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Genetic Diversity of Bacillus thuringiensis from Different Geo-Ecological Regions of Ukraine by Analyzing the 16S rRNA and gyrB Genes and by AP-PCR and saAFLP
N. V. Punina1, 2*, V. S. Zotov1, A. L. Parkhomenko3, T. U. Parkhomenko3, A. F. Topunov1 1Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospect, 33, bld. 2, Moscow, Russia, 119071
2Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moskvorechje str.,1, Moscow, Russia, 115478
institute of Agriculture of Crimea, National Academy of Agrarian Sciences of Ukraine, Kievskaya str., 150, Simferopol, Ukraine, 95453 *E-mail: hin-enkelte@yandex.ru Received 02.10.2012
Copyright © 2013 Park-media, Ltd. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT The Bacillus cereus group consists of closely related species of bacteria and is of interest to researchers due to its importance in industry and medicine. However, it remains difficult to distinguish these bacteria at the intra- and inter-species level. Bacillus thuringiensis (Bt) is a member of the B. cereus group. In this work, we studied the inter-species structure of five entomopathogenic strains and 20 isolates of Bt, which were collected from different geo-ecological regions of Ukraine, using various methods: physiological and biochemical analyses, analysis of the nucleotide sequences of the 16S rRNA and gyrB genes, by AP-PCR (BOX and ERIC), and by saAFLP. The analysis of the 16S rRNA and gyrB genes revealed the existence of six subgroups within the B. cereus group: B anthracis, B. cereus I and II, Bt I and II, and Bt III, and confirmed that these isolates belong to the genus Bacillus. All strains were subdivided into 3 groups. Seventeen strains belong to the group Bt II of commercial, industrial strains. The AP-PCR (BOX and ERIC) and saAFLP results were in good agreement and with the results obtained for the 16S rRNA and gyrB genes. Based on the derived patterns, all strains were reliably combined into 5 groups. Interestingly, a specific pattern was revealed by the saAFLP analysis for the industrial strain Bt 0376 p.o., which is used to produce the entomopathogenic preparation “STAR-t”.
KEYWORDS Bacillus cereus group; B. thuringiensis; 16S ribosomal RNA; gyrB; saAFLP; taxonomy; phylogeny. ABBREVIATIONS Bt - Bacillus thuringiensis; ICPs - 8-endotoxins; b.p. - base pair; MLST - multilocus sequence typing; MEE - multilocus enzyme electrophoresis; saAFLP, AFLP - single adapter amplified fragment length polymorphism; RFLP - restriction fragment length polymorphism; AP-PCR - arbitrarily primed polymerase chain reaction; rep-PCR - repetitive sequence-based PCR; BOX, ERIC - DNA repeats: ME - minimum evolution; NJ - neighbor joining.
INTRODUCTION
Bacillus thuringiensis (Bt) are gram-positive bacteria that exhibit bioinsecticide activity due to their ability to produce 6-endotoxins (ICPs), or Cry proteins, during sporulation [1]. These toxins are active for a wide range of insect species and genera, including agricultural pests and human parasites [2, 3]. Due to the high specificity of ICPs, entomopathogenic Bt bacteria can be used, instead of pesticides, and are widely employed in designing bioengineered crop protection agents [4, 5].
Based on a phenotypic and genotypic analysis, Bt species were attributed to the B. cereus group. This group also comprises the closely related species
B. cereus, B. anthracis, B. mycoides, B. pseudomycoides, and B. weihcnstephanensis. The B. сereus and Bt species cannot be distinguished using the morphological [6], phenotypic [7], or genetic methods [8-11]. It has been hypothesized that these species can belong to the same species, B. cereus sensu lato [12, 13]. Since this group of closely related bacteria is of significant interest for agriculture and medicine, a thorough investigation into their taxonomy, as well as an elaboration of new tools and technologies for their differentiation and isolation, remains a rather urgent task.
Bt strains were conventionally isolated and further divided into subspecies according to either the pres-
ence or absence of ICP crystals or the genes encoding them (cry and cty) [1, 3]. However, this method has a drawback: the ICP’s genes are localized on the plasmid, and bacteria can lose them or pass them to the other Bt strains or closely related bacterial species during conjugation [14]. Over 82 Bt serovars were revealed by a serological analysis of the flagellar antigen (H-serotyping) [15, 16]. However, such classification did not always correlate with the actual phylogenetic relationships for this species [17-19].
The genetic diversity of Bt bacteria and the possibility to distinguish between the two species, Bt and B. cereus, were studied using different methods: DNA-DNA hybridization [20] and the analysis of the nucleotide sequences of 16S rRNA, 23S rRNA, 16S-23S rRNA [8, 11], MLST [21], MEE [12, 18], AFLP [22-24], RFLP [25], AP-PCR [26-29], etc. However, the actual phylogenetic relationships between Bt have not been determined by these methods.
This work was aimed at assessing how the modified genomic fingerprinting technique (saAFLP) could be applied to reveal the phylogenetic differences between Bacillus sp. isolates and strains from various geo-ecological regions of Ukraine. The nucleotide sequences of the 16S rRNA and gyrB genes were analyzed in order to determine the taxonomic relationships at the genus-species level. The saAFLP method, along with other informative methods (rep-PCR), was used to study the structure at the intra-species level. This complex diagnostics, together with the results of physiological and biochemical assays, offers broad opportunities for studying the taxonomic structure of these closely related organisms. However, it should be borne in mind that the sampling of Bt strains requires further broadening.
