Larvicidal activity of crystal-forming strains of Brevibacillus laterosporus

ISSN 0003�6838, Applied Biochemistry and Microbiology, 2010, Vol. 46, No. 8, pp. 755–762. © Pleiades Publishing, Inc., 2010.Original Russian Text © M.V. Zubasheva, L.A. Ganushkina, T.A. Smirnova, R.R. Azizbekyan, 2009, published in Biotekhnologiya, 2009, No. 6, pp. 46–56.

755

Brevibacillus laterosporus (previously Bacillus late�rosporus [1]) has recently become more and moreattractive for researchers. This is connected with therevelation of a number of their important characteris�tics. Presently, it has been shown that some strains ofB. laterosporus are able to produce a number of antibi�otics [2–4], insecticides, and fungicide factors [5–8]and they possess toxicity against phyto� and zoonema�todes, as well as snails [9, 10]. Adding of bacteriaB. laterosporus in soil increases productivity of cerealcrop [11]. It is possible to use B. laterosporus as probi�otics [12]. A number of compounds that is synthesizedby B. laterosporus are of significant importance formedicine [13].

Taking into consideration the fact that a half ofpopulation of the earth lives in a risk zone that is con�nected with the possibility of contact with mosquitoesand blackflies—carriers of causative agents of a num�ber of dangerous diseases (malaria, encephalitis, andelephantiasis)—the search for new biological meansdipterous insects control is of great importance. Pres�ently, bacteria Bacillus thuringiensis spp. israelensis(BTi) and Bacillus sphaericus, the acting larvicidal fac�tors of which are protein crystals, are used as biologicalmeans for mosquito control [14, 15]. A number ofauthors have already reported the weak larvicidalactivity of bacteria B. laterosporus; however, the factorsthat determine activity were not revealed [16]. On the

ground that mosquitoes obtain persistence to bioprep�arations based on B. sphaericus [15], the problem ofbroadening the spectrum of bacteria that possess larvi�cidal effect has apparently become urgent.

EXPERIMENTAL

Bacterial strains. We used the following strains ofB. laterosporus in the study: IGM 16�92, IGM 16�931,IGM 16�932 and IGM 16�13 isolated in the Labora�tory of Biologically Active Conditions (State ResearchInstitute for Genetics) from natural samples (soils anddead animals) on selective media with polymyxin(100 μg/ml) [17]. Strains LAT001�LAT011 were pro�vided by Dr. Lecadet from the collection of ento�mopathogenic bacteria IEBC (Paster Institute, Paris,France) and Dr. Shida (Japan).

Cultivation media. As main nutritious media, weused native BP medium for entomopathogenic bacte�ria that contains: bacto�pepton (Difco)—0.75%,KH2PO4—0.068%, MgSO4 ⋅ 7H2O—0.012%,MnSO4 ⋅ 4H2O—0.00017%, ZnSO4 ⋅ 7H2O—0.0014%,Fe2(SO4)3 ⋅ 7H2O—0.002%, CaCl2 ⋅ 2H2O—0.0147%,glucose—1%; pH 7.4 [18].

A number of nutritious media were prepared on thebasis of NBY medium (0.8% broth (Difco, UnitedStates), 0.3% yeast extract (Difco), pH 6.8) by addingvarious salts, glucose (1%), and growth factors (thia�mine and methionine) [19, 20]. To prepare solidmedium, 1.8% bactoagar (Difco) was added in corre�sponding liquid medium. D�methionine and thiaminethat are necessary as growth factors were prepared sep�arately and added in the medium up to the concentra�

Larvicidal Activity of Crystal�Forming Strainsof Brevibacillus laterosporus

M. V. Zubashevaa, L. A. Ganushkinab, T. A. Smirnovaa, and R. R. Azizbekyana

aState Research Institute for Genetics and Selection of Industrial Microorganisms, Moscow, 117545 RussiabMartsinovskii Institute for Parasitology and Tropical Medicine, Sechenov Moscow Medical Academy, Moscow, 119435 Russia

e�mail: [email protected]; [email protected]

Abstract—The optimum conditions for growth, sporulation, and crystal�formation in four isolated crystal�forming strains of Bacillus laterosporus were determined. It was shown that culture broth and pellets of bac�terial culture liquid possess larvicidal activity against larvae of mosquitoes A. stephensi and A. aegypti. The pro�tein nature of crystal was shown. Crystals are monocomponent containing a protein with MM of 68 or130 kDa. Purified protein crystals demonstrated larvicidal activity. Specific larvicidal activity of crystals ofvarious strains essentially differed. High larvicidal activity of B. laterosporus strains allows for them to be rec�ommended as producers of antimosquito biological preparations.

