Molecular Characterization of Mercury Resistant Bacteria Inhabiting Polluted Water Bodies of Different Geographical Locations in India

Mercury pollution is a major environmental problem that arises as a result of natural processes as well as from anthropogenic sources. In response to toxic mercury compounds, microbes have developed astonishing array of resistance systems to detoxify them. To address this challenge, this study was aimed in screening bacterial isolates for their tolerance against varied concentrations of phenylmercuric acetate. Mercury transformation by bacteria being sensitive to factors such as available carbon source, etc. that affect mer-mediated transformation, screened mercury tolerant bacteria were also studied for their tolerance to different antimicrobials and carbon sources, followed by identification using biochemical as well as 16S rRNA approach. Following identification, gene encoding organomercurial lyase catalyzing protonolytic cleavage of C-Hg bond of organic mercury was amplified using gene specific primers, cloned in pGEMT(?) easy vector and sequenced. Microbe-based approach using organomercurial lyase encoded by merB gene being potentially economic, provides foundation to facilitate genetic manipulation of this environmentally important enzyme to remove high concentrations of obstinate mercury using holistic, multifaceted approach for use in bioremediation through generation of transgenics or as catalyst for use in bioreactors.     

Isolation and Partial Characterization of a Virulent Bacteriophage IHQ1 Specific for Aeromonas punctata from Stream Water

Aeromonas punctata is the causative agent of septicemia, diarrhea, wound infections, meningitis, peritonitis, and infections of the joints, bones and eyes. Bacteriophages are often considered alternative agents for controlling bacterial infection and contamination. In this study, we described the isolation and preliminary characterization of bacteriophage IHQ1 (family Myoviridae) active against the Gram-negative bacterial strain A. punctata. This virulent bacteriophage was isolated from stream water sample. Genome analysis indicated that phage IHQ1 was a double-stranded DNA virus with an approximate genome size of 25–28 kb. The initial characterization of this newly isolated phage showed that it has a narrow host range and infects only A. punctata as it failed to infect seven other clinically isolated pathogenic strains, i.e., methicillin-resistant Staphylococcus aureus 6403, MRSA 17644, Acinetobacter 33408, Acinetobacter 1172, Pseudomonas aeruginosa 22250, P. aeruginosa 11219, and Escherichia coli. Proteomic pattern of phage IHQ1, generated by SDS-PAGE using purified phage particles, showed three major and three minor protein bands with molecular weights ranging from 25 to 70 kDa. The adsorption rate of phage IHQ1 to the host bacterium was also determined, which was significantly enhanced by the addition of 10 mM CaCl₂. From the single-step growth experiment, it was inferred that the latent time period of phage IHQ1 was 24 min and a burst size of 626 phages per cell. Moreover, the pH and thermal stability of phage IHQ1 were also investigated. The maximum stability of the phage was observed at optimal pH 7.0, and it was totally unstable at extreme acidic pH 3; however, it was comparatively stable at alkaline pH 11.0. At 37°C the phage showed maximum number of plaques, and the viability was almost 100%. The existence of Aeromonas bacteriophage is very promising for the eradication of this opportunistic pathogen and also for future applications such as the design of new detection and phage typing (diagnosis) methods. The specificity of the bacteriophage for A. punctata makes it an attractive candidate for phage therapy of A. punctata infections.     

Cloning, characterization and molecular docking of a highly thermostable β-1,4-glucosidase from Thermotoga petrophila

A genomic DNA fragment, encoding a thermotolerant β-glucosidase, of the obligate anaerobe Thermotoga petrophila RKU-1 was cloned after PCR amplification into Escherichia coli strain BL21 CodonPlus. The purified cloned enzyme was a monomeric, 51.5?kDa protein (by SDS-PAGE) encoded by 1.341?kb gene. The estimated K (m) and V (max) values against p-nitrophenyl-β-D-glucopyranoside were 2.8?mM and 42.7?mmol?min(-1)?mg(-1), respectively. The enzyme was also active against other p-nitrophenyl substrates. Possible catalytic sites involved in hydrolyzing different p-nitrophenyl substrates are proposed based on docking studies of enzyme with its substrates. Because of its unique characters, this enzyme is a potential candidate for industrial applications.     

Peripheral regulation by ecdysteroids of olfactory responsiveness in male Egyptian cotton leaf worms, Spodoptera littoralis

Physiological and behavioral plasticity allows animals to adapt to changes in external (environmental) and internal (physiological) factors. In insects, the physiological state modulates adult behavior in response to different odorant stimuli. Hormones have the potential to play a major role in the plasticity of the olfactory responses. To explore if peripheral olfactory processing could be regulated by steroid hormones, we characterized the molecular, electrophysiological, and behavioral response to changes in endogenous hormone levels in adult male Spodoptera littoralis. The expression of the receptor complex (EcR/USP) was localized by in situ hybridization in the olfactory sensilla of antennae. Injections of 20-hydroxyecdysone (20E) induced an ecdysteroid signaling pathway in antennae and increased expression of the nuclear receptors EcR, USP and E75. Diacylglycerol kinase (DGK) and CaM expression were also up-regulated by 20E. Taken together, these molecular, electrophysiological, and behavioral results suggest a hormonal regulation of the peripheral olfactory processing in S. littoralis.     

