Pranab Roy

Degradation of Trichloroethylene by Bacillus sp.: Isolation Strategy, Strain Characteristics, and Cell Immobilization

A novel isolate of a bacterium, capable of degrading trichloroethylene (TCE) and growing on this as the sole carbon source is reported. The test strain was isolated by an enrichment technique with trichloroethylene as the substrate. The isolated strain belongs to the genus Bacillus. The practical utility of cleaning up oil spillage by bioremediation could be extended to this bacterium to degrade the environmental pollutant, which is used in metal degreasing in industries. Cells of the novel bacterium immobilized on calcium alginate were found to have better trichloroethylene degrading activity than the ones which were immobilized on agar-agar or free cells.

Copper Enhanced Monooxygenase Activity and FT-IR Spectroscopic Characterisation of Biotransformation Products in Trichloroethylene Degrading Bacterium: Stenotrophomonas maltophilia PM102

Stenotrophomonas maltophilia PM102 (NCBI GenBank Acc. no. JQ797560) is capable of growth on trichloroethylene as the sole carbon source. In this paper, we report the purification and characterisation of oxygenase present in the PM102 isolate. Enzyme activity was found to be induced 10.3-fold in presence of 0.7 mM copper with a further increment to 14.96-fold in presence of 0.05 mM NADH. Optimum temperature for oxygenase activity was recorded at 36°C. The reported enzyme was found to have enhanced activity at pH 5 and pH 8, indicating presence of two isoforms. Maximum activity was seen on incubation with benzene compared to other substrates like TCE, chloroform, toluene, hexane, and petroleum benzene. K m and V max for benzene were 3.8 mM and 340 U/mg/min and those for TCE were 2.1 mM and 170 U/mg/min. The crude enzyme was partially purified by ammonium sulphate precipitation followed by dialysis. Zymogram analysis revealed two isoforms in the 70% purified enzyme fraction. The activity stain was more prominent when the native gel was incubated in benzene as substrate in comparison to TCE. Crude enzyme and purified enzyme fractions were assayed for TCE degradation by the Fujiwara test. TCE biotransformation products were analysed by FT-IR spectroscopy.

Host-Range Dynamics of Cochliobolus lunatus: From a Biocontrol Agent to a Severe Environmental Threat

We undertook an investigation to advance understanding of the host-range dynamics and biocontrol implications of Cochliobolus lunatus in the past decade. Potato (Solanum tuberosum L) farms were routinely surveyed for brown-to-black leaf spot disease caused by C. lunatus. A biphasic gene data set was assembled and databases were mined for reported hosts of C. lunatus in the last decade. The placement of five virulent strains of C. lunatus causing foliar necrosis of potato was studied with microscopic and phylogenetic tools. Analysis of morphology showed intraspecific variations in stromatic tissues among the virulent strains causing foliar necrosis of potato. A maximum likelihood inference based on GPDH locus separated C. lunatus strains into subclusters and revealed the emergence of unclustered strains. The evolving nutritional requirement of C. lunatus in the last decade is exhibited by the invasion of vertebrates, invertebrates, dicots, and monocots. Our results contribute towards a better understanding of the host-range dynamics of C. lunatus and provide useful implications on the threat posed to the environment when C. lunatus is used as a mycoherbicide.

Cloning, Sequencing and Expression of Novel Trichloroethylene Degradation Genes from Stenotrophomonas maltophilia PM102: A Case of Gene Duplication

A bacterium capable of growth on trichloroethylene as the sole carbon source was identified as Stenotrophomonas maltophilia PM102 by 16S rDNA sequencing (GenBank acc. No. JQ797560). Genomic DNA of this bacterium was amplified using primers specific for the original todC1 gene of Pseudomonas putida F1. Two PCR products were obtained: 300 bp and 350 bp respectively, designated as tce300 and tce350 genes. These novel genes were separately cloned into p-GEMT Easy vector: PR300 and PR350 and sequenced. The sequences have been deposited at NCBI GenBank under accession nos. JX910450 and JX910451.

Identification and Typing of Aeromonas Hydrophila through 16S rDNA-PCR Fingerprinting

16S rRNA is a conserved biomolecule within a cell. Sequencing data was analyzed and examined by sequencing the corresponding 16s rDNA regions of isolates from different sources. Specific culture media and different biochemical tests primarily confirmed isolates as Aeromonas hydrophila; those were designed and tested following PCR assays and identified by 16S rDNA gene sequence analysis. To automate the method few online and offline computational tools have been used. The method has proven useful for identification of Aeromonas species. Our results emphasize the need to take into account the intragenomic diversity of the 16S rRNA gene. Different software and programme used in this study are freely available online in different website.

Production, Partial Purification and Characterization of an Extracellular Psychrotrophic Lipase from Pseudomonas Sp. ADT3

Psychrotrophic Pseudomonas ADT3 (NCBI GenBank Acc.no.JX914667) is capable of growth on lipid as the sole carbon source. In this paper, we report the purification and characterization of an extracellular lipase from psychrotroph, isolated from soil sample of Ny- Alesund, Svalbard, Arctic region. The Pseudomonas ADT3 isolate produces lipase enzyme in the extracellular minimal media with only 1% olive oil. The lipase was purified from the concentrated culture supernatant. The crude enzyme was partially purified by saturated ammonium sulphate precipitation followed by extensive dialysis. Enzyme activity was found to be induced 6-folds in presence of 1.2 mM lead ion but strongly inhibited by heavy metals Hg 2+ as well as EDTA and β-mercaptoethanol. The purified lipase has activity at two pH optima of pH i.e. pH 3.5 and 8.5. Optimum temperature for lipase activity was recorded at 22cC. The purified active fraction of lipase exhibits specific activity of 527.8 U/mg. The Vmax and Km was 144.93 U/mg/min and 0.260 mM respectively determined using Lineweaver-Burk plot. Zymogram analysis revealed prominent lipase band at 13.9 kDa range in the 80% saturated ammonium sulphate purified enzyme fraction.