Key words: Brevibacillus laterosporus, entomocidal crystal, larvicidal activity, mosquito.

DOI: 10.1134/S0003683810080041

Abbreviations: DTT—dithiotreitol, DFP—diisopropylfluoro�phosphate, CL—cultural liquid, CFU—colony�forming unit,OD—optical density, PAAG—polyacrylamidegel, PMSF—phe�nylmethylsolfonylfluoride, EDTA—ethylendiaminetetraaceticacid, SDS—sodium dodecylsulphat.

756

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 46 No. 8 2010

ZUBASHEVA et al.

tions 20 and 10 μg/ml, respectively. The solutions ofaminoacids, vitamins, glucose, and salt mixtures in10� or 100�fold concentration were prepared and ster�ilized separately.

Cultivation conditions. Synchronous cultures ofyoung B. laterosporus cells while being cultivated inliquid nutritious media were obtained by the methoddescribed in [21]. A suspension of spores (OP640 =0.8 units) that was heated for 20 min up to 80°C wasused as inoculate. Incubation temperature of B. late�rosporus strains was 30 or 37°C. The duration of culti�vation varied from 12 to 96 h depending on the aims ofthe experiment. On solid nutritious media the strainswere cultivated at 30 or 37°C for 18–96 h.

Cell titer of the strains was estimated by the numberof colony�forming units in 1 ml of the sample(CFU/ml), sporulation was estimated by quantity offorming thermo stable spores in 1 ml of the sample(spores/ml), and crystal formation was estimated bycorrelation of spores and crystals that are visible undera microscope.

Obtaining of fractions of cultural supernatant andcells. Bacterial cells in different time of cultivationwere precipitated by centrifugation at 10000 g for15 min at 4°C. The pellet was washed three times fromthe nutrient medium by centrifugation in saline at10000 g for 15 min at 4°C and resuspended in physio�logical solution up to initial volume. Obtained super�natant was sterilized by filtration through bacterialmembrane filters (Millipore, United States) with porediameter 0.45 μm.

Extraction and purification of crystal proteinsB. laterosporus. Strains LAT 006, LAT 011, and IGM16�92 were grown in liquid nutritious medium BP at37°C up to the stage of mature sporangia formation (itwas controlled by light microscope). Supernatant wasseparated by centrifugation (15000 g, 15 min). Pustulewas resuspended in 0.005 M in tris�HCl�buffer,pH 7.2; 1 mM phenylmethylsulfonylfluoride(PMSP), 1 mM EDTA, and 2% Triton X�100 wereadded up to the final concentration of 0.02% andincubated for 1 h at 20°C [22]. Spore�crystal suspen�sions that were prepared in such a way were subjectedto ultrasound disintegration in the cold (2 cycles of30 s with pauses of 30 s, frequency 40 kHz) on aVCD16�850 device (BANDELIN electronicGmbH&Co, KG, Germany). The purification ofB. laterosporus crystals and spores was conducted byultracentrifugation in spore�crystal suspension ofstrains in nonlinear density gradient 20, 30, 40, and50% sodium bromide (150000 g, 2h, 4°C) [23, 24].Obtained fractions were washed from sodium bromideby three�time centrifugation (25000 g, 15 min) in dis�tilled water, resuspended in initial volume of distilledwater and microscoped. The level of purification ofcrystals from spores was controlled by optical andphase�contrast microscopy of smears that were dyedby the Smirnov method [25]. Form and size of larvi�

cidal crystals were determined by electron micros�copy [17].

Separation of proteinkinases admixture from crys�tals was conducted as described in [22, 26]. B. lat�erosporus crystal suspension was incubated in 1 MNaCl at 20°C and mixed. Crystals were washed fromsalt using three�fold centrifugation in distilled water at25000 g for 10 min at 4°C; after that the precipitatewas placed in 0.05 M tris�HCl�buffer, pH 8.5–9.5,and incubated in the presence of 1 mM diisopropylflu�orophosphate (DFP) and 1 mM EDTA on magneticstirrer for 30 min at 20°C.

Protein concentration in the samples was deter�mined by the Bradford method [27] using bovineserum albumin (Serva, Gemany) as a standard.

To analyze the content of proteins of purified crys�tals of B. laterosporus strains, the method was used thatwas provided by the authors for crystal proteins [28].Solution of crystals and protein extraction from crystalsolution were conducted by one of the followingschemes:

(1) with 0.05 M tris�HCl�buffer, pH 8.5, that con�tained 8 M urea, 0.01 M DFP, and 0.001 M EDTA at20°C for 1 h;

(2) in the same buffer at 100°C for 5 min (“hot”extraction);

(3) in 0.05 M NaOH, pH 12.0, at 20°C for 1 h; and

(4) in 0.1 N Na2CO3–NaHCO3�buffer, pH 9.5,that contains 0.01 M DFP at 20°C for 1 h.

