ORIGINAL ARTICLE

A Bacillus thuringiensis host strain with high melaninproduction for preparation of light-stable biopesticides

Fengxia Liu & Wenjun Yang & Lifang Ruan & Ming Sun

Received: 22 February 2012 /Accepted: 5 November 2012# Springer-Verlag Berlin Heidelberg and the University of Milan 2012

Abstract The bacterium Bacillus thuringiensis produces acrystal protein with insecticidal properties; however, crystalproteins can be damaged by ultraviolet (UV) radiation. Theaim of this study was to improve the stability of the insecti-cidal crystal protein (ICP) by constructing a mutant line thatexpresses high levels of the UV light-protecting pigment,melanin. BMB181, a B. thuringiensis mutant with high mel-anin production, was obtained after sub-culturing BMB171for several generations at 42 °C. The melanin yield byBMB181 (without tyrosine supplementation) reached8.55 mg/ml. The electroporation efficiency of BMB181reached 106 CFU/μg when a 6.7-kb foreign plasmid was used.Microscopic and SDS-PAGE analyses revealed that ICP(CryIAc10; GenBank: AAA73077.1), which is highly toxicto Lepidoptera, was synthesized efficiently by strainBMB181. The insecticidal properties of a recombinant linederived from strain BMB181, designated BMB32 (cry1Ac10/BMB181), was tested against the cotton bollworm, Helico-verpa armigera. After UV irradiation for 4 h, BMB32 had ahalf maximal inhibitory concentration value of 1.37 μg/ml,whereas the control line BMB31 (cry1Ac10/BMB171) had amedian lethal dose value of 25.85 μg/ml. These results indi-cate that the B. thuringiensis mutant is a candidate for indus-trial scale production of light-stable insecticides.

Keywords Bacillus thuringiensis . Host . Melanin .

Ultraviolet radiation . Biopesticide

Introduction

Bacillus thuringiensis is a ubiquitous Gram-positive, spore-forming bacterium that produces a parasporal crystal duringthe stationary phase of its growth cycle (Schnepf et al.1998). This bacterium is of interest to biotechnology be-cause it can produce a crystal protein with multiple insecti-cidal properties. In fact, 460 different types of crystal proteintoxins (68 distinct classes) have been reported to date.Although as an extensively applied biopesticide, B. thurin-giensis has a number of drawbacks, the most important ofwhich is that the insecticidal crystal protein (ICP), which isthe primary source of the insecticidal activity, is easilydestroyed by ultraviolet (UV) radiation in sunlight. Despitethis drawback, diversity in both the type of toxins producedand the range of insect specificities makes B. thuringiensisan important biological agent. Thus, the identification of arecombinant strain with multiple pesticidal activities thatcan be tailored towards a given purpose is highly desirable.

Melanin is known to absorb radiation. It can absorb light ofany wavelength, but its optimum capacity is in the UV range.Given this characteristic, a number of studies have beenperformed with the aim of producing melanin or increasingmelanin production in B. thuringiensis cells in order to im-prove photoprotection. In one study, the Pseudomonas malto-philia mel gene was expressed in B. thuringiensis, and thepercentage survival of B. thuringiensis was shown to increasein the absence of ICP (Ruan et al. 2002). In another study, amelanin-producing B. thuringiensis mutant was shown tohave higher UV radiation resistance than the wild-type bacte-rium (Saxena et al. 2002). Interestingly, when the B. cereusmel gene and B. thuringiensis ICP gene were simultaneouslyexpressed inB. thuringiensis (acrystalliferous strain 4Q7), UVradiation resistance was conferred to the recipient strain(Zhang et al. 2008). However, Ruan et al. (2004) found thatmost B. thuringiensis strains have the potential to produce

F. Liu :W. Yang : L. Ruan :M. SunState Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University, Wuhan 430070, China

L. Ruan (*)State Key Laboratory of Agricultural Microbiology, College ofLife Science and Technology, Huazhong Agricultural University,Wuhan 430070, Chinae-mail: ruanlifang@mail.hzau.edu.cn

Ann MicrobiolDOI 10.1007/s13213-012-0570-0

melanin in the presence of L-tyrosine at an elevated tempera-ture (42 °C). Disappointingly, ICP could not be simultaneous-ly synthesized at this temperature (Ruan et al. (2004).

