Application of a multiplex PCR assay for the detection of Shigella, Escherichia coli and Shiga toxin-producing Esch. coli in milk.

A multiplex PCR (mPCR) assay using previously known genetic markers of Shigella, Escherichia coli and Shiga-toxic Esch. coli was standardized. uidA gene was targeted for the common detection of Esch. coli and Shigella, whereas ipaH and stx1 genes were used as markers for the detection of Shigella and shiga-toxin producing strains, respectively. The standardized assays detected the target organism specifically and selectively. The mPCR developed by combining all the three reactions generated specific products. The inclusivity and exclusivity tests depicted the precise specificity of the mPCR assay. Results were interpreted on the basis of the pattern of amplicons generated: amplifications of the ipaH and uidA gene fragments indicated the presence of Shigella spp., amplification of uidA alone revealed the presence of Esch. coli and additional presence of verotoxin gene amplicon indicated verotoxinogenic nature of the strain. Specific patterns of bands were obtained when different strains of Esch. coli and Shigella spp. were subjected to this assay. The reactions, individually as well as in the mPCR, could detect approximately 1 cell per 20-microl PCR assay. The protocols were validated by analyzing the coded samples of full fat milk spiked with different pathogens. In naturally contaminated raw milk samples (n=100), Esch. coli were detected in all samples and verotoxinogenic Esch. coli in 15 samples. Shigella, however, was not detected in any of the samples. When DNA purified from the samples found positive for Shiga-toxic Esch. coli was directly used as template for the mPCR, the results showed agreement with the enrichment based detection. The mPCR assay, standardized in this study, may be used for rapid microbiological evaluation of milk samples. Further, the study emphasizes the need for better hygienic conditions in dairies

Molecular cloning of carboxylesterase gene and biochemical characterization of encoded protein from Bacillus subtilis (RRL BB1)

An isolated strain of Bacillus subtilis identified by 16S rDNA sequence analysis produces an enantioselective ester hydrolase.Whole cells of B. subtilis (RRL BB1) and enzyme derived from it was capable of enantioselective hydrolysis of several racemates including drug intermediates with moderate to high enantioselectivity as already reported by us. In this communication, we describe cloning of the gene encoding the enantioselective esterase designated as estBB1. The primary structure of the enzyme determined from the nucleotide sequence indicated that esterase estBB1 has Mw ∼52 kDa and pI ∼5.2 and belongs to the family of type B carboxylesterases with 50–60% similarity at amino acid level. Alignment studies of sequences of the estBB1 and Pnb esterase 56C8 from B. subtilis showed that estBB1 has an �/� hydrolase fold with catalytic triad formed by Ser190, Glu305 and His394 at active site and Ser190 is located in the conserved motif –G–X–S–X–G–

Molecular cloning of enantioselective ester hydrolase fromBacillus pumilusDBRL-191

A gene from Bacillus pumilus expressed under its native promoter was cloned in Escherichia coli. Recombinant B. pumilus esterase(BPE) affects the kinetic resolution of racemic mixtures such as unsubstituted and substituted 1-(phenyl)ethanols (E � 33–103), ethyl 3-hydroxy-3-phenylpropanoate (E � 45–71), trans-4-fluorophenyl-3-hydroxymethyl-N-methylpiperidine (E � 10–13) and ethyl 2- hydroxy-4-phenylbutyrate (E � 7). The enzyme is composed of a 34-amino acid signal peptide and a 181-amino acid mature protein corresponding to a molecular weight of �19.2 kD and pI � 9.4. 3-D the structural model of the enzyme built by homology modelling using the atomic coordinates from the crystal structure of B. subtilis lipase (LipA) showed a compact minimal a/b hydrolase fold

Community Structure of Bacteria and Archaea Associated with Geotextile Filters in Anaerobic Bioreactor Landfills

Landfills are an example of an environment that contains highly complex communities of microorganisms. To evaluate the microbial community structure, four stainless steel pilot-scale bioreactor landfills with single- and double-layered geotextile fabric were used. Two reactors (R-1 and R-2) contained municipal solid waste (MSW) and sewage sludge, while the other two reactors (R-3 and R-4) contained only MSW. A single layer of geotextile fabric (R2GT3 and R3GT3) was inserted in the drainage layers of the two reactors (R-2 and R-3), while a double layer of geotextile fabric (R4GT2 and R4GT1) was inserted in one of the reactors (R-4). Scanning electron microscopy demonstrated that biomass developed on the geotextile fabrics after 540 days of bioreactor operation. The metagenomics analyses of the geotextile samples by 16S rRNA gene sequencing indicated that the geotextile bacterial communities were dominated by the phyla Firmicutes, Bacteroidetes, and Thermotogeae, while Proteobacteria were detected as the rarest bacterial phylum in all the geotextile samples. Treponema, Caldicoprobacter, and Clostridium were the most dominant anaerobic and fermentative bacterial genera associated with the geotextile fabric in the bioreactors. Euryarchaeota was the predominant archaean phylum detected in all the geotextile samples. In the archaeal communities, Methanosarcina, and Vadin CA11 were identified as the predominant genera. The diversity of microorganisms in landfill bioreactors is addressed to reveal opportunities for landfill process modifications and associated operational optimization. Thus, this study provides insights into the population dynamics of microorganisms in geotextile fabrics used in bioreactor landfills

Tubulin Inhibitors from an Endophytic Fungus Isolated from <i>Cedrus deodara</i>

From an endophytic fungus, a close relative of <i>Talaromyces</i> sp., found in association with <i>Cedrus deodara</i>, four compounds including two new ones (<b>2</b> and <b>4</b>) were isolated and characterized. The structures of two compounds (<b>1</b> and <b>4</b>) were confirmed by X-ray crystallography. The compounds displayed a range of cytotoxicities against human cancer cell lines (HCT-116, A-549, HEP-1, THP-1, and PC-3). All the compounds were found to induce apoptosis in HL-60 cells, as evidenced by fluorescence and scanning electron microscopy studies. Also, the compounds caused significant microtubule inhibition in HL-60 cells