MATERIALS AND METHODS
Bacterial strains
Five entomopathogenic strains and 20 isolates of Bt bacteria exhibiting unique biochemical properties from a collection of useful microorganisms of various ukrainian and Russian research institutions (Institute of Agriculture of Crimea, National Academy of Agrarian Sciences of ukraine, Simferopol, Autonomous Republic of Crimea, Ukraine; Institute of Agricultural Microbiology, National Academy of Agrarian Sciences of Ukraine, Chernigov, Ukraine; All-Russian Collection of Industrial Microorganisms “GosNIIGenetika”, Moscow, Russia) were used in this study. Five strains from the collection of the All-Russian Collection of Industrial Microorganisms “GosNIIGenetika” were used as standard strains. Isolates from the collection of the Institute of Agriculture of the Crimea, National Acad-
emy of Agrarian Sciences of Ukraine, were isolated in different geo-ecological regions of Ukraine.
DNA isolation
The overall cellular DNA specimens were isolated from strains cultured on agarized TY medium (g/l): yeast extract - 1.0; peptone - 10.0; CaCl2 - 0.4; agar - 20.0. DNA was isolated from fresh cultures on days 1-2 of growth via sorption onto magnetic particles (Mini-prep kit, Silex, Russia).
Phenotypic characterization
The morphological and physiological-biochemical characteristics of the pure bacterial cultures were determined based on the general strategy of phenotypic differentiation described in A Guide for Bacterial Identification [30] and Methods for General Bacteriology [31].
PCR amplification and sequencing of the 16S rRNA gene
The PCR analysis and subsequent determination of the nucleotide sequences of the 16S rRNA gene [32] were conducted on a genetic analyzer using the universal primers 27f (5’-GTTTGATCMTGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’)
[33]. The amplified fragments were detected by electrophoresis in 1.5% agarose gel. Sequencing was carried out on a Genetic Analyzer 3130xl ABI automated sequencing machine (Applied Biosystems, USA).
PCR amplification and sequencing of the gyrB gene The gyrB gene was amplified and sequenced using the previously constructed primer systems UP1 and UP2r
[34], the Bacillus genus-specific primers designed by us, gyrB_F (5’-CTTGAAGGACTAGARGCAGT-3’) + gyrB_Rf (5’-CCTTCACGAACATCYTCACC-3’) and gyrB_Fr (5’-GGTGARGATGTTCGTGAAGG-3’) + gyrB_R (5’-TGGATAAAGTTACGACGYGG-3’), and the protocol. The temperature-time profile of the reaction was as follows: the initial denaturation at 940C - 2 min; then, 30 cycles: 940C - 30 s, 620C - 30 s, 720C -1 min; final elongation - 5 min at 720C. The amplified fragments were revealed by electrophoresis in 1.5% agarose gel. Sequencing was carried out on a Genetic Analyzer 3130xl ABI (Applied Biosystems, USA).
PCR using primers to different repeating elements (rep-PCR)
The previously described primer systems [26, 27] ERIC1R 5’-ATGTAAGCTCCTGGGGATTCAC-3’; ERIC2 5’-AAGTAAGTGACTGGGGTGAGCG-3’; and BOXA1R 5’-CTACGGCAAGGCGACGCTGACG-3’ were used for rep-PCR.
Amplification was carried out in 25 |^l of the following mixture: 1x polymerase buffer BioTaq (17 mM (NH4)2SO4, 6 mM Tris-HCl, pH 8.8, 2 mM MgCl2), 5 nM dNTP, 50 ng of DNA template, 12.5 pM of the primer, and 1.25 AU of BioTaq DNA polymerase (Dialat Ltd., Russia). The temperature-time profile of the reaction: first cycle - 940C, 2 min; subsequent 40 cycles - 940C, 20 s; 400C, 30 s and 720C, 90 s; final elongation - 7 min at 720C. The PCR products were analyzed by electrophoresis in a 1.5% agarose gel stained with ethidium bromide at a field intensity of 6 V/cm and documented using the BioDoc Analyze system (Biometra, Germany).
saAFLP analysis [35]
We had modified the AFLP method developed and patented by M. Zabeau and P. Vos [36]; its suitability for the analysis of closely related Bt strains was assessed in this study. The phylogenetic relationships between closely related strains of various species belonging to the genus Rhizobium had been successfully analyzed using this modified saAFLP method [35]. The saAFLP procedure comprises three steps: (I) simultaneous treatment of the extracted bacterial DNA in the same tubes using one of the restriction endonucleases (XmaJI, XbaI, PstI) and ligation with a singlestranded adapter Ad.CTAG1; (II) PCR amplification with a single primer complementary to the Ad.CTAG1 sequence; (III) electrophoretic separation of the PCR products in agarose gel. The fundamentally new aspects for this saAFLP method include conducting the restriction analysis and the ligase reaction in the same tube, using restriction endonucleases XmaJI (XbaI, PstI) to study the phylogenetic relationships between the Bt strains isolated in various geo-ecological regions of Ukraine, and using only the single-stranded adapter Ad.CTAG1.
