Quorum sensing inhibitors as antipathogens: biotechnological applications

The mechanisms through which microbes communicate using signal molecules has inspired a great deal of research. Microbes use this exchange of information, known as quorum sensing (QS), to initiate and perpetuate infectious diseases in eukaryotic organisms, evading the eukaryotic defense system by multiplying and expressing their pathogenicity through QS regulation. The major issue to arise from such networks is increased bacterial resistance to antibiotics, resulting from QS-dependent mediation of the formation of biofilm, the induction of efflux pumps, and the production of antibiotics. QS inhibitors (QSIs) of diverse origins have been shown to act as potential antipathogens. In this review, we focus on the use of QSIs to counter diseases in humans as well as plants and animals of economic importance. We also discuss the challenges encountered in the potential applications of QSIs.     

Determination of serum triglycerides using lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase co-immobilized onto alkylamine glass beads

A method for determination of serum triglycerides (Tgs) using lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase co-immobilized onto alkylamine glass beads (pore diameter 55 nm) through glutaraldehyde coupling was developed and evaluated. The minimum detection limit of the method was 0.54 mM. The analytical recovery of added triolein in the serum was 97.55 +/- 1.5% (mean +/- S.D.). The mean value of serum Tgs, determined by the present method showed a good correlation (r = 0.984) with the Bayer's kit method, employing free enzymes. The within and between batch coefficients of variation (CV) were < 2.25% and < 1.35% respectively. No significant loss of activity was observed, when co-immobilized enzymes were reused for about 200 times and stored at 4 degrees C in distilled water. The cost of Tg determination for 200 serum samples was less, as compared with Bayer's kit method.     

In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting

Water-in-oil (w/o) emulsions can be used to compartmentalize and select large gene libraries for a predetermined function. The aqueous droplets of the w/o emulsion function as cell-like compartments in each of which a single gene is transcribed and translated to give multiple copies of the protein (e.g., an enzyme) it encodes. While compartmentalization ensures that the gene, the protein it encodes, and the products of the activity of this protein remain linked, it does not directly afford a way of selecting for the desired activity. Here we show that re-emulsification of w/o emulsions gives water-in-oil-in-water (w/o/w) emulsions with an external (continuous) water phase through which droplets containing fluorescent markers can be isolated by fluorescence-activated cell sorting (FACS). These w/o/w emulsions can be sorted by FACS, while the content of the aqueous droplets of the primary w/o emulsion remains intact. Consequently, genes embedded in these water droplets together with a fluorescent marker can be isolated and enriched from an excess of genes embedded in water droplets without a fluorescent marker. The ability of FACS instruments to sort up to 40000 events per second may endow this technology a wide potential in the area of high-throughput screening and the directed evolution of enzymes.     

Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes

Autophagy is a lysosomal degradative pathway that has diverse physiological functions and plays crucial roles in several viral infections. Here we examine the role of autophagy in the life cycle of JEV, a neurotropic flavivirus. JEV infection leads to induction of autophagy in several cell types. JEV replication was significantly enhanced in neuronal cells where autophagy was rendered dysfunctional by ATG7 depletion, and in Atg5-deficient mouse embryonic fibroblasts (MEFs), resulting in higher viral titers. Autophagy was functional during early stages of infection however it becomes dysfunctional as infection progressed resulting in accumulation of misfolded proteins. Autophagy-deficient cells were highly susceptible to virus-induced cell death. We also observed JEV replication complexes that are marked by nonstructural protein 1 (NS1) and dsRNA colocalized with endogenous LC3 but not with GFP-LC3. Colocalization of NS1 and LC3 was also observed in Atg5 deficient MEFs, which contain only the nonlipidated form of LC3. Viral replication complexes furthermore show association with a marker of the ER-associated degradation (ERAD) pathway, EDEM1 (ER degradation enhancer, mannosidase α-like 1). Our data suggest that virus replication occurs on ERAD-derived EDEM1 and LC3-I-positive structures referred to as EDEMosomes. While silencing of ERAD regulators EDEM1 and SEL1L suppressed JEV replication, LC3 depletion exerted a profound inhibition with significantly reduced RNA levels and virus titers. Our study suggests that while autophagy is primarily antiviral for JEV and might have implications for disease progression and pathogenesis of JEV, nonlipidated LC3 plays an important autophagy independent function in the virus life cycle.     

The insecticidal activity of some norditerpene dilactones

The effects on housefly (Musca domestica) of six norditerpene lactones (nagilactones B, D, E, podolide, hallactone B and 14-epi-ponalactone A) in a defined diet were tested. Nagilactone D was the most active, with an LD₅₀ of 0.7 ppm. Nagilactones C and D were also toxic to light-brown apple moth (Epiphyas postvittana) and codling moth (Laspeyresia pomonella).The relationship between lactone structure and toxicity to housefly is discussed.