In this study, we obtained a B. thuringiensis strain thatis characterized by high UV radiation resistance, highelectroporation efficiency, and ICP synthesis. This strainhas the potential to serve as the basis for a geneticallyengineered strain for the industrial production of B. thur-ingiensis with a broad insecticidal spectrum and improvedUV radiation stability.

Materials and methods

Strains and plasmids The B. thuringiensis acrystalliferousstrain BMB171 used in this study was constructed previously(Li et al. 2000; Peng et al. 2009; He et al. 2010). B. thurin-giensis BMB181, a mutant strain of BMB171, can producemelanin without requiring L-tyrosine and an elevated temper-ature. B. thuringiensis was grown at 28 °C with shaking(200 rpm) in Luria broth (LB), ICPM medium (Song et al.2008), or fermentation medium (in g/l: soy powder, 22.5; cornsyrup, 20; starch, 15; CaCO3, 1.5). Culture media were sup-plemented with erythromycin at 25 μg/ml.

The plasmid pBMB31-304 was obtained as follows. ThecryIAc10 gene under the control of the cryIIIA promoter wassubcloned into pHT304 (Arantes and Lereclus 1991) usingBamHI and SphI restriction enzymes. Plasmid pBMB31-304was electroporated into BMB171 and BMB181 to giveBMB31 and BMB32, respectively.

Melanin production The amount of melanin produced bythe bacterial cells was determined according to the methodsdescribed by Ruan et al. (2002).

Infrared spectrum scanning Infrared (IR) spectrum scan-ning was carried out as described by Hoti and Balaraman(1993). The melanin and potassium bromide powders were

mixed together and the mixture moulded into a pellet invacuo. The IR spectrum of this pellet was measured andrecorded on a Perkin-Elmer 783 IR spectrophotometer(Perkin-Elmer, Boston, MA).

Electroporation Electroporation experiments were carriedout as described by Peng et al. (2009). Electroporationwas carried out at 200 Ω and 25 μF in 2-mm cuvettes(voltage range up to 15 kV/cm) where 100 μl of cellswas combined with 1 μg DNA, or in 1-mm cuvettes(voltage range up to 30 kV/cm) where 50 μl of cellswere combined with 500 ng DNA, using a Bio-RadGene Pulser apparatus equipped with a Bio-Rad pulsecontroller (Bio-Rad, Hercules, CA). After electroporation,cell suspensions were diluted with 1 ml of LB andincubated for 2 h at 28 °C, 200 rpm. Aliquots werespread onto LB agar plates supplemented with an appro-priate antibiotic. Transformants harboring antibiotic resis-tance were counted following overnight incubation. Theelectroporation efficiency was expressed as the number ofCFU (transformants) per microgram DNA. Nanodrop UVspectrophotometry (GeneQuant 100; GE Healthcare,Little Chalfont, UK) was used to quantify DNAconcentrations.

UV irradiation of B. thuringiensis Samples were irradiatedwith a UV radiation luminaire box delivering a wavelengthof 254 nm; this process caused damage to the cells andcrystal protein. Fermentation liquid (10 μl) was sprayedonto 20-cm radius Petri dishes that were left open andplaced 50 cm from the UV radiation source. Cultures wereirradiated for 2 or 4 h, during which time the Petri disheswere shaken at 60 rpm.

Insect bioassays Qualitative insect toxicity testing wascarried out on spore-crystal preparations. The activitiesof BMB31 and BMB32 were tested against the cottonbollworm Helicoverpa armigera first-instar larvae. The

Fig. 1 Pattern of melanin production on different growth media.Top Bacillus thuringiensis acrystalliferous strain BMB171, bottomB. thuringiensis strain BMB181 (mutant strain of BMB171 that

can produce melanin without requiring L-tyrosine and an elevatedtemperature). a ICPM medium, b LB medium, c LB+1 % tyro-sine medium

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spore–crystal mixture containing 1 mg/ml crystal pro-tein was suspended in phosphate buffered saline (PBS)buffer (TaKaRa, Otsu, Japan) for the bioassays (Liu etal. 2002).

