Mutations in the N‐terminal region of the Schizosaccharomyces pombe glutathione transporter pgt1+ allows functional expression in Saccharomyces cerevisiae

Pgt1p encodes a glutathione transporter in Schizosaccharomyces pombe, orthologous to the Saccharomyces cerevisiae glutathione transporter, Hgt1p. Despite high similarity to Hgt1p, Pgt1p failed to display functionality during heterologous expression in S. cerevisiae. In the present study we employed a genetic strategy to investigate the reason behind the non‐functionality of pgt1⁺ in S. cerevisiae. Functional mutants were isolated after in vitro mutagenesis. Several mutants were obtained and four mutants analysed. Among these, three yielded different point mutations in the N‐terminal region (301–350 bp) of the transporter before the first transmembrane domain, while one mutant contained a deletion of 42 nucleotides within the same region. The mutant pgt1⁺ proteins not only expressed and localized correctly, but displayed high‐affinity glutathione transport capabilities in S. cerevisae. Comparison of wild‐type pgt1⁺ with the functional mutants revealed that a loss in protein expression was responsible for lack of functionality of wild‐type pgt1⁺ in S. cerevisiae. The mRNA levels in wild‐type and mutants were comparable, suggesting that the block was in translation. The formation of a strong stem–loop structure appeared to be responsible for inefficient translation in pgt1⁺ and disruption of these structures in the mutants was probably permitting translation. This was confirmed by making silent mutations in this region of wild‐type pgt1⁺, which led to their functionality in S. cerevisiae. This genetic strategy to relieve functional blocks in expression should greatly facilitate the study of these and other transporters from more intractable genetic organisms in a heterologous expression system. Copyright © 2012 John Wiley & Sons, Ltd.     

Genome Subtyping of Autochthonous Bacillus Species Isolated from Iru,a Fermented Parkia biglobosa Seed

Phylogenetic relationship and strains sub-typing of Bacillus species isolated from iru, a traditional fermented condiment in Africa were studied using polyphasic genomic approaches and the profiles compared with bacilli isolated from similar Asian condiments. The 16S rRNA gene sequencing identified the strains as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus pumilus, and Brevibacillus formosus. The phylogenetic analysis conducted showed five distinct clusters with genetic relatedness among B. subtilis and B. amyloliquefaciens strains from Africa and Asia. Amplified ribosomal DNA restriction analysis (ARDRA) successfully differentiated species of B. subtilis phylogeny from B. cereus. Combined analyses of ARDRA, internal transcribed spacer-polymerase chain reaction (ITS-PCR), ITS-PCR-restriction fragment length polymorphism (ITS-PCR-RFLP) and randomly amplified polymorphic DNA (RAPD-PCR) further confirmed B. subtilis and B. amyloliquefaciens as the dominant Bacillus species associated with fermentation of iru, and revealed high strains genetic diversity, while multilocus sequence analysis (MLSA) data distinguished B. cereus from B. thuringiensis. This information is essential for selection of starter cultures with desirable functional attributes to guarantee product consistency and safety quality of traditional fermented foods.     

Characterization of constitutive microflora of biofilms in dairy processing lines

The biofilm status of pasteurization lines of a commercial plant (CP) and an experimental dairy plant (EDP) was evaluated. Samples were collected after cleaning-in-place systems from different segments of pasteurization lines over a period of 6 months. A total of 105 and 36 isolates from CP and EDP, respectively, were selected, on the basis of colony morphology and Gram's reaction. Biochemical differentiation of organisms in biofilms revealed the predominance of the genus Bacillus in both plants (59 and 64% in the CP and EDP, respectively). The other Gram-positive microflora of the commercial plant included Lactobacillus spp., Streptococcus spp. and Staphylococcus spp., while the EDP only had Micrococcus spp. in addition to Bacillus spp. The incidence of Gram-negative genera also revealed the presence of Shigella spp. Escherichia coli and Enterobacter aerogenes in both plants. On the other hand, Klebsiella spp. were only present in samples from the EDP. Further characterization of the isolates indicated the presence of psychrotropic microflora at a level of 38 and 75% in post-pasteurization segments of the CP and EDP, respectively. In addition, the percentage of heat-resistant organisms in the pre-pasteurization segment was 17%, as compared to 21% in post-pasteurization segments. Antibiotic susceptibility of selected isolates indicated maximum resistance of the isolates against carbenicillin (90% in CP and 75% in EDP), followed by cloxacillin (86% in CP and 89% in EDP), cephaloridine, novobiocin and vancomycin, indicating the presence of multiple antibiotic-resistant microflora that can be shed into the final product. Thus, the study reveals that all plants should be regularly evaluated for the prevalence of biofilms.     