Protein electrophoresis in the presence of sodiumdodecylsuphate was conducted in a flat layer of 7.5%polyacrylamide gel by Laemmli [29]. The proteinswere incubated with 1% SDS and 0.01 M DFP at100°C for 5 min. As a standard kit for identification ofmolecular weight, the protein mixture for SDS�gel�electrophoresis was used (Sigma (United States),stock No. SDS�6H): myosin (205 kDa), β�galactosi�dase (116 kDa), phosphorylase b (97.4 kDa), bovineserum albumin (66 kDa), ovalbumin (45 kDa), andcarboanhydrate from bovine erythrocytes (29 kDa).

Gel dye was conducted by 0.25% solution coomassiebrilliant blue R�250 (Sigma) for 5 min at 100°C.

Gel ablution was conducted by 7% acetic acid at65°C for 15 min.

Larvicidal activity of the studied samples wasassessed using larvae of mosquitoes Anophelis stephensi(Liston) and Aedes aegypti (L.) [30]. Concentration ofthe preparation that induces death of 50% of testinsects (LC50) was determined after 48�h expositionand was expressed in the final dilution of CL or quan�tity of CFU in 1 ml of the sample.

Statistical treatment of the results of the conductedstudies was carried out using the Probit computer pro�gram. The values LC50 and 95% confidence intervalare average for three experiments.


LARVICIDAL ACTIVITY OF CRYSTAL�FORMING STRAINS 757

RESULTS AND DISCUSSION

Crystal�Forming Strains B. laterosporus

The ability of bacteria B. laterosporus for crystalformation had not been established until recently [31].B. thuringiensis and B. sphaericus were previouslythought to be the only species Bacillus that producecrystal protein toxins [32]. Working with a huge collec�tion of B. laterosporus that were selected in our labora�tory as well as provided by Dr. Lecadet and Dr. Shide,we have revealed strains that were able to form crystalsof various morphology and size [33, 34]. It was inter�esting to study characteristics of the mentionedstrains, reveal optimal conditions for bacteria cultiva�tion, assess insecticide activity, and determine physi�cal�chemical and biological characteristics of crystals.

This work presents the thorough study of the fol�lowing features and characteristics of B. laterosporusstrains IGM 16�92, IGM 16�13, LAT 006, and LAT011: morphology, influence of various cultivation fac�tors on ability to form spores and crystals, dynamics ofsynthesis and localization of larvicidal factors of crys�tal�forming strains B. laterosporus, toxicity of paraspo�ral crystals of B. laterosporus strains for various speciesof mosquitoes, and protein content of crystals.

Morphological Characteristics of Crystal�Forming Strains

Strains form ellipsoid spores with canoe�like inclu�sions attached to a spore, as well as parasporal crystalinclusions. Inclusion in the form of a canoe—a char�acteristic morphological structure B. laterosporus [35,36]—usually remains bound with a spore, whereascrystals separate from spores during lysis of spo�rangium as it occurs in B. thuringiensis [37] (but not inB. shpaericus). Because crystals of strains IGM 16�92,IGM 16�13, and LAT 006 have been thoroughlydescribed before, here, only their separate characteris�tics are presented (Table 1). Crystals of B. laterosporusstrains are smaller and they are surrounded by mem�

branes. Strain IGM 16�92 forms small rhombic crys�tals with clearly delineated corners with the size 0.4 ×0.3 μm (Fig. 1).

Influence of Cultivation Conditions on the Ability of B. laterosporus for Growth, Sporulation, Crystal

Formation and Level of Larvicidal Activity

To reveal conditions that influence the ability ofB. laterosporus strains for sporulation and crystal for�mation, liquid and solid nutritious media of variouscontent that are recommended for cultivation of ento�mopathogen bacillus were used [18, 31], cultivationtemperature was also varied (30 and 37°C). LAT 006served as a model strain. It is known that level ofsporulation and crystal formation of B. thuringiensisand B. sphaericus significantly depends on the contentof medium that is used for cultivation [20]. The stud�ied strain LAT 006 was grown on liquid and solidnutritious media that contain peptones, triptone, meatextract, glucose, yeast extract, and mineral salts in var�ious compositions. In some media, thiamine and L�methionine were added because bacteria B. lat�erosporus are auxotrophs by these growth factors [39].

