Whole genome sequencing of a novel Bacillus thuringiensis isolated from Assam soil.

BACKGROUND: Bacillus thuringiensis (Bt) is a gram-positive ubiquitous saprophytic bacterium that produces proteins (Crystal protein, Vegetative insecticidal protein, and Secreted insecticidal protein) toxic to insects during its growth cycle. In the present study, the whole genome of a locally isolated B. thuringiensis strain BA04 was sequenced to explore the genetic makeup and to identify the genes responsible to produce insecticidal proteins including the virulence factors. The strain was isolated from the soil sample of the Kaziranga National Park, Assam, North-Eastern part of India (Latitude: 26°34'39.11''N and Longitude: 93°10'16.04''E). RESULTS: The whole genome sequencing (WGS) of the BA04 strain revealed that it has a circular genome of size 6,113,005 bp with four numbers of plasmids. A total of 6,111 genes including two novel crystal protein-encoding genes (MH753362.1 and MH753363.1) were identified. The BLASTn analysis of MH753362.1 showed 84% similarities (maximum identity) with Cry1Ia (KJ710646.1) gene, whereas MH753363.1 exhibited 66% identity with Insecticidal Crystal Protein (ICP)-6 gene (KM053257.1). At the protein level, MH753362.1 and MH753363.1 shared 79% identity with Cry1Ia (AIW52613.1) and 40% identity with Insecticidal Crystal Protein (ICP)-6 (AJW76687.1) respectively. Three-dimensional structures of these two novel protein sequences revealed that MH753362.1 have 48% structural similarity with Cry8ea1 protein, whereas MH753363.1 showed only 20% structural similarity with Cry4Aa protein. Apart from these insecticidal genes, the strain was also found to contain virulence and virulence-associated factors including the antibiotic resistance genes and Clustered regularly interspaced short palindromic repeat (CRISPR) sequences. CONCLUSION: This is the first report on the whole genome sequence of Bt strain BA04 isolated from Assam, a North-Eastern state of India. The WGS of strain BA04 unveils the presence of two novel types of insecticidal crystal protein-encoding genes which can be used for the development of insect-resistant transgenic crops. Additionally, the strain could be used for the formulations of effective biopesticides. The WGS provides the fastest and cheapest platform for a better understanding of the genetic makeup of a strain and helps to explore the role of virulence genes in pathogenicity against the insect host.

Multilocus sequence typing for phylogenetic view and vip gene diversity of Bacillus thuringiensis strains of the Assam soil of North East India.

An agriculturally important insecticidal bacterium, Bacillus thuringiensis have been isolated from the soil samples of various part of Assam including the Kaziranga National Park. Previously, the isolates were characterized based on morphology, 16S rDNA sequencing, and the presence of the various classes' crystal protein gene(s). In the present study, the phylogenetic analysis of a few selected isolates was performed by an unambiguous and quick method called the multiple locus sequence typing (MLST). A known B. thuringiensis strain kurstaki 4D4 have been used as a reference strain for MLST. A total of four the MLST locus of housekeeping genes, recF, sucC, gdpD and yhfL were selected. A total of 14 unique sequence types (STs) was identified. A total number of alleles identified for the locus gdpD and sucC was 12, followed by locus yhfL was 11, however, only 6 alleles were detected for the locus recF. The phylogenetic analysis using MEGA 7.0.26 showed three major lineages. Approximately, 87% of the isolates belonged to the STs corresponding to B. thuringiensis, whereas two isolates, BA07 and BA39, were clustered to B. cereus. The isolates were also screened for the diversity of vegetative insecticidal protein (vip) genes. In all, 8 isolates showed the presence of vip1, followed by 7 isolates having vip2 and 6 isolates for vip3 genes. The expression of Vip3A proteins was analyzed by western blot analyses and expression of the Vip3A protein was observed in the isolate BA20. Thus, the phylogenetic relationship and diversity of Bt isolates from Assam soil was established based on MLST, in addition, found isolates having vip genes, which could be used for crop improvement.

Isolation and characterization of Bacillus thuringiensis strains native to Assam soil of North East India.

We have identified both crystalliferous and acrystalliferous Bt isolates from the Assam soil of North East India for the first time. A total of 301 Bacillus type colonies were selected based on their appearance and colony morphology. Out of these colonies, 42 isolates had characteristics similar to Bt isolates on MYP (Mannitol Egg Yolk Polymyxin) agar base medium. The ERIC-PCR and 16S rDNA analyses confirmed that 42 isolates are Bacillus thuringiensis. Phase contrast microscopy showed that 37 isolates produced crystal endospore during the sporulation phase and 5 acrystalliferous isolates were also found. Amplification of cry gene was carried out using general Cry primers along with one cry2 gene specific primer. Out of 42 isolates, 50% of the isolates showed presence of cry2 gene followed by cry9 (40.47) and cry1 (40.47). Moreover, 21.42% of isolates showed the presence of more than one cry genes. We also screened these isolates for the possibility of having new Bt genes using universal primer and found two strains having a new type of Cry1I gene with 82 and 85% similarities with the available Cry1I gene sequences. Thus, these new types of Bt gene could be useful for Bt-based bioformulations and generation of transgenic plants.