Persistent Organic Pollutants Induced Protein Expression and Immunocrossreactivity by Stenotrophomonas maltophilia PM102: A Prospective Bioremediating Candidate

A novel bacterium capable of growth on trichloroethylene as the sole carbon source was identified as Stenotrophomonas maltophilia PM102 by 16S rDNA sequencing (accession number of NCBI GenBank: JQ797560). In this paper, we report the growth pattern, TCE degradation, and total proteome of this bacterium in presence of various other carbon sources: toluene, phenol, glucose, chloroform, and benzene. TCE degradation was comparatively enhanced in presence of benzene. Densitometric analysis of the intracellular protein profile revealed four proteins of 78.6, 35.14, 26.2, and 20.47 kDa while the extracellular protein profile revealed two distinct bands at 14 kDa and 11 kDa that were induced by TCE, benzene, toluene, and chloroform but absent in the glucose lane. A rabbit was immunised with the total protein extracted from the bacteria grown in 0.2% TCE + 0.2% peptone. Antibody preadsorbed on proteins from peptone grown PM102 cells reacted with a single protein of 35.14 kDa (analysed by MALDI-TOF-mass-spectrometry) from TCE, benzene, toluene, or chloroform grown cells. No reaction was seen for proteins of PM102 grown with glucose. The PM102 strain was immobilised in calcium alginate beads, and TCE degradation by immobilised cells was almost double of that by free cells. The beads could be reused 8 times.

Switching between Heat Shock Proteins and Cold Inducible Proteins under Temperature Fluctuation in Solanum tuberosum L. Cultivars in In Vivo Condition

Cross-talking between heat shock proteins (HSPs) and cold inducible proteins (CIPs) subsequent to combinational mild heat (35°C) and cold (8°C) stress was investigated in vivo for four cultivars of Solanum tuberosum L. viz. Kufri Pukhraj (PO), Kufri Jyoti (GS), Kufri Ashoka (KF) and Kufri Chandramukhi (CM) in the order of their decreasing thermotolerance, to understand how this economic crop adapts to extreme temperature fluctuation. We showed a time-course differential genotypic expression pattern for HSPs at 35°C for 10h and CIPs at 8°C for 12h time-lapse. Remarkably, we noted the disappearance of a housekeeping protein (HKP) of about 19.8KD at 2h, 35°C in GS absent in CM, KF and PO; but strongly expressed as CIPs at 8°C for all the cultivars. Furthermore, heat-stress led to an outstanding transient induction of HSP95.9, HSP83.6, HSP78.7, HSP70.7, HSP66.0, HSP54.1, HSP48.6, HSP43, sHSP38.3, sHSP35.3, sHSP29, sHSP22.5, sHSP17.8 and sHSP9.5 in GS at 6h, while HKP58.7, HKP55.5 and HKP43.7 were stably overexpressed in CM, KF and PO. Temperature switching from 35°C to 8°C upregulated HKP43.4, HKP54.6, CIP14.1 and HKP19.9 for all the cultivars. The recovery process 24h subsequent to this archetype switching was governed by overexpression of small(s)HSPs of about 25.4KD-14.1KD, HKP58.7 and HKP43.5 for all cultivars. Results suggest crosstalk protection for this paradigm-shift in temperature is chiefly conferred by isoforms of constitutively expressed HKPs, CIP19.9 and CIP14.1 in S. tuberosum L. Explicitly, this differential proteome change within 22h signify HKPs may not participate in thermotolerance as HSPs, but participate in cold acclimation as CIPs, recovery as sHSPs and even switch-off during heat-stress in some cultivars as depicted in GS. Research Article British Biotechnology Journal, 1(3): 101-112, 2011 102

Invasive Aspergillus terreus morphological transitions and immunoadaptations mediating antifungal resistance

Background and aims Aspergillus terreus Thom is a pathogen of public health and agricultural importance for its seamless abilities to expand its ecological niche. The aim of this study was holistically to investigate A. terreus morphological and immunoadaptations and their implication in antifungal resistance and proliferation during infection. Materials and methods In-depth unstructured mining of relevant peer-reviewed literature was performed for A. terreus morphological, immune, resistance, and genetic diversity based on the sequenced calmodulin-like gene. Results Accessory conidia and phialidic conidia produced by A. terreus confer discrete anti-fungal resistance that ensures survivability during therapies. Interestingly, by producing unique metabolites such as Asp–melanin and terretonin, A. terreus is capable of hijacking macrophages and scavenging iron, respectively. As such, A. terreus has established a rare mechanism to mitigate phagocytosis and swing the interaction dynamics in favor of its proliferation and survival in hosts. Conclusion It is further unraveled that besides A. terreus genetic diversity, morphological, biochemical, and immunologic adaptations associated with conidia germination and discharge of chemical signals during infection enable masking of the host defense as an integral part of its strategy to survive and rapidly colonize hosts.

Black List of Unreported Pathogenic Bambusicolous Fungi Limiting the Production of Edible Bamboo

Edible bamboo species are now domesticated and commercialized because of their nutraceutical values. The production of edible bamboo species are restrained by diseases caused by pathogenic bambusicolous fungi valued at 40% losses of the total