As main criteria for medium choice growth (pro�ductivity), ability for sporulation, crystal formation,and larvicidal activity were assessed. Analysis of 13nutritious medium let us reveal BP medium (see“Conditions of Experiment”) that respond with theaims: provided maximal productivity (>108 CFU/ml),a significant level of sporulation (more than 90%), anda high level of crystal formation; furthermore, the cor�relation between sporulation and crystal formationwas revealed. Obtained data confirmed the value ofmineral salts for the processes of sporulation and crys�tal formation [20]. Microscopic observations enabledus to establish that the cultures of LAT 006 strains thatwere grown on complex nutritious media with nutri�tious broth, salts, growth factors, and glucose were themost homogeneous by morphological content to theend of growth.

Table 1. Crystal�forming strains B. laterosporus

Strain Crystals form in the plane Size of crys�tals, μm

LC50 of purified crystals, μg/ml when tested on Molecular weight

of crystals, kDaA. stephensi A. aegypti

LAT 006 0.4 × 0.40.005 0.003 68

0.2 × 0.7

IGM 16�92 0.4 × 0.3 2.0 4.0 68

IGM 16�13 0.7 × 0.5 was not identified was not identified was not identified

LAT 011 0.5 × 0.2 0.5 was not identified 130

758


ZUBASHEVA et al.

The differences in insecticide activity of spore�crystal preparations of LAT 006 strains that wereobtained on tested nutritious media were revealed.Maximal toxicity was revealed when bacteria weregrown on BP medium for 96 h; LC50 value of such cul�ture in relation to A. aegypti larvae was ≤104 CFU/ml.When cultivated on other media, strain LAT 006 didnot posses a higher level of sporulation and crystal for�mation in comparison with complex medium, but bio�logical activity of the strain that is determined by tox�icity against A. aegypti larvae was high (data is not pre�sented). This data, from our point of view, indicatesthat larvicidal activity of strain LAT 006 is conditionednot only by its ability for sporulation and crystal for�mation but other factors (soluble toxins, bacterio�cines, hemolysisis, proteases) also have a significantcontribution to toxicity.

According to studied features (productivity by bio�mass, sporulation, crystal formation, and larvicidalactivity) the following LAT 006 strain cultivation con�ditions turned out to be optimal: BP medium, incuba�tion duration 96 h, and incubation temperature 37°C.

For some strains, productivity (spore titer) turned outto be higher when they were cultivated at 30°C. Culti�vation method—liquid or solid medium—was insig�nificant. Table 2 presents indices of the studied char�acteristics of strain LAT 006 under optimal cultivationconditions.

It is interesting that spore : crystal ratio is a speciescharacteristics of spore�forming bacillus. For exam�ple, this value for B. thuringiensis spp. on the final stageof cultivation is ≤1 [40], whereas, for B. laterosporus, itis 1.0–1.5 (see Table 2).

The influence of temperature on growth and ento�mocide activity of strain LAT 006 was studied (Table 3).It was shown that larvicidal activity of strains at 37°C ishigher than at 30°C.

Study of Dynamics of Synthesis and Localization of Larvicidal Factor of Crystal�Forming

B. laterosporus Strains

It was interesting to determine dynamics of appear�ance of larvicidal activity in the process of growth ofcrystal�forming strains B. laterosporus and reveallocalization of this activity under its maximal value.The toxicity of strain LAT 006 was compared for mos�quito A. stephensi at different stages of strain growth(vegetative and sporulation and synthesis of crystaltoxin). The results are presented in Fig. 2.

Exponential phase of the studied culture isobserved between 8 and 20 h after seeding (see Fig. 2).It is known that culture is homogeneous at the expo�nential growth phase, especially in its first half, inten�sive cell death is not detected, and the quantity ofincoming products of autolysis in nutritious medium isinsignificant [41]. However, at early exponential growthphase (8 h) the culture does not possess detected toxicity

(a) (b)

Fig. 1. Spore and crystal inside sporangium of LAT 011 strain: (a) is ultrathin section, instrumental magnification power ×30000and (b) is negative staining, instrumental magnification power ×10 000.

Table 2. Characteristics of LAT 006 strain under optimalconditions of cultivation

Characteristics Index

Spores, CFU/ml ≥108

Sporulation frequency, % 90–95

Frequency of crystal formation spore : crystal ratio = 1.0–1.5

Incubation time, h 96

Incubation temperature, °C 37

Larvicidal activity (LC50), CFU/ml For A. stephensi ≤104



for insects (see Fig. 2). Vegetative culture (18 h) LAT 006with titer more than 107 CFU/ml and quantity of spores4.4 ×104 in 1 ml (less than 0.3%) possesses the least toxic�ity for mosquitoes A. stephensi. Value LC50 of 18�h culturewas 2.0 × 105 CFU/ml. Larvicidal activity of 96�h cul�ture was approximately two degrees higher than 18�hculture of LAT 006, which could be due to increase ofspore and crystal quantity: culture LAT 006 titer was≥108 CFU/ml, level of sporulation reached 80%, andLC50 value was approximately 1.0 × 104 CFU/ml.

