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 characterization of bacterial population in the forest soil of Kashmir, India

The bacterial diversity in the forest soil of Kashmir, India was investigated by 16S rDNA-dependent molecular phylogeny. Small subunit rRNA (16S rDNA)from forest soil metagenome were amplified by polymerase chain reaction (PCR) using primers specific to the domain bacteria. 30 unique phylotypes were obtained by PCR based RFLP of 16S rRNA genes using endonucleases Hae 111 and Msp 1, which were most suitable to score the genetic diversity. The use of 16S rRNA analysis allowed identification of several bacterial populations in the soil belonging to the following phyla: Firmicutes (33.3%),Bacteroidetes (13.3%), Proteobacterium (6.6%), Planctomycete(3.3%), and Deferribacteraceae (3.3%) in addition to the others that were not classified, beyond Archaea domain, However, 36.6% of the retrieved bacterial sequences could not be grouped with any phylum/lineage.The large amount of unclassified clone sequence could imply that novel groups of bacteria were present in the forest soil

Phylogenetic Characterization of Archaea in Saltpan Sediments

A study was undertaken to investigate the presence of archaeal diversity in saltpan sediments of Goa, India by 16S rDNA-dependent molecular phylogeny. Small subunit rRNA (16S rDNA) from saltpan sediment metagenome were amplified by polymerase chain reaction(PCR) using primers specific to the domain archaea. 10 unique phylotypes were obtained by PCR based RFLP of 16S rRNA genes using endonuclease Msp 1, which was most suitable to score the genetic diversity. These phylotypes spanned a wide range within the domain archaea including both crenarchaeota and euryarcheaota. None of the retrieved crenarchaeota sequences could be grouped with previously cultured crenarchaeota however; two sequences were related with haloarchaea. Most of the sequences determined were closely related to the sequences that had been previously obtained from metagenome of a variety of marine environments. The phylogenetic study of a site investigated for the first time revealed the presence of low archaeal population but showed yet unclassified species, may specially adapted to the salt pan sediment of Goa

A novel esterase from Bacillus subtilis (RRL 1789): Purification and characterization of the enzyme

An esterase (EC 3.1.1.1) produced by an isolated strain of Bacillus subtilis RRL 1789 exhibited moderate to high enantioselectivity in the kinetic resolution of several substrates like aryl carbinols, hydroxy esters, and halo esters. The enzyme named as B. subtilis esterase(BSE), was purified to >95% purity with a specific activity of 944 U/mg protein and 12% overall yield. The purified enzyme is �52 kDa monomer, maximally activity at 37 �C and pH 8.0 and fairly stable up to 55 �C. The enzyme does not exhibit the phenomenon of interfacial activation with tributyrin and p-nitrophenyl butyrate beyond the saturation concentration. The enzyme showed preference for triacyglycerols and esters of p-nitrophenol with short chain fatty acid. Presence of Ca2+ ions increases the activity of enzyme by �20% but its presence does not have any influence on the thermostability of the enzyme. The enzyme is not a metalloprotein and belongs to the family of serine proteases. The N-terminal amino acid sequence of BSE determined, as Met-Thr-Pro-Glu-Iso-Val-Thr-Thr-Glu-Tyr-Glyrevealed similarity with the N-terminal amino acid sequence of p-nitrobenzylesterase of B. subtilis

Phylogenetic Analyses of Archaeal Ribosomal DNA Sequences from Salt Pan Sediment of Mumbai, India

The archaeal diversity in salt pan sediment from Mumbai, India, was investigated by 16S rDNAdependent molecular phylogeny. Small-subunit rRNA (16S rDNA) from salt pan sediment metagenome were amplified by polymerase chain reaction (PCR) using primers specific to the domain archaea. Thirty-two unique phylotypes were obtained by PCR-based RFLP of 16S rRNA genes using endonucleases Hae111 and Msp1, which were most suitable to score the genetic diversity. These phylotypes spanned a wide range within the domain Archaea including both Crenarchaeota and Euryarcheaota. However, none of the retrieved Crenarchaeota sequences could be grouped with previously cultured Crenarchaeota. Of all the Euryarcheaota sequences, three sequences were related to Haloarchaea, two were related to cultured Methanosarcina sp., and the remaining sequences were affiliated with uncultured Methanosarcina sp., Methanogenium sp., and Methanolobus sp. Most of the sequences determined were closely related to the sequences that had been previously obtained from metagenome of a variety of marine environments. The phylogenetic study of a site investigated for the first time revealed the presence of a highly diverse archaeal population and may represent novel sequences and organisms specially adapted to the salt pan sediment of Mumbai. These findings are of fundamental value for understanding the complexity of salt pan ecosystems

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

Purification and characterization of a cold active alkaline protease from Stenotrophomonas sp., isolated from Kashmir, India

A Psychrotolerant alkaline protease producing bacterium IIIM-ST045 was isolated from a soil sample collected from the Thajiwas glacier of Kashmir, India and identified as Stenotrophomonas sp. on the basis of its biochemical properties and 16S ribosomal gene sequencing. The strain could grow well within a temperature range of 4–37�C however, showed optimum growth at 15�C. The strain was found to over-produce proteases when it was grown in media containing lactose as carbon source (157.50 U mg-1). The maximum specific enzyme activity (398 U mg-1) was obtained using soya oil as nitrogen source, however, the inorganic nitrogen sources urea,ammonium chloride and ammonium sulphate showed the lowest production of 38.9, 62.2 and 57.9 U mg-1. The enzyme was purified to 18.45 folds and the molecular weight of the partially purified protease was estimated to be *55 kDa by SDS-PAGE analysis. The protease activity increased as the increase in enzyme concentration while as the optimum enzyme activity was found when casein (1% w/v) was used as substrate. The enzyme was highly active over a wide range of pH from 6.5 to 12.0 showing optimum activity at pH 10.0. The optimum temperature for the enzyme was 15�C. Proteolytic activity reduced gradually with higher temperatures with a decrease of 56% at 40�C. The purified enzyme was checked for the removal of protein containing tea stains using a silk cloth within a temperature range of 10–60�C. The best washing efficiency results obtained at low temperatures indicate that the enzyme may be used for cold washing purposes of delicate fabrics that otherwise are vulnerable to high temperatures

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

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