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Zusammenfassung Viele aus Podocarpus-Arten isolierte Norditerpenlactone sind für Stubenfliegen toxisch. Es wird über die Resultate der Fütterung von sechs Lactonen an die Stubenfliege in einem definierten Nährboden berichtet und die LD₅₀ von zwölf Verbindungen werden verglichen. Der aktivste Stoff ist Nagilacton mit einer LD₅₀ von 0,7 ppm. Nagilacton C und D sind auch toxisch für Epiphyas postvittana und für den Apfelwickler. Die wirksamsten Verbindungen haben eine kurze nichtpolare Seitenkette und eine elektronenreiche, funktionelle Gruppe bei C-8, ferner sind sie Epoxyalkohole in Ring-A. Diese in der Natur vorkommenden Lactone spielen wahrscheinlich eine Schutzrolle in der Pflanze.

     

Hydrogen and polyhydroxybutyrate producing abilities of microbes from diverse habitats by dark fermentative process

Thirty five bacterial isolates from diverse environmental sources such as contaminated food, nitrogen rich soil, activated sludges from pesticide and oil refineries effluent treatment plants were found to belong to Bacillus, Bordetella, Enterobacter, Proteus, and Pseudomonas sp. on the basis of 16S rRNA gene sequence analysis. Under dark fermentative conditions, maximum hydrogen (H₂) yields (mol/mol of glucose added) were recorded to be 0.68 with Enterobacter aerogenes EGU16 followed by 0.63 with Bacillus cereus EGU43 and Bacillus thuringiensis EGU45. H₂ constituted 63–69% of the total biogas evolved. Out of these 35 microbes, 18 isolates had the ability to produce polyhydroxybutyrate (PHB), which varied up to 500 mg/l of medium, equivalent to a yield of 66.6%. The highest PHB yield was recorded with B. cereus strain EGU3. Nine strains had high hydrolytic activities (zone of hydrolysis): lipase (34–38 mm) – Bacillus sphaericus strains EGU385, EGU399 and EGU542; protease (56–62 mm) – Bacillus sp. strains EGU444, EGU447 and EGU445; amylase (23 mm) – B. thuringiensis EGU378, marine bacterium strain EGU409 and Pseudomonas sp. strain EGU448. These strains with high hydrolytic activities had relatively low H₂ producing abilities in the range of 0.26–0.42 mol/mol of glucose added and only B. thuringiensis strain EGU378 had the ability to produce PHB. This is the first report among the non-photosynthetic microbes, where the same organism(s) – B. cereus strain EGU43 and B. thuringiensis strain EGU45, have been shown to produce H₂ – 0.63 mol/mol of glucose added and PHB – 420–435 mg/l medium.     

Biorefinery for Glycerol Rich Biodiesel Industry Waste

The biodiesel industry has the potential to meet the fuel requirements in the future. A few inherent lacunae of this bioprocess are the effluent, which is 10 % of the actual product, and the fact that it is 85 % glycerol along with a few impurities. Biological treatments of wastes have been known as a dependable and economical direction of overseeing them and bring some value added products as well. A novel eco-biotechnological strategy employs metabolically diverse bacteria, which ensures higher reproducibility and economics. In this article, we have opined, which organisms and what bioproducts should be the focus, while exploiting glycerol as feed.     

Metabolic engineering of Synechococcus elongatus for photoautotrophic production of mannitol

With multiple applications in food, pharmaceutical, and chemical industries as antioxidant or nonmetabolizable sweetener; the bioproduction of d -mannitol is gaining global attention, especially with photosynthetic organisms as hosts. Considering the sustainability prospects, the current work encompasses metabolic engineering of a widely used cyanobacterial strain, Synechococcus elongatus PCC 7942, and two newly isolated fast-growing cyanobacterial strains; S. elongatus PCC 11801 and S. elongatus PCC 11802, for mannitol production. We engineered these strains with a two-step pathway by cloning genes for mannitol-1-phosphate dehydrogenase (mtlD) and mannitol-1-phosphatase (mlp), where the mtlD expression was under the control of different promoters from PCC 7942, namely, P_rbc225, P_cpcB300, P_cpcBm1, P_rbcLm17, and P_rbcLm15. The strains were tested under the “switch conditions,” where the growth conditions were switched after the first 3 days, thereby resulting in differential promoter activity. Among the engineered strains of PCC 11801 and PCC 11802, the strains possessing P_rbc225-mtlD module produced relatively high mannitol titers of 401 ± 18 mg/L and 537 ± 18 mg/L, respectively. The highest mannitol titer of 701 ± 15 mg/L (productivity 60 mg/L.d, yield 895 µM/OD₇₃₀) was exhibited by the engineered strain of PCC 7942 expressing P_cpcB300-mtlD module. It is by far the highest obtained mannitol yield from the engineered cyanobacteria.