about tained on nutrient agar for serological activity tests. 1,500 bp. In addition, for the detection of other Cry genes, we used some primers described by Ben-Dov et al. (1997) 2. H-serotype classification (Table 1). The total PCR volume was 10.0 μL. The final B. thuringiensis isolates were H-serotyped using the concentrations of the family-specific primers were 0.5 μM slide agglutination test (Ohba and Aizawa, 1978). H-anti- (+) and 1.0 μM (−). The final concentration of the type- serum against the H-serotype 1-55 reference strains of B. specific primers was also 0.5 μM. SP-Taq DNA Poly- thuringiensis were prepared in rabbits (Lecadet et al., merase (Cosmo Genetech Co., Ltd.) was used for PCR, 1999). In this study, we classified isolates according to 14 and DNA was amplified for 30 cycles: denaturing at 94°C separation types that have been previously reported in for 60 s, annealing at 45°C for 45 s, and extending at 72°C Japanese sera also: H3abc, 3ac, 4ab, 4ac, 6, 15, 16, 17, for 120 s. In Cry4A, 4B and Cry11A, we changed anneal- 18ab, 19, 24ab, 25, 27, and 29. All sera were obtained ing to 54°C for 45 s. PCR products were separated on an through the courtesy of Dr. Shin-ichiro Asano of Hokkaido agarose gel by electrophoresis. Each band was classified University. The slide agglutination test was performed by according to fragments representing Cry genes. PCR prod- mixing one drop of the flagellated bacterial broth culture ucts were purified by a Fast GeneTM Gel/PCR Extraction on a glass slide with one drop of a 100-fold dilution of kit (NIPPON Genetics Co, Ltd). Then, DNA was cloned H-antiserum. In the current study, isolates with strong mo- using a Dyna Express PCR TA Cloning kit (pTAC-2) tility, or no reaction to the reference H-antisera, have been (BioDynamics Laboratory. Inc.) and ECOSTM Competent E. referred to as untypable (UTY); those with poor or no coli DH5α (NIPPON GENE CO., LTD.). The purified motility and/or strong auto-agglutination have been re- plasmid DNA was sequenced by Fasmac Co., Ltd. Analy- ferred to as untestable (UTE). sis of each Cry gene sequence was performed using the Isolated strains were classified according to the H-sero- Basic Local Alignment Search Tool (BLAST) from the type by using the experimental method described in National Center for Biotechnology Information (NCBI) Lecadet et al. (1999). (http://www.ncbi.nlm.nih.gov/). Then, these sequences were aligned by ClustalW ver. 2.1 on the website of Na- 3. Detection of Cry genes tional Institute of Genetics (http://clustalw.ddbj.nig.ac.jp/). To detect the Cry1 gene group, we performed PCR, which can be used to specifically detect Cry genes, with a

Table 1. Primer list used Cry gene Primer name Sequence Cry1A All cry1 genes (−) 5’- MDA TYT CTA KRT CTT GAC TA -3’ All cry1 genes (+) 5’- TRA CRH TDD BDG TAT TAG AT -3’ 1A-s cry1A 5’- CAA TAG TCG TTA TAA TGA TT -3’ Cry2 Un2 (d) 5’- GTT ATT CTT AAT GCA GAT GAA TGG G -3’ Un2 (r) 5’- CGG ATA AAA TAA TCT GGG AAA TAG T -3’ Cry3 Un3 (d) 5’- CGT TAT CGC AGA GAG ATG ACA TTA AC -3’ Un3 (r) 5’- CAT CTG TTG TTT CTG GAG GCA AT -3’ Cry4 Un4 (d) 5’- GCA TAT GAT GTA GCG AAA CAA GCC -3’ Un4 (r) 5’- GCG TGA CAT ACC CAT TTC CAG GTC C -3’ EE-4A (d) 5’- GGG TAT GGC ACT CAA CCC CAC TT -3’ EE-4B (d) 5’- GAG AAC ACA CCT AAT CAA CCA ACT -3’ Cry7-8 Un7-8 (d) 5’- AAG CAG TGA ATG CCT TGT TTA C -3’ Un7-8 (r) 5’- CTT CTA AAC CTT GAC TAC TT -3’ Cry7 EE-7Aa (d) 5’- GCG GAG TAT TAC AAT AGA ATC TAT CC -3’ Cry8 EE-8A (d) 5’- GAA TTT ACT CTA TAC CTT GGC GAC -3’ EE-8B (d) 5’- GAC CGC ATC GGA AGT TGT GAG -3’ EE-8C (d) 5’- GGT GCT GCT AAC CTT TAT ATT GAT AG -3’ Cry11 EE-11A (d) 5’- CCG AAC CTA CTA TTG CGC CA -3’ EE-11A (r) 5’- CTC CCT GCT AGG ATT CCG TC -3’ These primers were described by Juárez-Pérez et al. (1997). The nucleotides are follows; B: C, G or T, K: G or T, Y: C or T, D: A, G or T, M: A or C, H: A, C or T, and R: A or G. Isolation and characterization of Bacillus thuringiensis 73

