CC BY
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AGRICULTURAL BIOLOGY, ISSN 2412-0324 ffngfeli ed. Online)
2015, V. 50, № 3, pp. 332-338
(SEL’SKOKHOZYAISTVENNAYA BIOLOGIYA) ISSN 0131-6397 (Russian ed. Print)
v_____________________________________' ISSN 2313-4836 (Russian ed. Online)
UDC 579.64:577.22 doi: 10.15389/agrobiology.2015.3.332rus
doi: 10.15389/agrobiology.2015.3.332eng
WHOLE GENOME SEQUENCING AND THE COMPARATIVE ANALYSIS OF HOUSEKEEPING LOCUSES AND VIRULENCE GENES FROM THE COMMERCIAL STRAINS OF Bacillus thuringiensis WITH INSECTICIDAL ACTIVITY
V.I. SAFRONOVA, A.L. SAZANOVA, I.G. KUZNETSOVA, Zh.P. POPOVA,
S.D. GRISHECHKINA, V.P. ERMOLOVA, E.E. ANDRONOV
All-Russian Research Institute for Agricultural Microbiology, Federal Agency of Scientific Organizations, 3, sh.
Podbel’skogo, St. Petersburg, 196608 Russia, e-mail v.safronova@rambler.ru
Acknowledgements:
Supported by the Ministry of Education and Sciences of the Russian Federation (Agreement № 14.604.21.0024, RFMEFI60414X0024)
Received March 30, 2015
Abstract
Russian collection of agricultural microorganisms (RCAM) supports 37 strains of Bacillus thuringiensis with pronounced insecticidal activities, which are used for the production of phytoprotective biopreparations. The effectiveness of biopreparations depends on the quality of the microbial material. Monitoring of the purity and authenticity of the commercial strains can be accomplished by their molecular-genetic certification. Currently, the most effective technology for detailed genetic characterization of strains is a whole genome sequencing. However, despite the obvious demand for whole genome sequencing for the certification of commercial strains, its widespread use is limited because of considerable labour-intensive and cost. Therefore, the developing of rapid methods for high-throughput sequencing is necessary to reduce the cost of analysis and the terms of its implementation. One possible solution might be to analyze the part of genome, which includes the most taxonomically and functionally important genetic loci (referent-complexes). The goal of this work was to detect the referent-complexes of 10 B. thuringiensis strains having different serotypes. After receiving whole genome sequences the comparative analysis was carried out to search for the most divergent genetic loci that determine the uniqueness of strains. To identify most divergent loci of housekeeping genes and virulence genes in the strains, we used BioNumerics («Applied Maths», USA) program and MEGA v. 5.0 program for clustering according to Neighbour-Joining method. As a result, 10 housekeeping genes (glpT, glpF, pyrE, purF, purH, pta, gyrB, ftsA, panC and isd), as well as 8 virulence genes (hblA, hblC, hblD, nheA, nheC, capA, capC and inA) were selected. For each strain two referent complexes were constructed, representing the concatenated sequences of selected housekeeping and virulence loci taken separately. Total length of the referent-complexes was 11809 and 10094 bp respectively. In the future the sets of primers will be created for multiplex analysis of referent complexes in the high-throughput DNA sequencing and the rapid method for genetic certification of the commercial B. thuringiensis strains will be developed.
Keywords: Bacillus thuringiensis, whole genome DNA sequencing, housekeeping and virulence genes, genetic certification.
Microorganism strains with a pronounced (target) practical value are the basis for any microbial technology. Cultivation of strains during their laboratory storage and manufacturing process is often accompanied by the loss of their target properties and contamination (pollution) up to the full replacement by contaminants. As a result, microbial material of poor quality can be used which causes the reduction of production efficiency and the risk of the use of pathogenic microorganisms. Monitoring of the purity and authenticity of commercial strains can be accomplished by their molecular genetic certification. For this purpose, the latest molecular genetic methods such as the amplified DNA fragment length polymorphism analysis (AFLP fingerprinting) [1], pulse-electro-
phoresis method [2, 3], and high-performance whole genome sequencing [4-8] are used currently. These techniques make it possible to identify the unique characteristics of microorganism cultures that can be used to create strain specific passports. Currently, the whole genome sequencing is considered the most effective technology for detailed genetic characterization of microorganism strains. However, despite the obvious demand for whole genome sequencing for the certification of commercial strains, its widespread use is limited because of considerable labor intensive and cost. Therefore, the developing of rapid methods for high-throughput sequencing is necessary to reduce the cost of analysis and the terms of its implementation. One possible solution might be to analyze not the whole genome but the most taxonomically and functionally important genetic loci (so called referent complexes).