Thus, as a result of the study of dynamics of larvi�cidal activity of crystal�forming strain LAT 006, it wasestablished that culture at the stage of sporulation(96 h) that produces thermo stable spores and crystalspossesses maximal toxicity.

To determine localization of larvicidal factor at thestage of the most active production (96 h), the activityof B. laterosporus LAT 006 strain culture (CL, super�natant, and precipitate) fraction activity was studied.The analysis indicated that practically all larvicidalactivity against A. stephensi under its maximal value—at the stage of sporulation—was in the precipitate, i.e.,it was connected with crystals and spores. In the frac�tion of supernatant, no CFU were revealed. Similarresult was obtained in the analysis of strain IGM 16�13.Cultural liquid and precipitate of spore cultures ofstrains IGM 16�13 and LAT 006 had the same level oftoxicity for larvae A. stephensi. Values LC50 of culturalsupernatant were approximately 10–1000 times lessthan LC50 values for CL and precipitate (Table 4).

This data indicates that larvicidal activity of B. lat�erosporus strains against mosquitoes A. stephensi wasmostly connected with CL and precipitate that con�tain crystals and spores but not with soluble substancesthat are present in cultural supernatant.

The highest insecticide activity that is present insupernatant of spore culture could be due to LAT 006cell production of some soluble factors (noncrystaltoxins).

It is known that vegetative cells of B. sphaericusproduce nonsecreted protein toxins Mtx that arelocalized inside or on the surface of bacterial cells [15,40]. Mtx�toxins of B. sphaericus may be present in thecells connected with membrane form. Larvicidal

activity of vegetative cells of B. laterosporus strain LAT006 is due to similar Mtx�toxins.

Thus, the study of insecticide activity of variousfractions of B. laterosporus that were obtained as aresult of centrifugation of cultures of crystal�formingstrains LAT 006 indicated that practically all insecti�cide activity against larvae A. stephensi at the stage ofsporulation was connected with precipitate fraction,i.e., with crystal inclusions or spores (see Table 4).

Table 3. Influence of cultivation temperature on growth and larvicidal activity of B. laterosporus strains LAT 006 and IGM16�92 against A. agypti larvae

Growth and activity parametersLAT 006 IGM 16�92

30°C 37°C 30°C 37°C

Titer, CFU/ml 2.0 × 107 2.0 × 108 2.0 × 108 1.0 × 108

Crude protein, µg/ml 150 320 340 400

LC50, CFU/ml 6.0 × 104 8.0 × 103 2.0 × 106 2.35 × 105

LC50, (crude protein), µg/ml 0.45 0.128 1.7 0.94

1.0E + 09

1.0E + 08

1.0E + 07

1.0E + 06

1.0E + 05

1.0E + 04

1.0E + 037230 96481880

Time, h

Titer, CFU/ml

1

2

Fig. 2. (1, 2) Dynamics of growth and (3) larvicidal activityof B. laterosporus strain LAT 006 against mosquitoesA. stephensi: 1 is cell concentration, 2 is spore concentra�tion, and 3 is LC50.

3

760


ZUBASHEVA et al.

Larvicidal Activity of Purified Crystals B. laterosporus

To establish true protein content of B. laterosporuscrystal, it is necessary to extract them under condi�tions that totally preclude the possibility of proteolysis.It is known that proteins of B. laterosporus crystalscould be subjected to hydrolysis by their own proteasesthat are adsorbed on crystals or included in defects ofcrystal lattice in the process of sporulation [26, 42].Thus, inter and extracellular serine proteinases (thatare characteristic for other subspecies of BT too),extracellular methalproteinases, and some other pro�teinases are the source of proteolytic activity in the BTipreparations. To prevent the contact of proteins ofB. laterosporus crystals with extracellular proteinases,the cultivation of microorganisms was stopped at thestage of mature sporangia (36–48 h). Before sporangiadisruption by ultrasound, inhibitors of serine protein�ases (PMSP) and methalproteinases (EDTA) wereadded to the cell suspension.

The spore and crystal inclusions of B. laterosporusstrains LAT 006, LAT 011, and IGM 16�92 were sep�arated under ultracentrifugation in nonlinear densitygradient of sodium bromide. As a result, the fractionswere obtained that correspond to 30% density of NaBrand contain only crystals of strains LAT 006, LAT 011,and IGM 16�92. Spore and crystal suspensions andfractions of pure crystals possessed toxicity against lar�vae A. aegypti and A. stephensi. Purification of proteincrystals of B. laterosporus was accompanied byincrease of their toxicity against larvae of two mos�quito species (data is not presented). It is known thatLC50 of purified BTi crystals for A. aegypti larvae is inthe range from 4 to 20 ng/ml and that for A. stephensiis a bit less [43].