The restriction analysis was carried out simultaneously with the ligation in 10 |^l of the mixture containing 80 ng of the DNA sample, the ligase buffer (Fermentas, USA), 10 pM of the single-stranded adapter Ad.CTAG1 (5’-ctagCTGGAATCGATTCCAG-3’), 5 AU of T4 DNA ligase (Fermentas, USA), and 1 AU of restrictase XmaJI (XbaI, PstI). The resulting mixture was incubated at 370C for 2 h. The reaction volume was then brought up to 100 |^l. PCR was carried out on a Mastercycler Gradient Eppendorf amplifier in 25 |^l of the mixture containing 1 x PCR buffer, 2.8 mM MgCl2, 0.2 mM dNTP, 2 ^l of the restrictase-ligase mixture as a DNA template, 0.4 ^l of primer Pr.CTAG1 (5’-CTGGAATCGATTCCAGctag-3’) complementary to the adapter, and 1 AU BioTaq DNA polymerase (Di-alat Ltd., Russia). PCR amplification was carried out in the following mode: initial denaturation - 940C, 2 min,
followed by 30 cycles - 940C, 30 s; 400C, 30 s; 720C, 3 min; final elongation - 5 min at 720C.
Analysis of nucleotide sequences
The primary comparative analysis of the nucleotide sequences determined in this study and represented in the GenBank database was carried out using the NCBI Blast software [37]. Sequence alignment was performed using the CLUSTALW 1.75v. software [38]; the sequences were verified and edited using BioEdit 7.0.5.3 [39] and Mega 3.1 [40] editors. The phylogenetic trees were constructed in the Mega 3.1 software [40] using the neighbor joining (NJ) [41] and minimum evolution (ME) [42] methods. The statistical significance of the branching order of the resulting trees was determined using the bootstrap analysis by constructing 1,000 alternative trees.
RESULTS AND DISCUSSION
Analysis of the nucleotide sequences of the 16S rRNA gene
The analysis of the nucleotide sequences of the 16S rRNA gene is frequently used for taxonomic localization and the identification of the bacterial genus/species. We amplified and sequenced the PCR fragments of the 16S rRNA gene (the size of the sequenced region was 1386 bp) of five typical strains of genus Bacillus and 20 Ukranian isolates to verify their taxonomic attribution to the genus Bacillus. Similar nucleotide sequences of the 16S rRNA gene of B. cereus, Bt, B. anthracis, B. my-coides, B. pseudomycoides, and B. weihcnstephanensis were obtained from the database of the National Center for Biotechnology Information (NCBI, USA) and used for comparative purposes. The nucleotide sequences of B. pumilus, B. licheniformis, and B. subtilis were selected as the remote control for the phylogenetic analysis. A phylogenetic tree representing the evolution of the analyzed gene was constructed based on the aligned sequences using the ME algorithm (Fig. 1). The pairwise genetic distances were calculated using the Kimura’s two-parameter model.
The topology of the resulting tree was consistent with the phylogenetic structure of the genus determined by DNA-DNA hybridization [43] and established for the B. сereus group by the analysis of the 16S rRNA, 23S rRNA gene fragments [8, 11], and the 16S-23S rRNA intergenic region [44], rep-PCR [29], and AFLP [23].
The attribution of the isolates to the genus Bacillus has been verified by analysing the nucleotide sequences of the 16S rRNA gene. However, this method did not allow one to reliably distinguish individual species within the B. cereus group due to the fact that the sequence of
62
99
EF210294. B. thuringiensis ser tohokuensis EBC-T17 AB617500 B thuringiensis ser neoleonensis B. thuringiensis H10 B. thuringiensis 5681st B. thuringiensis ser israelensis B-5246 B. thuringiensis 1b B. thuringiensis 1a B. thuringiensis 0371-2 B. thuringiensis 411
- EU429669 B thuringiensis ser toumanoffi NZ ACNF01000156 B thuringiensis ser berliner ATCC 10792 B. thuringiensis 994 EF210300 B thuringiensis ser alesti ÍEBC-T0 B. thuringiensis 0409 B. thuringiensis 072 D16281 B thuringiensis ÍAM12077 B. thuringiensis 14 B. thuringiensis 408 B. thuringiensis 787 B. thuringiensis A/N
B. thuringiensis subsp. thuringiensis B-1223
JQ669397 B. thuringiensis ser israelensis SB67
Bt II
NZ ACNA01000167 B. thuringiensis subsp. thuringiensis T01001 B. thuringiensis 0371-1 B. thuringiensis 410 B. thuringiensis A/M
CP00190/ B. thuringiensis ser chinensis CT-43 : EF210314 B. thuringiensis ser ostriniae ÍEBC-T08A001 B. thuringiensis 0376 p.o.