3. Detection of Cry genes RESULTS Agarose gel electrophoresis of the PCR products showed 1. Detection of B. thuringiensis from an urban specific bands representing Cry genes from 12 strains environment (isolate 4-2, 4-4, 6-1, 6-2, 6-5, 7-1, 7-2, 7-4, 7-6, 8-1, 8-2 We investigated 20 sites at the campus of the college of and 8-5). The sizes of the bands obtained by primer sets Bioresource Sciences at Nihon University, for the pres- were used (Fig. 3). It is evident that the bands of each ence of B. thuringiensis (Fig. 1). We detected Bacillus-like strain were confirmed at a roughly nearby position to a B. colonies, which were isolated from a total of 53 colonies, thuringiensis-type strain. For example, the 1,529 bp DNA at 12 sites (Table 2). Based on microscopic examination fragment from isolate 4-4 was similar to Cry11A (Acces- of the 53 colonies, 30 isolates were determined to be B. sion Number JQ228567). Also, the 3 fragments of isolate thuringiensis. The BT index was calculated using the fol- 6-5 (445 bp, 1,951 bp and 1,529 bp) were similar to Cry4A lowing ratio: number of B. thuringiensis colonies/total (Acc. No. JQ228565), Cry4B (Acc. No. D00247) and number of Bacillus colonies. The BT index in this study Cry11A (Acc. No. JQ228567), respectively (Fig. 4). As was 56.6%. The sites with the highest levels of B. with the results of the serotype classification, differences thuringiensis were the bench and the floor of a men’s toi- were observed in the Cry genes from strains that were ob- let on campus (Table 2). tained from the same location. Incidentally, we detected up to 3 different putative Cry genes from the same strain 2. H-serotype classification (isolate 6-5). We performed an agglutination serotyping test for the classification of the 30 isolated strains; and 17 of those DISCUSSION strains were classified into 9 serotypes (Table 3). The site from which the greatest number of isolates was obtained Previous surveys have reported a BT index of 3% for B. was the floor of the men’s toilet. From this site, 5 strains thuringiensis from soil (Hyakutake and Mizuki, 2008), that could be classified into 4 serotypes (serovar entomo- 4.4% from river water (Ichimatsu et al., 2000), and 3.4% cidus, dakota, indiana and neoleonensis) were obtained. from plant surfaces (Mizuki et al., 1999). These results

Fig. 1. Description of survey site (Campus of College of Bioresource Sciences, Nihon University). Separation places were follows; 1: water supply button, 2: vending machine button, 3: water of a biotope 1, 4: water of a biotope 2, 5: rear surface of the rose leaf, 6: bench, 7: floor of men’s toilet, 8: surface of cherry leaf, 9: water of a fountain, 10: back of the hand, 11: faucet, 12: mobile phone, 13: elevator button 1, 14: elevator button 2, 15: doorknob of a track, 16: en- ter key, 17: handrail, 18: farm soil, 19: cash dispenser, 20: rose petal. These places were chosen as the screening locations at random. 74 Yamamoto et al.

Table 2. The number of Bacillus thuringiensis isolates obtained at each site Separation place Bacillus like colony B. thuringiensis 1 Water supply button 3 0 2 Vending machine button 3 1 3 Water of a biotope 1 1 1 4 Water of a biotope 2 4 4 5 Rear surface of the rose leaf 2 1 6 Bench 11 6 7 Floor of men’s toilet 12 6 8 Surface of cherry leaf 6 5 9 Water of a fountain 4 3 10 Back of the hand 1 1 11 Faucet 3 0 12 Mobile phone 3 2 total 53 30 Each number refers to Fig. 1.