Protein Content of Purified Crystals of B. laterosporus Strains

It is known that one of the factors that determinerevealed plurality of protein forms of B. thuringiensiscrystals is the presence of admixtures of bacterial pro�teinases [26]. Under certain conditions and especiallyunder full or partial denaturation of carrier protein,

doped proteinases, in spite of their low content, areable to effectively split protein forming more or less bigfragments. Degradation of initial crystal protein isusually observed at pH 8.5 (20°C) or at pH 12.5(37°C). To establish true protein content, it is neces�sary to solve crystals under conditions that totally pre�clude the possibility of proteolysis [28]. Protein con�tent of purified crystals of B. laterosporus strains LAT006, LAT 011, and IGM 16�92 were analyzed with thehelp of various disintegrating reagents with prelimi�nary inactivation of proteinases in 1 M of NaCl for30 min at 20°C as well as in 1mM DFP and 1 mMEDTA for 30 min at 20°C.

It was established that B. laterosporus crystals aswell as B. thuringiensis do not solve in water, buffersolutions, and organic solvents under usual condi�tions. It was possible to solve crystals by two ways: (1)using reagent that causes denaturation of proteins(urea) and recovery of disulfide bridges (dithiotreitol);(2) in high alkaline medium (pH 12 and higher). Toanalyze the protein content of purified crystals ofB. laterosporus strains, the method of “hot” extractionwas used that is introduced for B. thuringiensis crystals[28] and makes it possible to inactivate proteinasesthat are adsorbed on the crystals surface (treatment at100°C).

As a result of the conducted experiments, it wasestablished that total solution of crystals of B. lat�erosporus strains occurred under simultaneous actionof denaturating and recovery agents, in particular 8 Murea and 0.01 M DFP, pH 8.5, at 20°C for 1 h and at100°C for 5 min (“hot” extraction) as well as in 0.01 MNaOH, pH 12.5, for 60 min on them. The crystal solu�tions that were obtained in such a way, according todata of electrophoresis in 7.5% SDS�PAAG, con�tained similar protein components (Fig. 3). At pH9.5–11.5, in the presence of recovery agents, partialloss of protein components of B. laterosporus crystalsoccurred (data is not presented), as was indicated forB. thuringiensis.

Electrophoretic analysis of crystal proteins ofB. laterosporus strains indicated the presence of onemajor peptide with molecular weight 68 kDa in the

Table 4. Larvicidal activity of fraction of spore culture B. laterosporus in relation to mosquitoes A. stephensi

SampleLAT 006 IGM 16�13

CFU/ml LC50* CFU/ml LC50*

CL 2.0 × 108 8.0 × 103 1.0 × 108 2.35 × 105

(4.0 × 10–5) (4.7 × 10–3)

Supernatant** spore were not revealed 1.0 × 10–2 spore were not revealed 4.0 × 10–2

Precipitate 2.0 × 108 8.0 × 103 1.0 × 108 2.4 × 105

(4. × 10–5) (4.7 × 10–3)

Notes: * Values LC50 are presented in terms of CFU/ml or dilution (are given in brackets).** Because no CFU was revealed in supernatant LC50, values for supernatant are presented in dilutions.



studied strain B. laterosporus. The crystals of LAT 006strain also contained one major peptide with molecu�lar weight 68 kDa (see Fig. 3). The preparation of crys�tals of LAT 011 strain demonstrated the presence ofone bond of weight 130 kDa, but protein production inthis strain was significantly less (in 50 times) in com�parison with strains IGM 16�92 and LAT 006.

Thus, the study of the content of crystal proteins ofB. laterosporus strains under conditions that precludeproteolysis revealed the presence in crystals of B. lat�erosporus strains IGM 16�92 and LAT 006 of onemajor protein with molecular weight 68 kDa and incrystals of LAT 011 strain of one protein with molecu�lar weight 130 kDa (see Fig. 3). The comparison ofdata of electrophoresis indicates that crystals of B. lat�erosporus strains are apparently one�component pro�tein systems with various level of toxicity against mos�quitoes. It makes the extraction and more detailedcomparative characteristics of crystal proteins (immu�nologic, by aminoacid content) necessary.

The summary data on physical chemical and larvi�cidal features of B. laterosporus crystals are presentedin Table 1.

The presented data indicate that specific larvicidalactivity of crystals of B. laterosporus strain LAT 006(3–5 ng/ml) is comparable with activity of B. thuring�

iensis and B. sphaericus crystals, which lets us recom�mend B. laterosporus as a potential producer of biolog�ical mean of insect control.