i--EF210311 B. thuringiensis ser wratislaviensis ÍEBC-T47
-87- EF210312 B. thuringiensis ser entomocidus ÍEBC-T06 i B. thuringiensis ser subtoxicus B-822 AB617494 B. thuringiensis ser kumamotoensis
EF210299 B. thuringiensis ser bolivia ÍEBC-T63 EF210295 B. mycoides ATCC 6462 NZ ACMU01000002 B. mycoides DSM 2048 _ EF210293 B. thuringiensis ser vazensis ÍEBC-T67 NR 024697 B. weihenstephanensis DSM11821 EF210284 B. thuringiensis ser oswaldocruzi ÍEBC-T38 EF210310 B. thuringiensis ser thompsoni ÍEBC-T12 JQ669398 B. thuringiensis ser kurstaki AB142 EF210309 B. thuringiensis ser sotto íEBC-T04001 B. thuringiensis 109
AB617476 B. thuringiensis ser sumiyoshiensis
EF210315 B. thuringiensis ser kenyae ÍEBC-T04B001
99
Bt + B. mycoides +
B. weihenstephanensis
EF210287 B. thuringiensis ser shadongiensis ÍEBC-T22
— EF210289 B. thuringiensis ser zhaodongensis ÍEBC-T62 = EF210308 B. thuringiensis ser higo ÍEBC-T44
EU429662 B. thuringiensis ser galleriae B. thuringiensis 836 B. thuringiensis 0293
-■ EU429670 B. thuringiensis ser morrisoni
------------------AB617495 B. thuringiensis ser tochigiensis BGSC 4Y1
AB617485 B. thuringiensis ser darmstadiensis
- EF210286 B. thuringiensis ser seoulensis ÍEBC-T35 i AB671487 B. thuringiensis ser kyushuensis EF210288 B. thuringiensis ser tolworthi ÍEBC-T09 ACNC01000278 B. thuringiensis ser pakistani T13001 AB617496 B. thuringiensis ser yunnanensis AY461760 B. thuringiensis ser aizawai
!= AE016877 B. cereus ATCC 14579
■ EF210303 B. thuringiensis ser azorensis BGSC 4CB1 AE017334 B. anthracis str. Ames Ancestor AE017225 B. anthracis str. Sterne AE016879 B. anthracis str. Ames
EF210304 B. thuringiensis ser roskildiensis ÍEBC-T45 - AE017194 B. cereus ATCC 10987 " B. thuringiensis ser finitimus B1162 -- AB617473 B. thuringiensis ser finitimus L _ EF210285 B. thuringiensis ser pulsiensis ÍEBC-T65 AB617504 B. thuringiensis ser monterrey CP001746 B. cereus biovar anthracis str. Cl ACNG01000003 B. thuringiensis ser andalousiensis BGSC 4AW1
---- EF210302 B. thuringiensis ser graciosensis ÍEBC-T66
NC 005957 B thuringiensis ser konkukian 97-27
— EF210305 B. thuringiensis ser poloniensis ÍEBC-T54
---- AY167879 B. pumilus SAFR-032
9S>- NC 006322 B. licheniformis ATCC 14580
Bt I
B. cereusI
B. anthracis
B. cereus II
CP002183 B. subtilis subsp. spizizenii W23
0.005
Fig. 1. Phylogenetic tree constructed based on the sequences of the 16S rRNA gene for bacteria of the B. cereus group using the ME algorithm. The scale corresponds to 0.5 substitutions per 100 bp (genetic distances). The bootstrap confidence values were generated using 1,000 permutations and showed in % under the branches. Branches absent in more than 50% of the trees are not shown
A
B
99
100 [
86
n
100
T_
92
NC 014335 B. cereus biovar anthracis str. Cl NC 005945 B. anthracis str. Sterne EU761168 B. thuringiensis ser monterrey IEBC-T28 NC007530 B. anthracis str. Ames Ancestor NC003997 B. anthracis str. Ames EF210265 B. thuringiensis ser oswaldocruzi IEBC-T38 EF210266 B. thuringiensis ser pulsiensis IEBC-T65 CM000755 B. thuringiensis ser pondicheriensis BGSC 4BA1 I I CM000754 B. thuringiensis ser andalousiensis BGSC 4AW1
i 1 NC 005957 B. thuringiensis ser konkukian 97-27
991 I 100r CM000746 B. thuringiensis ser tochigiensis BGSC 4Y1 AAEK01000032 B. cereus G9241
B. thuringiensis ser finitimus B-1162 ,
AE017194 B. cereus ATCC 10987 '
1 EF210281 B. thuringiensis ser poloniensis IEBC-T54 J
EF210274 B. thuringiensis ser graciosensis IEBC-T66 EF210270 B. thuringiensis ser bolivia IEBC-T63 EF210271 B. thuringiensis ser finitimus IEBC-T02
1001— ^ AE
Bt + Ba
B. cereus II
Bt III
100
76
Bt I
97
73
■ CM000756 B. thuringiensis ser huazhongensis BGSC 4BD1 i ■ EF210277 B. thuringiensis ser tolworthi IEBC-T09
■ = EU761180 B. thuringiensis ser canadensis IEBC-T05A001
CM000750 B. thuringiensis ser pakistani T13001 AE016877 B. cereus ATCC 14579 EF210272 B. thuringiensis ser azorensis IEBC-T64 EF210278 B. thuringiensis ser zhaodongensis IEBC-T62 EU761170 B. thuringiensis ser nigeriensis IEBC-T08B001 EU761172 B. thuringiensis ser entomocidus IEBC-T06A001 EU761182 B. thuringiensis ser jegathesan IEBC-T28A001 B. thuringiensis subsp. subtoxicus B-822 AY461770 B. thuringiensis H-10