Correlated Electrostatic Mutations Provide a Reservoir of Stability in HIV Protease

HIV protease, an aspartyl protease crucial to the life cycle of HIV, is the target of many drug development programs. Though many protease inhibitors are on the market, protease eventually evades these drugs by mutating at a rapid pace and building drug resistance. The drug resistance mutations, called primary mutations, are often destabilizing to the enzyme and this loss of stability has to be compensated for. Using a coarse-grained biophysical energy model together with statistical inference methods, we observe that accessory mutations of charged residues increase protein stability, playing a key role in compensating for destabilizing primary drug resistance mutations. Increased stability is intimately related to correlations between electrostatic mutations - uncorrelated mutations would strongly destabilize the enzyme. Additionally, statistical modeling indicates that the network of correlated electrostatic mutations has a simple topology and has evolved to minimize frustrated interactions. The model's statistical coupling parameters reflect this lack of frustration and strongly distinguish like-charge electrostatic interactions from unlike-charge interactions for [Formula: see text] of the most significantly correlated double mutants. Finally, we demonstrate that our model has considerable predictive power and can be used to predict complex mutation patterns, that have not yet been observed due to finite sample size effects, and which are likely to exist within the larger patient population whose virus has not yet been sequenced.     

Psychosine-induced alterations in peroxisomes of twitcher mouse liver

Krabbe disease is a neuroinflammatory disorder in which galactosylsphingosine (psychosine) accumulates in nervous tissue. To gain insight into whether the psychosine-induced effects in nervous tissue extend to peripheral organs, we investigated the expression of cytokines and their effects on peroxisomal structure/functions in twitcher mouse liver (animal model of Krabbe disease). Immunofluorescence analysis demonstrated TNF-α and IL-6 expression, which was confirmed by mRNAs quantitation. Despite the presence of TNF-α, lipidomic analysis did not indicate a significant decrease in sphingomyelin or an increase in ceramide fractions. Ultrastructural analysis of catalase-dependent staining of liver sections showed reduced reactivity without significant changes in peroxisomal contents. This observation was confirmed by assaying catalase activity and quantitation of its mRNA, both of which were found significantly decreased in twitcher mouse liver. Western blot analysis demonstrated a generalized reduction of peroxisomal matrix and membrane proteins. These observations indicate that twitcher mouse pathobiology extends to the liver, where psychosine-induced TNF-α and IL-6 compromise peroxisomal structure and functions.     

Chromium-tolerant bacteria isolated from industrial effluents and their use in detoxication of hexavalent chromium

Three bacterial strains were isolated from effluents of leather (CMBL Cr13, CMBL Cr14) and steel (CMBL Cr15) industries for their possible use in chromium(VI) detoxication of industrial waste. CMBL Cr13 was found to tolerate chromium(VI) up to a concentration of 45 g/L in the medium, while CMBL Cr14 and CMBL Cr15 could tolerate up to 40 g/L. These bacteria were also checked for resistance to other metals. They resisted a lead concentration of 1 g/L and cadmium concentration of 550 mg/L in the medium. They showed optimum growth at pH 7.3–7.5 at a temperature of 35–37°C. Cr^VI-reducing ability of the three strains ranged from 70 to 80% after 3 d of incubation. The possible use of these bacteria in environmental cleanup is discussed.     

Reactive centre loop mutants of α-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway

The common severe Z mutation (E342K) of α₁-antitrypsin forms intracellular polymers that are associated with liver cirrhosis. The native fold of this protein is well-established and models have been proposed from crystallographic and biophysical data for the stable inter-molecular configuration that terminates the polymerization pathway. Despite these molecular ‘snapshots’, the details of the transition between monomer and polymer remain only partially understood. We surveyed the RCL (reactive centre loop) of α₁-antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P₁₄P₁₂ and P₄, but not P₁₀P₈ or P₂P_1′, resulted in a decrease in detectable polymer in a cell model that recapitulates the intracellular polymerization of the Z variant, consistent with polymerization from a near-native conformation. We have developed a FRET (Förster resonance energy transfer)-based assay to monitor polymerization in small sample volumes. An in vitro assessment revealed the position-specific effects on the unimolecular and multimolecular phases of polymerization: the P₁₄P₁₂ region self-inserts early during activation, while the interaction between P₆P₄ and β-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P₆P₄, but not P₁₄P₁₂, yield an increase in the overall apparent activation energy of association from ~360 to 550 kJ mol⁻¹.     

Methoprene modulates the effect of diet on male melon fly,Bactrocera cucurbitae,performance at mating aggregations

The effect of access to dietary protein (P) (hydrolyzed yeast) and/or treatment with a juvenile hormone analogue, methoprene (M), (in addition to sugar and water) on male aggregation (lekking) behaviour and mating success was studied in a laboratory strain of the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Six‐day‐old males were treated with (1) protein and methoprene (M+P+), (2) only protein (M?P+), or (3) only methoprene (M+P?), and compared with 14‐day‐old sexually mature untreated males (M?P?). The lekking behaviour of the four groups of males when competing for virgin sexually mature females (14 –16 days old) was observed in field cages. The following parameters were measured at male aggregations: lek initiation, lek participation, males calling, male–male interaction, female acceptance index, and mating success. For all these parameters, the M+P+ males significantly outperformed the other males. Moreover, for all parameters, there was a similar trend with M+P+ > M?P+ > M?P? > M+P?. More M+P+ males called and initiated and participated in lek activities than all other types of male, which resulted in higher mating success. They had also fewer unsuccessful copulation attempts than their counterparts. Whereas treatment with methoprene alone had a negative effect in young males with only access to sugar, access to dietary protein alone significantly improved young male sexual performance; moreover, the provision of methoprene together with protein had a synergistic effect, improving further male performance at leks. The results are of great relevance for enhancing the application of the sterile insect technique (SIT) against this pest species. The fact that access to dietary protein and treatment of sterile males with methoprene improves mating success means that SIT cost‐effectiveness is increased, as more released males survive to sexual maturity.