Table 3. The number of H-serotype obtained from the isolates at each site Separation place* Total H-antigen H-serotype 2. 3. 4. 5. 6. 7. 8. 9. 10. 12. 4ac kenyae – – – – – – – 1 – – 1 6 entomocidus – – – – – 2 – – – – 2 15 dakota – – – – – 1 – – – – 1 16 indiana – – 1 – 1 1 – – 1 – 4 17 tohokuensis – – – – – – 1 – – – 1 18ab kumamotoensis – – – – 1 – – – – – 1 19 tochigiensis – – – 1 – – – – – – 1 24ab neoleonensis – – – – 1 1 1 – – – 3 27 mexicanensis 1 1 – – – – – – – 1 3 UTY – – 1 – 1 – 1 2 – 1 6 UTE – – 2 – 2 1 2 – – – 7 Total 1 1 4 1 6 6 5 3 1 2 30 *: Each number refers to Table 2. The number of samples for which agglutination was confirmed is presented. H-antigen represents the corresponding H-serotype. H-serotype represents the subspecies name of Bacillus thuringiensis. UTY: Untypable by the reference antisera against H4ac, 6, 15, 16, 17, 18ab, 19, 24ab and 27. UTE: Poor or no motility and/or strong auto-agglutination have been referred to as untestable.

Fig. 2. Isolating situation on each sample location. a: Floor of men’s toilet. MYP agar plate was used by stamping on the sample location. b: water of a biotope 2. After sampling by a cotton swab, each materials were applied on the surface of MYP agar plate. Isolation and characterization of Bacillus thuringiensis 75

Fig. 3. Gel electrophoresis of PCR products obtained with specific primers for Cry genes. Each lane No. from left were follows, M: gene marker, each type strain: DNA of B. thuringiensis serovar kurstaki, serovar islaerensis and soro- ver dakota, our isolates strain: 6-1, 6-2, 6-5, 4-2, 7-1, 7-2, 7-4, 7-6, 8-1, 4-4, 8-2 and 8-5. Duplication strain No. were omit. All type strains were obtained by the courtesy of Dr. Michio Ohba. The black arrow heads shown the target band.

Fig. 4. Alignment of our Cry gene sequences with known Cry genes on the DNA database. Each alignment data were follows; a: isolate 4-4 was corresponded with a portion of Cry11A of B. thuringiensis serovar israelensis (Acc. No. JQ228567), b: isolate 6-5 was corresponded with a portion of Cry4A of serovar israelensis (Acc. No. JQ228565), c: isolate 6-5 was corresponded with a portion of Cry4B of serovar israelensis (Acc. No. D00247), d: isolate 6-5 was corresponded with a portion of Cry11A of serovar israelensis (Acc. No. JQ228567). 76 Yamamoto et al. suggest that the BT index may often be low in the natural part by a Grant-in-Aid from the Nihon University College environment. In the present study, by surveying an envi- of Bioresource Sciences Research Fund for 2011 and 2012. ronment that has not been frequently investigated, we suc- ceeded in obtaining a high BT index, that is, >50%. 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(1978) Serological identification of ACKNOWLEDGEMENTS Bacillus thuringiensis and related bacteria isolated in Japan. J. Invertebr. Pathol., 32, 303-309. We are grateful to Dr. Shin-ichiro Asano (Associate Ohba, M., Wasano, N. and Mizuki, E. (2000) Bacillus thuring- Professor, Graduate School of Agriculture, Hokkaido Uni- iensis soil populations naturally occurring in the Ryukyus, a versity) for supplying the B. thuringiensis H-serotype an- subtropic region of Japan. Microbiol. Res., 155, 17-22. tigens. Furthermore, we are grateful to Dr. Michio Ohba Rampersad, J. and Ammons, D. (2005) A Bacillus thuringiensis isolation method utilizing a novel stain, low selection (Former Professor, Kyusyu University) for supplying the and high throughput produced atypical results. BMC Micro- B. thuringiensis-type strains. This study was supported in biol., 5, 52.