EU761194 B. thuringiensis ser kumamotoensis IEBC-T18 EU761190 B. thuringiensis ser yunnanensis IEBC-T20 EU761192 B. thuringiensis ser galleriae BGSC-4G2 rAY461777 B. thuringiensis H18 EF210257 B. thuringiensis ser kenyae IEBC-T04B001 EU761169 B. thuringiensis ser londrina IEBC-T10A001 B. thuringiensis 836
r- B. thuringiensis ser galleriae B-197 B. thuringiensis 0293 IIAY461778 B. thuringiensis ser aizawai
EF210260 B. thuringiensis ser thompsoni IEBC-T12 I EU761176 B. thuringiensis ser kurstaki BGSC-4D2
■ EF210256 B. thuringiensis ser higo IEBC-T44 EU761183 B. thuringiensis ser yosoo IEBC-T18A001
AF090331 B. thuringiensis IAM12077 CP001907 B. thuringiensis ser chinensis CT-43 CM000748 B. thuringiensis ser thuringiensis T01001 B. thuringiensis subsp. thuringiensis B-1223 B. thuringiensis H10 B. thuringiensis A/N B. thuringiensis A/M B. thuringiensis 994 B. thuringiensis 787 B. thuringiensis 5681st B. thuringiensis 410 B. thuringiensis 411 B. thuringiensis 409 B. thuringiensis 408 B. thuringiensis 1b B. thuringiensis 1a B. thuringiensis 14 B. thuringiensis 072 B. thuringiensis 0371-2 B. thuringiensis 0371-1 B. thuringiensis subsp. israelensis B-5246 CM000753 B. thuringiensis ser berliner ATCC 10792
- B. thuringiensis 0376 p.o.
EF210275 B. thuringiensis ser ostriniae IEBC-T08A001 = EF210276 B. thuringiensis ser tohokuensis IEBC-T17
- AY461779 B. thuringiensis ser galleriae AY461780 B .thuringiensis ser israelensis EU761175 B. thuringiensis ser alesti BGSC-4C1
- B. thuringiensis 109 EU761186 B. thuringiensis ser sotto T04001 EU761185 B. thuringiensis ser darmstadiensis IEBC-T10 EU761196 B. thuringiensis ser mirrisoni BGSC-4K1
99 ■ EU761173 B. thuringiensis ser sumiyoshiensis IEBC-T03B001 —I EF210279 B. thuringiensis ser fukuokaensis IEBC-T03C001
■ ■ EU761174 B. thuringiensis ser toumanoffi IEBC-T11 100 ' EF210261 B. thuringiensis ser wratislaviensis IEBC-T47 i,- EU761191 B. thuringiensis ser neoleonensis IEBC-T24 i— EF210268 B. thuringiensis ser shandongiensis IEBC-T22 72 EF210258 B. thuringiensis ser seoulensis IEBC-T35
87l EU761167 B. thuringiensis ser kyushuensis IEBC-T11A001
100 ■ EF210267 B. thuringiensis ser roskildiensis IEBC-T45
■ CP000001 B. cereus E33L
99 r AF090332 B. mycoides ATCC 6462 B. mycoides
B. cereus I
84
81
77
100
99
\
78
Bt II
100 ' NZ ACMU01000094 B. mycoides DSM2048
97,
1 EF210269 B. thuringiensis ser vazensis IEBC-T67 99 g NC010184 B. weihenstephanensis KBAB4
1 EU761171 B. thuringiensis ser novosibirsk IEBC-T24A001 NC009848 B. pumilus SAFR-032
■ NC006270 B. licheniformis ATCC 14580
}
Bt + Bw
NC014479 B. subtilis subsp. spizizenii W23
0.05
Fig. 2. Phylogenetic tree constructed based on the sequences of the gyrB rRNA gene for bacteria of the B. cereus group using the ME algorithm. The scale corresponds to 5 substitutions per 100 bp (genetic distances). The bootstrap confidence values were generated using 1,000 permutations and showed in % under the branches. Branches absent in more than 50% of trees are not shown
II
the 16S rRNA gene was highly conserved (99.7-100.0% homology), which has also been repeatedly mentioned in other studies [8, 29].
The B. anthracis strains were grouped into a single cluster; however, the level of significance was low. Bt strains were also attributed to this cluster B. anthracis. We distinguished two B. cereus groups (I and II), identically to the study by Bavykin et al. [45]. This branching has not been statistically confirmed (statistical significance of the branching order < 50%). The B. cereus I group included the pathogenic B. cereus strain ATCC 14579T and a number of nonpathogenic Bt serovars. The B. cereus II group consisted of various Bt serovars and nonpathogenic B. cereus strain ATCC 10987T. Most of the Bt strains with a low significance level of branching formed a single cluster, which brought together different serovars of this species. The B. mycoides and B. weihenstephanensis strains were attributed to a separate subgroup.