The All-Russian Research Institute of Agricultural Microbiology (AR-RIAM) departmental collection of beneficial agricultural microorganisms (RCAM) supports 37 strains of Bacillus thuringiensis with pronounced insecticidal activities used for the production of phytoprotective biopreparations (bitoxibacillin, bactoculicid, batsikol).
The goal of this study was to detect the whole genome sequencing of 10 Bacillus thuringiensis strains having different serotypes, to perform their comparative analysis and to search for the most divergent genetic loci to further develop a rapid method of reliable authentication of these species commercial strains.
Technique. Bacillus thuringiensis var. thuringiensis, B. thuringiensis subsp. darmstadiensis and B. thuringiensis var. israelensis strains were cultured on meat-peptone agar (MPA) at 28 °С [9]. The strains are placed at Station of Low Temperature Automated Storage of Biological Samples of RCAM [10]. Information about the strains is available online in the RCAM database at AR-RIAM web-site (http://www.arriam.spb.ru).
To isolate bacterial DNA, 3 ml of overnight culture of strains in meat-peptone broth (MPB) were taken [9], then the cells were pelleted by centrifugation at 13,400 rev/min for 2 min. The pellet was washed with 1 ml of buffer 1 (25 mM TrisHCl pH = 8.0; 10 mM EDTA) and centrifuged again. The pellet was re-suspended in 100 pl of lysing mixture containing 960 pl of buffer 1, 3 mg of lysozyme, and 25 pl of RNase (10 mg/ml) and incubated for 20 min at 37 °С. Then, 500 pl of proteinase K in buffer 2 (10 mM TrisHCl pH = 8.0; 5 mM EDTA; 0.5 % SDS) was added to the mixture and the latter was incubated for 30 minutes at 55 °С. After that, 60 pl of 3 M sodium acetate and 750 pl of phe-nol:chloroform mixture (1:1) was added, vortexed for 5 min and centrifuged at 14,000 rev/min for 10 minutes. Supernatant was transferred into clean tubes, and 750 ml of phenol:chloroform (24:1) mixture was added, vortexed for 5 min and centrifuged again. Supernatant was transferred into clean tubes, an equal volume of isopropyl alcohol was added, the mixture was gently stirred for 5 min and centrifuged at 13,400 rev/min for 10 minutes. Supernatant was separated, the pellet was washed with 70 % ethanol and dried. H2O MQ (50 pl) was added to the residue, stirred and heated at 65 °С for 5 min. Additional cleaning of the isolated DNA was performed using the AxyPrep Multisource Genomic DNA miniprep Kit (Axygen, USA) according to the manufacturer’s recommendations.
Concentration and purity of DNA was assessed using the SpectroStar Nano spectrophotometer (BMG Labtech GMbH, Germany) at four wavelengths of 230, 260, 280 and 340 nm. The nucleotide sequence of genomic DNA was determined using the Junior GS pyrosequencer (Roche Applied
Science, Germany) according to manufacturer’s recommendations. The work on assembling reeds was performed using the CLC Genomics Workbench 7.5.1 program (CLC bio QIAGEN, Germany, http://www.clcbio.com/pro-ducts/clc-genomics-workbench/).
To identify the most divergent loci of housekeeping and virulence genes in B. thuringiensis strains, we used the BioNumerics (Applied Maths, USA) program. To create dendrograms reflecting the phylogenetic relationship between the studied strains, the clusters of selected housekeeping gene sequences and separate virulence genes were processed using the MEGA v. 5.0 (NeighbourJoining method).