BIOGRAPHIES

Zubasheva Margarita Vladimirovna, GanushkinaLyudmila Alimp’evna, Smirnova Tatyana Aleksan�drovna, Azizbekyan Rudolf Rubenovich

REFERENCES

1. Shida, O., Takagi, H., Kadowaki, K., and Komagata, K.,Int. J. Syst. Bacteriol., 1996, vol. 46, pp. 939–946.

2. Orlova, M.V., Smirnova, T.A., Shamshina, T.N.,Kovalenko, N.A., and Azizbekyan, R.R., Biotekh�nologiya, 1995, nos. 1–2, pp. 22–26.

3. Gerard, J.M., Haden, P., Kellyk, M.T., andAndersen, R.J., J. Nat. Prod., 1999, vol. 62, no. 1,pp. 80–85.

4. Desjardine, K., Pereira, A., Wright, H., Matainaho, T.,Kelly, M., and Andersen, R.J., J. Nat. Prod., 2007,vol. 70, no. 12, pp. 1850–1853.

5. Barsby, T., Kelly, M.T., and Andersen, R.J., N. Nat.Prod., 2002, vol. 65, no. 10, pp. 1447–1451.

6. Favret, M.E. and Yousten, A.A., J. Invertebr. Pathol.,1985, vol. 45, pp. 195–203.

7. Orlova, M.V., Smirnova, T.A., Ganushkina, L.A., andAzizbekyan, R.R., Appl. Environ. Microbiol., 1998,vol. 64, pp. 2723–2725.

8. Ruiu, L., Delrio, G., Ellar, D., Floris, I., Paglietti, B.,Rubino, S., and Satta, A., Entomol. Exper. Appl., 2006,vol. 118, pp. 137–144.

9. de Oliveira, E.J., Rabinovitch, L., Monnerat, R.G.,Konovaloff, L., Jannotti, P.J., and Zahner, V., Appl.Enriron. Microbiol., 2004, vol. 70, no. 11, pp. 6657–6664.

10. Huang, X., Tian, B., Niu, Q., Yang, J., Zhang, L., andZhang, K., Res. Microbiol., 2005, vol. 156, nos. 5–6,pp. 719–727.

11. Donnell, B., US Patent no. 5702701, C12N001/29,1997.

12. Sanders, M.E., Morelli, L., and Tompkins, T.A., Comp.Rev. Food Sci. Food Saf., 2006, vol. 2, pp. 101–110.

13. Kamiyama, T., Umino, T., Nakamura, Y., Itezono, Y.,Sawairi, S., Saton, T., and Yokose, K., J. Antibiot.,1994, vol. 47, pp. 959–968.

14. Bachtel, D.B. and Bulla, L.A., J. Bacteriol., 1979,vol. 127, pp. 1472–1481.

15. Charles, J.F., Nielsen�Leroux, C., Delecluse, A., andCharles, J.F., Ann. Rev. Entomol., 1996, vol. 41,pp. 451–472.

16. Rivers, D.B., Vann, C.N., Zimmack, H.L., andDean, D.H., J. Invertbr. Pathol., 1991, vol. 58,pp. 444–447.

17. Smirnova, T.A., Shamshina, T.N., Konstantinova, G.E.,Gannushkina, L.A., Kuznetsova, N.I., Nikolaenko, M.A.,and Azizbekyan, R.R., Biotekhnologiya, 1993, no. 9,pp. 11–15.

kDa 1 2 3 4 5 6 7

205

116

97.4

66

45

29

130

68

kDa

Fig. 3. SDS�PAAG�electrophoresis (7.5% gel) of proteinsthat were extracted by various solvents from crystals ofB. laterosporus strains : 1 is standards of molecular weight;2 is solution of crystals of LAT 011 strain in 0.01 M tris�HCl�buffer, pH 8.5, that contains 8 M urea and 0.01 Mdithioerythrite at 100°C for 5 min (“hot” extraction); 3 issolution of crystals of strain LAT 011 in 0.01 M NaOH at20°C for 60 min; 4 is solution of crystals of strain IGM 16�92in 0.01 M tris�HCl�buffer, pH 8.5, that contains 8 M ureaand 0.01 M dithioerythrite at 100°C for 5 min (“hot”extraction); 5 is solution of crystals of strain IGM 16�92 in0.01 M NaOH at 20°C for 60 min; 6 is solution of crystalsof strain LAT 006 in 0.01 M tris�HCl�buffer, pH 8.5, thatcontains 8 M urea and 0.01 M dierythrite at 100°C for5 min (“hot” extraction); and 7 is solution of crystals ofstrain LAT 006 in 0.01 M NaOH at 20°C for 60 min.