The potential commercial strains and the typical strain Bt ser. berliner ATCC 10792T were put together and attributed to the Bt II group with a branching significance of 56%. This group comprised seventeen Ukrainian isolates of different serotypes isolated from different host insects, mostly from the Lugansk and Kherson regions, and the Krasnogvardeisk and Simferopol districts. Strain Bt 0376 p.o. (serotype 1) was proposed for the production of the eco-friendly en-tomopathogenic preparation “STAR-t” (OOO Simbi-tor) intended to control the number of Colorado potato beetle (Leptinotarsa decemlineata) larvae, potato tuber moth (Phtorimea operculella Zel.), and chickpea leaf-miner (Liriomiza cicerina Rd.) during vegetation and storing potato and chickpea and was attributed to this group and had the group-specific substitutions A/G77, T/C90, T/A92, C/T192, C/A1015 in the 16S rRNA gene. All the investigated isolates from the Bt II group had completely identical nucleotide sequences of the 16S rRNA gene. Strain Bt var. thuringiensis 994 (serotype 1, analogue of the bioagent of bacterial preparation “Bitoxybacillin”) used to produce the preparation “Akbitur,” strain Bt 408 (serotype 3) exhibiting high entomopathogenic activity against L. decemlineata, and strain Bt var. darmstadiensis H10 (serotype X) were also attributed to the Bt II group.
Strains Bt 836 (serotype 4), Bt var. kurstaki 0293 (serotype 3, analogue of the strain used as a bioagent in the preparation “Lepidocid”), and Bt var. mor-risoni 109 (serotype X) were attributed to the Bt I group. Both specific nucleotide substitutions typical and unique for the Bt strains were found within each group. A total of 16, 30, 32, 28, and 21 substitutions were found in B. anthracis, B. cereus I, B. cereus II, Bt
I, and Bt II, respectively. However, it should be men-
tioned that most nucleotide substitutions were random and strain-specific.
Thus, the 16S rRNA gene cannot be used to assess and study the phylogenetic relationships of the B. cereus group at a levels below genus/species, since it does not allow one to determine the species-specific nucleotide substitutions for this group.
Genetic diversity of the sequences of the gyrB gene The nucleotide sequence of the gyrB gene is used along with the 16S rRNA gene in taxonomic studies and for bacterial identification [34]. A number of studies have recently been published where the variability of the sequence of this gene in different bacterial species belonging to the genus Bacillus was studied (e.g., B. sub-tilis [46], B. cereus groups [47]). The universal primers proposed earlier [34] and the primer systems constructed by us and specific for the 3’-terminus of the gyrB gene of bacteria belonging to the B. cereus group were used to amplify and sequence the PCR fragments of this gene (the size of the sequenced region was 1800 bp, 81.82% of the entire gene). We selected this fragment of the gene on the basis of the distribution of the polymorphism (entropy) level of the gyrB nucleotide sequence using the DNAsp v. 5 software [48]. The level of polymorphism was above average on the regions 150-700 and 1650-200 bp from the beginning of the gene (data not shown). However, due to the fact that there was a limited number of gyrB DNA sequences of a certain length of strains belonging to the B. cereus group in GenBank, we selected the region from 385 to 1507 bp from the beginning of the gene (the annotation is provided for the strain Bt ser. berliner ATCC 10792T), which comprised 60% of the total length of the gene, for the analysis. The phylogenetic tree shown in Fig. 2 was constructed using the ME algorithm for 25 investigated strains, isolates, and reference sequences of the Bacillus sp. strains included in GenBank. The nucleotide sequences of species B. pumilus, B. licheniformis, and B. subtilis were used as remote controls for the phylogenetic analysis.
The topology of the constructed tree was similar with that of the phylogenetic trees constructed earlier for the 16S rRNA gene, and for the intergenic region 16S-23S rRNA; it showed no dependence on the algorithms used for the construction (NJ, ME). The inter- and intraspecies differences between the species B. anthracis and the B. cereus - Bt group have been identified. The results of the studies demonstrated that the nucleotide sequence of the gyrB gene possesses a higher resolving power than the 16S rRNA gene and the intergenic region 16S-23S rRNA sequences [34, 46] and, hence, is more suitable for the taxonomic studies of closely related species.
Fig. 3. Electrophoretic analysis of the saAFLP products obtained for the DNA samples of B. thuringiensis strains. Lanes: 17, 18, 29 — 1 kb GeneRuler™ DNA molecular mass marker (Fermentas); 1 — Bt Н10, R-type; 2 — Bt A/N; 3 — Bt 408; 4,19 - Bt 5681st; З, 23 - Bt 0376 p.o.; 6 - Bt 787; 7 - Bt 411; 8 - Bt 72; 9 - Bt 0371-1; 10, 27 - Bt 109; 11 - Bt 14; 12
- Bt 994; 13 - Bt 1b; 14 - Bt A/M; 13 - Bt 836; 16, 23 - Bt 0293; 20 - Bt sbsp. israelensis B-5246; 21 - Bt 0371; 22
- Bt sbsp. thuringiensis B-1223; 24 - Bt sbsp. subtoxicus B-822; 26 - Bt sbsp. galeriae B-197; 28 - Bt sbsp. finitimus B-1162; 30 - control PCR without template DNA
Identically to the data obtained for the 16S rRNA gene (but with a higher significance level), five subgroups can be distinguished within the B. cereus group: B. anthracis, B. cereus I and II, Bt I and II. Another group, Bt III, was distinguished in the phylogenetic cladogram with a 92% statistical significance of the branching order. It comprised the following strains: Bt ser. bolivia IEBC-T63 and Bt ser. finitimus IEBC-T02. With a high significance level, most strains formed the Bt II group, which also comprised the strains used for the production of entomopathogenic preparations. However, as previously assumed based on published data, the B. cereus and Bt species were indistinguishable [45]. Thus, the Bt strains, along with the strains belonging to the species B. anthracis, B. сєгєш, and B. weihenstephanensis, were grouped into the subgroups B. anthracis, B. cereus I and II and B. weihenstephan-ensis. The gyrB gene of strain Bt 0376 р.о. and other strains of this group were compared; due to the higher resolving power and variability of the gyrB nucleotide sequence, two substitutions specific to this strain (A/G861 and A/G1149) have been identified. In general, the level of similarity between the nucleotide and amino acid sequences in the B. сєгєш group was 87.1 — 95.2% and 95.1 — 99.2%, respectively.