Results. Commercially valuable strains of B. thuringiensis with pronounced insecticidal action were selected for the study. Biological properties of strains are summarized in Table 1.
1. Biological characteristics of the compared Bacillus thuringiensis strains of different serogroups
№ strain Isolated object Spore titer, x 109/ml of LC Exotoxin content (LC50, |rl/g of feed for house fly L2) Insecticidal activity LC50, %
to Colorado potato beetle L2 to yellow fever mosquito L4 (x10-3)
B. thuringiensis var. thuringiensis
12 Dead Colorado potato
beetle 2.79 3.40 0.22 -
20 Sick cabbage moth
caterpillar 2.58 4.00 0.26 -
40 Caddage field soil 2.42 4.29 0.32 -
800/19 Dead cabbage pierid
caterpillar 2.72 3.78 0.28 -
B. thuringiensis subsp. darmstadiensis
79 Dead Colorado potato
beetle 1.98 5.86 0.42 -
109 Colorado potato beetle
larvae 2.78 4.28 0.36 -
109/4 Reisolation from Colo-
rado potato beetle larvae 3.08 3.80 0.32 -
109/57 Reisolation from Colo-
rado potato beetle larvae 3.25 3.60 0.27 -
B. thuringiensis var. israelensis
38 Aedogenic water reser-
voir (water) 2.81 - - 0.24
44 Anophelogenic water
reservoir (soil) 3.28 - - 0.16
Note. LC — liquid culture The dashes mean that the studies were not performed.
B. thuringiensis var. thuringiensis strains are active against Colorado potato beetle and lepidopteran pests, while B. thuringiensis subsp. darmstadiensis is effective primarily against coleopteran insect herbivores (Colorado potato beetle, flea beetles, rape blossom beetle, oriental mustard leaf beetle, raspberry and strawberry weevil, etc.). Representatives of B. thuringiensis var. israelensis have larvicidal activity and are effective against mosquitoes. Two strains of the analyzed ones (109/4 и 109/57) are variants of the B. thuringiensis subsp. darmstadiensis 109 strain chosen as a result of selection (see Table 1).
Upon obtaining the whole genome sequences of B. thuringiensis strains having different serotypes, their comparative analysis and the search for the most divergent genetic loci were performed. The genes that determine strain virulence and the genes required to sustain the essential life functions of microorganism cells (named housekeeping genes) were compared. As a result, 10 housekeeping genes (glpT, glpF, pyrE, purF, purH, pta, gyrB, ftsA, panC and isd), as well as 8 virulence genes (hblA, hblC, hblD, nheA, nheC, capA, capC and inA) were selected (Table 2).
2. Housekeeping and virulence genes studied in comparative analysis of whole genome sequences in 10 Bacillus thuringiensis strains having insecticidal activity
Gene Fragment size, bp Coded protein Reference
glpT 1350 Housekeeping genes glycerol-3-phosphate permease [11]
glpF 837 glycerol uptake facilitator protein [12, 14]
pyrE 633 orotate phosphoribosyltransferase [11]
purF 1416 glutamine phosphoribosylpyrophosphate amidotransferase [13]
purH 1536 phosphoribosylaminoimidazole carboxamide formyltransferase [14]
pta 972 phosphate acetyltransferase [12, 14]
gyrB 1883 DNA gyrase subunit B [15]
AsA 1308 cell division protein [11]
panC 849 pantoate-beta-alanine ligase [13]
isd 1025 iron-regulated surface determinant protein This article
hblA 1388 Virulence genes hemolytic enterotoxin hemolysin A [16, 17]
hblC 1340 hemolytic enterotoxin hemolysin C [16, 17]
hblD 1221 hemolytic enterotoxin hemolysin D [17]
nheA 1161 nonhemolytic enterotoxin A [16]
nheC 1080 nonhemolytic enterotoxin C [16]
capA 748 capsular polysaccharide A [18]
capC 768 capsular polysaccharide C [18]
inA 2388 metalloprotease [18]
3. Number of different nucleotides in housekeeping gene referent complexes in 10 Bacillus thuiingiensis strains having insecticidal activity at pairwise comparisons
Strain var. thuringiensis subsp. darmstadiensis var. israelensis
40 12 20 800/19 109 I 109/4 109/57 | 79 38 | 44
40
12 12
20 27 15
800/19 18 6 21
109 352 345 360 350