Results

Screening B. thuringiensis mutants for melanin production

Bacillus thuringiensis strain BMB171 was cultured to theexponential phase at 28 °C in LB medium, then subculturedin triplicate at 42 °C for 8 h, after which the cultures werediluted and spread onto LB plates (supplemented with 1 % L-tyrosine). A clone that produced pigment, which we namedBMB181, was obtained after overnight incubation at 28 °C.The pigment produced by this clone was certified as melaninfollowing IR scanning (data not shown). The characteristic IRabsorbance peak of the pigment produced by BMB181 wasidentical to that of the commercially available source sample(Sigma Chemical Co, St. Louis, MO). Strain BMB181 wastested for melanin production on different growth media, andthe results of these experiments are shown in Fig. 1. Quanti-tative assays of melanin production for strain BMB181showed that the maximum yield of the protein obtained was3.91, 4.81, 8.55, and 4.66 mg/ml in ICPM medium, LBmedium, LB medium with 1 % tyrosine, and fermentationmedium, respectively. The BMB181 strain has been depositedin the Cultures of China Center for Type Culture Collection(CCTCC: WDCM611) under accession number M2011016.

Melanin production

Bacillus thuringiensis strains BMB181 and BMB171 weregrown in LB media at 28 °C. The optical density of thecultures and the time when melanin production first com-menced were determined using a UV spectrophotometer at400, 600, and 721 nm, respectively. The optical density ofthe culture supernatant measured at 400 nm after centrifu-gation (12,000 rpm, 1 min) was used for measuring melaninconcentrations. The quantitative melanin assays were per-formed in triplicate. Figure 2 shows the growth and melaninconcentration curves for the cultures. The melanin curveswere calculated using a standard curve based on purifiedmelanin (Sigma Chemical Co.). The growth curves shownin Fig. 2 indicated that the two strains grew synchronouslyin LB medium. Such curves revealed that strain BMB181started to produce melanin from the late logarithmic phasewith subsequent increases during the stationary phase (inLB medium). In contrast, melanin production was notdetected in the culture supernatant of strain BMB171.

Electroporation efficiency of B. thuringiensis strain BMB181

The BMB181 strain shown to have high melanin productionis a derivative of the B. thuringiensis host strain acrystallif-erous BMB171. We next sought to determine the electro-poration efficiency of BMB181 because of its candidacy asan optimal host for biopesticide preparation. Plasmids withdifferent molecular weights were electroporated intoBMB181, and the electroporation efficiency of the processwas calculated (Table 1). The results of these experimentsindicated that BMB181 retained the high electroporationefficiency of BMB171.

Expression of insecticidal crystal protein in B. thuringiensisstrain BMB181

The ICP (CryIAc10) is highly toxic to Lepidoptera speciesof insect pests (Peng et al. 2009). The recombinant strainBMB32 that carries the cryIAc10 gene was used to expressthe crystal protein. Recombinant strain BMB32 was incu-bated to the sporulation phase at 28 °C in ICPM medium,and light microscopy confirmed that the characteristic para-sporal crystal was efficiently synthesized (Fig. 3). Quantifi-cation of the ICP, as determined by sodium dodecyl sulfate-

Table 1 Electroporation effi-ciency of Bacillus thuringiensisstrain BMB181

Plasmid name(molecular weight)

pHT304(6.7 kb)

pBMB1251(10 kb)

pBMB292(17 kb)

pBMB625(38 kb)

Electroporation efficiency(CFU/μg)

7.12±0.14×106 8.53±0.37×104 5.78±0.54×103 4.37±0.33×102

0 5 10 15 20 25 30 35

Time (h)

0

1

2

3

4

5

6

Melanin (mg/ml)/OD600

Fig. 2 Growth and melanin production curves of B. thuringiensisstrains BMB171 and BMB181 in LB medium. Filled diamondBMB171, open square BMB181. Melanin yield: open diamondBMB181, filled triangle BMB171. Optical density was measured at600 nm

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polyacrylamide gel electrophoresis (SDS-PAGE) using bo-vine serum albumin (BSA) as the standard and a previouslydescribed method (Song et al. 2008) revealed that the yieldof the protein reached up to 0.469 mg/ml.