Clusterization of the strains into two groups with a high statistical significance of the branching order is worth mentioning. Cluster I was formed by groups A and B. Group A consisted of the reliably grouped pathogenic strains B. anthracis, the nonpathogenic strain B. cereus ATCC 10987Т, entomopathogenic strains Bt ser. finitimus B1162 and Bt ser. poloniensis IEBC-T54 belonging to the B. cereus II group, and entomopatho-genic strains belonging to the Bt III group. Group B was formed by strain B. cereus ATCC 14579Т (pathogenic for humans), entomopathogenic strains Bt belonging to the B. cereus I group, and entomopathogenic strains belonging to the Bt I group. Cluster II included bacteria belonging to the species B. weihenstephanensis and B. mycoides, and the Bt II group comprising most of the strains used for industrial production of entomopatho-genic preparations. This clusterization of bacteria probably attests to a paraphyletic structure of both the B. cereus group in general and the separate species of this group.
Polymorphism among Bt detected using saAFLP and rep-PCR markers
Along with the housekeeping genes, genomic fingerprinting methods are used to reveal the differences be-
1 2 З 4 5 6 7 8 9 10 11 12 1З 14 15 16 17 1819 20 21 22 2З 24 25 26 27 28 29 ЗО
1 2 З 4 5 6 7 8 9 10 11 12 1З 14 15 16 17 18 19 20 21 22 2З 24 25 26 27 28 29 ЗО
А
B
Fig. 4. Electrophoretic analysis of the PCR products obtained for DNA samples of B. thuringiensis strains with primers ERÍC (A) and BOX (B). Lanes: 17, 19, 30 - 1 kb GeneRuler™ DNA molecular mass marker (Fermentas); 1 - Bt Н10, R-type; 2 - Bt A/N; 3 - Bt 408; 4,19 - Bt 5681st; 5, 23 - Bt 0376 p.o.; 6 - Bt 787; 7 - Bt 411; 8 - Bt 72; 9 - Bt 0371-1; 10, 28 - Bt 109; 11 - Bt 14; 12 - Bt 994; 13 - Bt 1b; 14 - Bt A/M; 15 - Bt 836; 16, 26 - Bt 0293; 18 - control PCR without template DNA; 21 - Bt sbsp. israelensis B-5246; 22 - Bt 0371; 22 - Bt sbsp. thuringiensis B-1223; 25 - Bt sbsp. sub-toxicus B-822; 27 - Bt sbsp. galeriae B-197; 29 - Bt sbsp. finitimus B-1162
tween closely related bacterial species and strains. Rep-PCR is the most frequently used. This method is based on using oligonucleotide primers homologous to the sequences of various intergenic repeats. In our study, the differences between the closely related Bt strains were identified using rep-PCR (BOX-, ERIC-PCR) and saAFLP. The results obtained are shown in Figs. 3,4.
All the strains under study were analyzed by saAFLP applying three restriction endonucleases (XmaJI, XbaI, and PstI). The informative spectra for all the Bt strains were recorded using XmaJI only. The modified saAFLP method allowed to distinguish the strains at the species-group level. All investigated Bt strains were divided into six group according to these spectra (Fig. 3). All strains presumably belonged to different subspecies of the Bt species. Group 1 comprised the typical strains Bt subsp. thuringiensis and Bt 0376 p.o. This strain contained a unique saAFLP pattern (1,000 bp long), which distinguished it from the other strains belonging to group 1 (marked with a white arrow in Fig. 3). Groups 2, 3, 4, and 5 were represented by either a small number of strains or a single strain. It
should be mentioned that this grouping corresponded to the data obtained previously by the analysis of 16S rRNA and gyrB genes sequences. It is significant that each strain of the same group was characterized by a group-specific saAFLP spectrum and pattern, which distinguished it from the strains belonging to the other groups. We also found patterns (markers) that are unique for individual strains (e.g., the commercial strain Bt 0376 p.o.), which distinguished them among all the strains belonging to group 1. Thus, the proposed method is more specific and can be used for a quick search for strain/group-unique markers and for the study of polymorphism in populations.
