Center for Advanced Research in Sciences

Bacillus thuringiensis (Bt) was investigated in three different habitats (vegetable and crops-cultivated soils, phylloplanes and insect guts) of Bangladesh. A total of 61 Bacillus cereus-like isolates were obtained by selective methods and 57 of those were identified as Bacillus thuringiensis isolates based on their hemolytic activity, presence of parasporal crystal proteins, plasmid profile and crystal protein profile. The prevalence of Bt was highest (60%) in soil samples followed by leaf and insects. Five different types of parasporal crystal proteins (spherical, bipyramidal, irregular pointed, cuboidal and irregular shaped) were observed among the isolates which indicates the diversity of the local Bt isolates examined. In addition, confirmation of the strain identification was done using 16S rDNA gene sequencing. Plasmid analysis recovered from the isolates yielded at least one 15kb DNA, which is well comparable to the reference strain, B. thuriginesis kurstaki var. HD-73. Further similarity between the test and standard strains was observed while analyzing crystal proteins on a SDS-polyacrylamide gel that produced major bands of Cry1, Cry2 and Cry9 type proteins. Bioassay performed with isolates Bt-01i, Bt-25f and reference strain, Bt kurstaki var. HD-73 against pulse beetles Callosobrochus chinensis resulted LC 50 values of 0.30, 0.72 and 0.22 mg/ml crude proteins respectively which demonstrated their competency in Biopesticide production and application.

Genetic diversity is highly relevant and significant in discovering novel insecticidal genes in Bacillus thuringiensis (Bt) strains and to deal with the problems of emerging insect resistance towards Bt biopesticides. In view of this, Random Amplified Polymorphic DNA (RAPD)-PCR analysis was performed with a decamer AGCTCAGCCA for molecular typing of 177 Bt strains of Bangladesh to determine their genetic diversity. These Bt strains were allocated into 15 genomic types with their binary matrices as determined from the dendrogram based on a standardized distance in scale bar. Genotype 9 and 11 were the largest among others, each containing more than 25% of the Bt strains. The average diversity index, as deduced for each group by cluster: isolate ratio at a specific distance, was higher for locations (0.27 ± 0.098) than that for biotypes (0.23 ± 0.046) which indicates an unmingled and vertical transfer of biochemical properties among the strains. Prevalence of agriculturally important subgroups of cry1 gene in indigenous Bt strains was also determined where cry1Aa and cry1Ca gene were found to be the most prevalent (21.74%). While analyzing the distribution pattern of cry genes, they were observed to be present in all RAPD- genotypes but genotype 10 and were most prevalent in genotypes 1, 6 and 9. The phylogeny reconstruction among the strains was performed by neighbor-joining method with the 16S rRNA gene sequences and the correlation among the phylogeny, RAPD genotypes, Biotypes and presence of cry genes were analyzed.

Proteolytic bacteria isolated from municipal solid wastes (MSW) were identified as Serratia marcescens A3 and Pseudomonas putida A2 based on 16S rDNA sequencing. Protease produced through fermentation of organic MSW by these bacteria under some optimized physicochemical parameters was partially purified and characterized. The estimated molecular mass of the partially purified protease from S. marcescens and P. putida was approximately 25 and 38 kDa, respectively. Protease from both sources showed low K m 0.3 and 0.5 mg ml À1 and high V max 333 and 500 mmole min À1 at 40 °C, and thermodynamics analysis suggested formation of ordered enzyme-substrate (E-S) complexes. The activation energy (E a) and temperature quotient (Q 10) of protease from S. marcescens and P. putida were 16.2 and 19.9 kJ/mol, and 1.4 and 1.3 at temperature range from 20 to 40 °C, respectively. Protease of the both bacterial isolates was serine and cysteine type. The protease retained approximately 97% of activity in the presence of sodium dodecyl sulphate. It was observed that the purified protease of S. marcescens could remove blood stains from white cotton cloth and degrade chicken flesh remarkably. Our study revealed that organic MSW can be used as raw materials for bacterial protease production and the protease produced by S. marcescens A3 might be potential for applications.

The scarcity of hygienic drinking water is a normal phenomenon in the coastal areas of Bangladesh due to the high salinity of ground water. The inhabitants of this locality, therefore, live on alternative supplies of water including rain-fed pond water, and rain-water with persistent complex microbial interactions therein, often contaminated with life-threatening pathogens. Hence, this study was aimed at analyzing the prevalence of Vibrio cholerae (Vc) in the alternative drinking waters of Mathbaria, a coastal subdis-trict neighboring the Bay of Bengal, the efficacy of pond sand filter (PSF) and the co-association among Bacillus-like spore formers (Sf) and Vc. Vc presumably entrapped into the membrane filter was enriched in alkaline peptone water medium and was isolated on selective thiosulfate-citrate-bile salts-sucrose and taurocholate-tellurite-gelatin agar media. They were finally identified by immunochromatographic one step rapid test and serology test. A total of 26% Vc positive samples were obtained out of 100 [ponds—48, household (HH)—29, and PSFs—23] where 13% cases were pathogenic (Vc O1) and 13% were non-pathogenic (Vc non-O1/non-O139). The distribution of Vc as observed was 33, 26, and 13.8% in waters derived from pond surface, PSF, and HH reservoirs, respectively, and for pathogenic type, it was 62.5%, 50%, and nil, respectively. Although none of the samples was identified with pathogenic Vc O139, the statistics represents a significant and augmentative risk of cholera outbreak in the focused area. The antibiotic sensitivity pattern in this study resembled the trend observed during last few years for Vc. The PSF demonstrated its inability to remove Vc from any of the samples and in addition, the filter itself was evidenced to be the source of pathogens and spores in further contamination and transmission. The development of biofilm in the PSF could be hypothesized as the reservoir in contaminating pathogen-free water samples. From the test of homogeneity, the risk levels of alternative water sources were estimated equal regarding Vc. Simultaneously, it was determined statistically that the prevalence of Vc, by no means, is influenced by Bacillus-like Sf be it for pond surface, HH, or PSF derived water.

Bacillus subtilis BAB-1, isolated from cotton rhizosphere soil, is an excellent biocontrol agent for tomato gray mold. The genome of B. subtilis strain BAB-1 was fully sequenced and annotated, genes encoding the antifungal active compound were identified, and multiple sets of regulatory systems were found in the genome.

Parasporin, another type of δendotoxin from Bacillus thuringiensis (Bt), renowned for its highly specific and preferential toxicity against different cancer cells, could be a potentially safe anticancer therapeutic. Considering the current cancer situation, isolation and molecular characterization of parasporin-producing Bt isolates were aimed. Hence, Bt isolates were obtained through a series of tests viz. heat treatment, acetate selection, starch hydrolysis, lecithinase activity, and microscopy for parasporal proteins. Then the biochemical properties, plasmid, parasporin gene, and δendotoxin protein profiles were checked. Primers were designed for ps gene detection and the molecular identification of the isolates was performed by 16S rRNA gene sequence analysis. From the tested samples (n=128), 91 Bt isolates were obtained and of them, 28 non-hemolytic Bt isolates were selected as potential parasporin producers. Plasmid profile analysis of these isolates revealed that nonhemolytic...