109/4 357 351 366 343 7
109/57 354 346 361 351 3 8
79 346 339 354 344 20 25 21
38 414 399 424 414 188 195 191 190
44 413 408 423 414 192 199 195 194 24
4. Number of different nucleotides in virulence gene referent complexes in 10 Bacillus thuringiensis strains having insecticidal activity at pairwise comparisons
Strain var. thuringiensis subsp. darmstadiensis var. israelensis
40 12 20 | 800/19 109 109/4 109/57 79 38 | 44
40
12 18
20 8 12
800/19 15 3 9
109 296 300 292 299
109/4 296 300 292 299 0
109/57 296 300 292 299 0 0
79 276 279 270 277 229 229 229
38 385 389 379 386 420 420 420 394
44 383 399 389 396 429 429 429 403 29
These loci, except for the isd gene, have been previously used by other authors for genotyping and studying of biodiversity of B. cereus and B. thuringiensis strains [11-18]. In this study, a high level of genetic divergence in the isd locus was identified. For each strain, two referent complexes were constructed, representing the sequences of selected housekeeping loci and virulence genes taken separately. Total length of referent complexes was 11,809 and 10,094 bp, respectively. The data on the number of different nucleotides in referent complexes in the studied strains of B. thuringiensis at pairwise compari-
sons are summarized in Tables 3 and 4. One can see that the most homologous housekeeping genes were typical for B. thuringiensis subsp. darmstadiensis strains 109, 109/4 and 109/57 (3-8 different nucleotides) and B. thuringiensis var. thuringiensis strains 12, 20 and 800/19 having from 6 to 21 different nucleotides (see Table 3). Strains 109, 109/4 and 109/57 that are the variants of the same strain, were identical while the 12, 20 and 800/19 closely related strains had from 3 to 12 different nucleotides (see Table 4).
Phylogenetic relationship of B. thuringiensis strains studied was shown more evident by dendrograms obtained based on the referent complex analysis of housekeeping and virulence genes (Fig. 1 and 2). One can see that in both cases, strains are grouped strictly according to their taxonomic position and serotype which indicates high taxonomic significance of the loci used.
100
B. thuringiensis subsp. darmstadiensis 100 B. thuringiensis subsp. darmstadiensis B. thuringiensis subsp. darmstadiensis " B. thuringiensis subsp. darmstadiensis
- B. thuringiensis var. israelensis 38 ' B. thuringiensis var. israeiensis 44 ~ B thuringiensis var. thuringiensis 40 B thuringiensis var. thuringiensis 12 ; B thuringiensis var. thuringiensis 20 B thuringiensis var. thuringiensis 800/19
100
100
99
0.002
Fig. 1. Dendrogram for Baciilus thuringiensis strains with insecticidal activity based on the referent complex analysis including housekeeping genes glpT, gpF, pyrE, purF, purH, pta, gyrB, ftsA panC and isd.
100
79
100
B thuringiensis var. thuringiensis 12 ' B thuringiensis var. thuringiensis 20 B thuringiensis var. thuringiensis 800/19 B thuringiensis var. thuringiensis 40
B. thuringiensis subsp. darmstadiensis 79
I B. thuringiensis subsp. darmstadiensis 109 Юё! B. thuringiensis subsp. darmstadiensis 109/4 B. thuringiensis subsp. darmstadiensis 109/57
______|~ B. thuringiensis var. israeiensis 38
100 B. thuringiensis var. israeiensis 44
0.005
Fig. 2. Dendrogram for Bacillus thuringiensis strains with insecticidal activity based on the referent complex analysis including virulence genes hblA, hblC, hblD, nheA, nheC, capA, capC and inA.
Thus, unique housekeeping and virulenceas genes have been identified as a result of a comparative whole genome sequence analysis of 10 Bacillus thur-ingiensis strains having different serotype, sequencing of which will allow the rapid authentication of commercial strains of the species for monitoring quality of microbial material in the manufacture of biologics with insecticidal effect.
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