Melanin produced by B. thuringiensis strain BMB181 protectsagainst UV radiation

The insecticidal activities of BMB32 and BMB31were assayedbefore and after UV radiation using H. armigera larvae. Thedata in Table 2 shows that the LC50 (concentration lethal to50% of the cells) for strains BMB32 (1.23 μg/ml) and BMB31(2.12 μg/ml) were similar before irradiation. The toxicity ofstrain BMB32 toH. armigera remained the same after a 2- and4-h exposure to UV radiation. In contrast, strain BMB31 tox-icity towards H. armigera decreased over time by 3.73- and12.19-fold at 2 and 4 h after UV exposure. We conclude,therefore, that the melanin produced by BMB181 improvedthe stability of the insecticidal activity after UV radiation.

Discussion

Melanin, pigments that can absorb radiation, is widely dis-tributed among fungi, plants, insects, and mammals. Asmany organisms are exposed to the damaging rays of thesun, the ability to synthesize melanin has the potential tobestow fitness advantages to those that can do this. Themajor biopesticides produced by B. thuringiensis suffer

from UV degradation. Previous researchers have tried tosolve this problem by heterologous expression (Ruan et al.2002) or heterogeneous expression (Zhang et al. 2008) ofthe melanin coding gene in B. thuringiensis or by addingmelanin to preparations of B. thuringiensis (Liu et al. 1993).However, low melanin yields (Ruan et al. 2002; Zhang et al.2008) or problems with its purification (Liu et al. 1993),among others have hindered progress in this field of re-search. Our own research has found that most B. thuringien-sis strains have the potential to produce melanin in thepresence of L-tyrosine at an elevated temperature (42 °C).The aim of our present study was to screen B. thuringiensisfor melanin production at 42 °C because this temperatureappeared to be better suited for melanin production. A B.thuringiensis BMB171 mutant, BMB181, was obtained andshowed steady melanin production in different media with-out additional L-tyrosine. Moreover, BMB181 was able tosynthesize melanin in fermentation broth.

BMB171 is an optimal host strain for ICP production andhas the capacity to carry foreign plasmids of large molecularweight (≤60 kb) as shown previously by our group (Guo et al.2008; Liu et al. 2009; Liu et al. 2010). B. thuringiensis strainBMB181 was shown to be a derivative of BMB171 throughexamination of the sequence of the csaB gene, which is essen-tial for S-layer assembly (Kern et al. 2010). The csaB gene hasbeen used to construct phylogenetic trees in the Bacillus cereusgroup (unpublished data). We found that the csaB gene in B.thuringiensis strains BMB171 and BMB181 had identicalsequences, so we deduced that the BMB181 is the derivantof BMB171(accession no.CP00193). Therefore, BMB181retains the characteristics of BMB171 at this locus.

In our study, the B. thuringiensis BMB171 mutant thathas high melanin production was obtained after being incu-bated at an elevated temperature. Interestingly, data fromSaxena et al. (2002) and Zhang et al. (2008) imply that amechanism exists for the regulation of melanin productionin B. thuringiensis. In this context, it is noteworthy that thehmgA gene, which encodes an oxidoreductase in a mutantVibrio cholerae, exhibited high melanin production. Furtherresearch using microarray analysis showed that the lack ofan hmgA gene in this mutant interrupted the pathway from

Fig. 3 Optical microscope graph of parasporal crystals and spores ofrecombinant B. thuringiensis strain BMB32 (magnification×1000)

Table 2 Toxicities of B. thurin-giensis strains against Helico-verpa armigera after ultravioletradiation

LC50, Concentration lethal to50% of the cells

B. thuringiensisstrains

Ultraviolet radiationexposure (h)

Regression equation Regressioncoefficient (R2)

LC50 (μg/ml)

BMB31 0 y00.8637x+0.2172 0.9941 2.12

2 y00.3322x+0.2019 0.9643 7.90

4 y00.4429x+0.1256 0.9231 25.85

BMB32 0 y00.4113x+0.4628 0.9956 1.23

2 y00.7198x+0.5068 0.9826 0.97

4 y00.4208x+0.4424 0.9627 1.37

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L-tyrosine to fumarate and affected metabolism to melanin(Valeru et al. 2009).

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