The ERIC-PCR (Fig. 4A) and BOX-PCR (Fig. 4B) methods were also used in this study to compare the results obtained using these reference primers and by saAFLP analysis modified by us. Based on the analysis of the obtained ERIC and BOX patterns, all investigated Bt strains were subdivided into six groups. However, no differences between the ERIC and BOX spectra detected within each group for every strain. The number of specific PCR markers and the total number
99
94
84
78
Bt 5681st
Bt subsp. thuringiensis B-1223
Bt subsp. israelensis B-5246
Bt A/M
Bt 1b
Bt 1a
Bt 994
Bt 14
Bt 0371-1
Bt 0371-2
Bt 411
Bt 410
Bt 787
Bt 072
Bt 409
Bt 408
Bt H10
Bt A/N
---------- Bt 0376 p.o.
-----Bt subsp. subtoxicus B-822
Bt subsp. galleriae B-197
52
100 | Bt0293 Bt 836 ----- Bt 109
Bt subsp. finitimus B-1162
3
4
; 5
Fig. 5. Dendrogram of 25 Bt strains isolated on the territory of Ukraine. The dendrogram was constructed using the NJ algorithm based on the total results of ERIC and BOX PCR and saAFLP for 36 polymorphic markers. The numbers denote the statistical significance of the branching order (%) determined by a bootstrap analysis of 1,000 permutations
0.1
2
of fragments obtained by saAFLP were greater than those obtained using the ERIC and BOX primers. This fact attests to the higher sensitivity, specificity, and informativity of the saAFLP method. The character of the results could be attributed to the fact that the ERIC and BOX-PCR allow one to analyze only separate genomic regions, which are rather conserved (promoter regions (ERIC1R-ERIC2, BOX, REP2-I-REP1R-I) or the regions of functional genes (e.g., tRNA)). Hence, the spectra obtained by these methods contain general, rather than strain-specific, information and could be more useful for passportization of strains. The spectra recorded by saAFLP, which is not confined to any particular genomic region, show the individuality of each microorganism. The differences between all the analyzed strains could be identified on the basis of fingerprints (patterns) based on this method [34]. The specificity of the spectra allows one to conclude that the saAFLP method is probably appropriate for investigating and distinguishing the true phylogenetic relationships between bacteria without using the data obtained through other primers or methods, with the exception of determining the genus of a microorganism using the 16S rRNA or gyrB gene.
However, in order to verify the reliability of these results and obtain a complete view of the genetic relationships between closely related bacteria, it is necessary to analyze the overall data obtained by both the saAFLP method and ERIC- and BOX-PCR.
In our study, we used three methods (ERIC-, BOX-PCR, and saAFLP) to identify 36 polymorphic markers (unique fragments) among the analyzed strains. The resulting data were used to construct a dendrogram (Fig. 5). The genetic distances between the strain pairs were determined using Pearson’s correlation, the Simple difference, and the Cosine distance (data not shown). The resulting matrix distances were used to conduct a cluster analysis using the NJ method. According to the results obtained, all the investigated strains were subdivided into five clusters. The statistical significance of the branching order varied from 58 to 99%. The clusters were isolated based on a similarity of >80%) and/or a significance level of branching >50%. Cluster 1 comprised the strains Bt H10 R-type, Bt A/N, Bt 408, Bt 409, Bt 410, Bt 5681st, Bt 787, Bt 411, Bt 072, Bt 0371-1, Bt 14, Bt 994, Bt 1a, Bt 1b, Bt A/M, Bt subsp. israelensis B-5246, Bt 0371-1, Bt 0371-2, and the typical strain Bt subsp. thuringiensis B-1223. Within this cluster, the strain Bt 0376 p.o. was isolated with a high statistical significance of the branching order. Cluster
2 was formed by two subspecies, Bt subsp. galleriae and Bt subsp. subtoxicus. A significance of branching of <50% demonstrates that these strains presumably belong to two different subspecies and may represent separate clusters if the strain sampling is enlarged. In order to verify or refute this hypothesis, the strain sample should be broadened. Cluster 3 consisted of the strains Bt 0293 and Bt 836; clusters 4 and 5 were rep-
resented by the strains Bt 109 and Bt subsp. finitimus B-1162, respectively.
Based on the published data and the results obtained by us, it can be concluded that a complex approach combining an analysis of both the biochemical properties of the strain and molecular-biological methods, is required to study and identify the Bt species belonging to the B. сereus group. The Bt species can be successfully studied using the nucleotide sequence of the gyrB gene. At the intraspecies level, it can be studied by saAFLP, along with the other AP-PCR methods (rep-PCR). These methods were used to subdivide the strain sample into five groups, which also corresponded
to their unique biochemical properties that had previously been determined in studies conducted by our colleagues [49]. The elaborated saAFLP method enabled to identify the DNA fragment, which is unique for the strain Bt 0376 p.o. ., isolated first by our colleagues from the Institute of Agriculture of the Crimea (National Academy of Agrarian Sciences of Ukraine) and used to produce the entomopathogenic preparation “STAR-t”. We intend to increase the size of the strain sampling, to study the composition of the cry genes, and to determine the nucleotide sequences of the unique DNA fragments revealed for the separate saAFLP groups and strains. •
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