762


ZUBASHEVA et al.

18. Zeigler, D.R., Bacillus Genetic Stock Center. Catalog ofStrains, 7th ed., Ohio: The Ohio State University, 1999,vol. 2, pp. 1–56.

19. Thorne, C., J. Virol., 1968, vol. 2, pp. 657–661.20. Nickerson, K.W. and Bulla, L.A., Appl. Microbiol.,

1974, vol. 28, pp. 124–128.21. Nakata, H.M. and Halvorson, H.O., J. Bacteriol.,

1960, vol. 80, pp. 801–810.22. Zalunin, I.A., Kostina, L.I., Chestukhina, G.G.,

Brmatova, M.E., Klepikova, F.S., Khodova, O.M., andStepanov, V.M., Biokhimiya, 1986, vol. 51, no. 3,pp. 449–457.

23. Pfannenstiel, M.A., Crey, W.C., Jr., Couche, G., andNickerson, K.W., Appl. Environ. Microbiol., 1990,vol. 56, no. 1, pp. 162–166.

24. Ibarra, J.E. and Fererici, B.A., J. Bacteriol., 1986,vol. 165, no. 2, pp. 527–533.

25. Smirnoff, W.A., J. Insect. Pathol., 1962, vol. 4, pp. 384–386.

26. Chestukhina, G.G., Zalunin, I.A., Kostina, L.I.,Kotova, T.S., Katrukha, S.P., Lyublinskaya, L.A., andStepanov, V.M., Biokhimiya, 1978, vol. 43, no. 5,pp. 857–864.

27. Bradford, M., Anal. Biochem., 1976, vol. 72, pp. 248–254.

28. Zalunin, I.A., Chestukhina, G.G., and Stepanov, V.M.,Biokhimiya, 1979, vol. 44, no. 4, pp. 693–698.

29. Laemmli, U.K., Nature (London), 1970, vol. 227,pp. 680–685.

30. Voitsik, A.A. and Rasnitsyn, S.P., Med. Parazitol., 1992,no. 4, pp. 55–57.

31. Claus, D. and Berkeley, R.C.W., in Bergey’s Manual ofSystematic Bacteriology, Sneath, P.H.A., Mair, N.S.,Sharpe, M.E., and Holt, J.G., Eds., Baltimore: Will�iams Wilkins, 1986, vol. 2, pp. 1105–1139.

32. Charles, J.F. and de Barjac, H., An. Microbiol. (Inst.Pasteur), 1982, vol. 133A, pp. 425–442.

33. Smirnova, T.A., Minenkova, I.B., Orlova, M.V., Leca�det, M.�M., and Azizbekyan, R.R., Res. Microbiol.,1996, vol. 47, pp. 343–350.

34. Smirnova, T.A., Poglazova, M.N., Orlova, M.V., andAzizbekyan, R.R., Biotekhnologiya, 1997, no. 1,pp. 29–36.

35. Smirnova, T.A. and Azizbekyan, R.R., Biotekhnologiya,2002, no. 5, pp. 8–13.

36. Fitz�James, P.C. and Young, I.E., J. Biochem. Cytol.,1958, vol. 4, pp. 639–649.

37. Lecadet, M.�M., Microbial. Toxins, 1970, vol. 3,pp. 437–440.

38. Insell, J.P. and Fitz�James, P.C., Appl. Environ. Micro�biol., 1985, vol. 50, pp. 56–62.

39. Grigor’eva, T.M. and Azizbekyan, R.R., Biotekh�nologiya, 2002, no. 4, pp. 31–37.

40. Charles, J.F. and Nielsen�Leroux, C., Mem. Inst.Oswaldo Cruz, 2000, vol. 95, pp. 201–206.

41. Grigor’eva, T.M. and Azizbekyan, R.R., in Mat. 2�goMezhdunarodnogo kongressa “Biotekhnologiya�sostoya�nie i perspektivy razvitiya” (Proc. 2nd Int. Congr. “Bio�technology: State and Prospects of Development”),Moscow, 2003.

42. Chestukhina, G.G., Kotova, T.S., Zalunin, I.A., andStepanov, V.M., Biokhimiya, 1979, vol. 44, pp. 796–802.

43. Federici, B.A., Luthy, P., and Ibarra, J.A., in BacterialControl of Mosquitoes and Black Flies: Biochemistry,Genetics and Application of Bacillus thuringiensisisraelensis and Bacillus sphaericus, de Barjak, H. andSazerland, D.J., Eds., Rutgers: University Press, 1990,pp. 16–44.

Documents

Larvicidal activity of crystal-forming strains of Brevibacillus laterosporus