Isolation, enumeration and PCR characterization of aflatoxigenic fungi from food and feed samples in India

A total of 54 market samples comprising nine different food and feed commodities from Mysore city were examined in order to isolate aflatoxin-producing fungi as well as to assess aflatoxins in the commodities. Thirty-two samples were contaminated with aflatoxigenic fungi and the total mycoflora and aflatoxigenic fungi in different food and feed commodities were in the range of 0.2–260 and 0–100 cfu×10³/g, respectively. In total, 136 fungi were isolated, of which 32 were Aspergillus flavus strains and 26 of them were found to produce aflatoxins. A. flavus group of fungi comprising A. flavus, A. parasiticus, A. oryzae, A. sojae were characterized by using Aspergillus differential medium and PCR. The PCR was performed using two different sets of primers specifically targeted to aflR and omt genes of aflatoxin biosynthesis pathway. Most of the fungi belonging to A. flavus group reacted positively with the primers resulting in expected size amplicons of 796 bp for aflR and 404 bp for omt. Among the nine commodities screened for aflatoxin only, groundnut and groundnut cake were contaminated with aflatoxins B₁ and B₂. The aflatoxin contamination in these commodities exceeded the Indian regulatory limit of 30 μg/kg.     

Antifungal activity of five different essential oils in vapour phase for the control of Colletotrichum gloeosporioides and Lasiodiplodia theobromae in vitro and on mango

Mango fruit has high commercial value; however, major postharvest losses are encountered throughout the supply chain due to postharvest diseases. These results lead to the search for natural fungicide for postharvest diseases control. The antifungal effects of five essential oils (thyme, clove, cinnamon, anise and vitex) were assessed by disc volatilisation method. Thyme oil vapours at 5 μL per Petriplate, and clove and cinnamon oil at 8 μL per Petriplate showed 100% growth inhibition of mango pathogens in vitro. GC/MS analysis of essential oil showed thymol (23.88), o‐cymol (23.88) and terpinolene (23.88) as the major constituents of thyme oil. Clove and cinnamon oils contain 3‐allyl‐2‐methoxyphenol (37.42%) and benzofuran 3‐methyl (17.97%), respectively. Thyme oil as a fumigant at 66.7 μL L^?1 showed a significant (P < 0.05) inhibition on postharvest pathogens of mango fruits stored at 25 °C for 6 days. Results of our study suggest the possibility of using thyme oil as an alternate natural fungicide to manage postharvest diseases in mango.     

Induction of pitting corrosion on stainless steel (grades 304 and 316) used in dairy industry by biofilms of common sporeformers

This study hypothesised that there may be induction of pitting corrosion or microbially‐induced corrosion on stainless steel (SS) dairy‐processing surfaces by biofilms of common milk sporeformers such as Bacillus sporothermodurans and Geobacillus stearothermophilus. Scanning electron microscopy (SEM) and micrographs generated from energy‐dispersive spectroscopy were used to observe pitting corrosion and to find the elemental composition and distribution on the control and pitted surfaces. From SEM images and energy‐dispersive spectroscopy micrographs, it was observed that pitting corrosion on SS could be induced by biofilms of both B. sporothermodurans and G. stearothermophilus.     

Saccharomyces cerevisiae S288C genome annotation: a working hypothesis

The S. cerevisiae genome is the most well-characterized eukaryotic genome and one of the simplest in terms of identifying open reading frames (ORFs), yet its primary annotation has been updated continually in the decade since its initial release in 1996 (Goffeau et al., 1996). The Saccharomyces Genome Database (SGD; www.yeastgenome.org) (Hirschman et al., 2006), the community-designated repository for this reference genome, strives to ensure that the S. cerevisiae annotation is as accurate and useful as possible. At SGD, the S. cerevisiae genome sequence and annotation are treated as a working hypothesis, which must be repeatedly tested and refined. In this paper, in celebration of the tenth anniversary of the completion of the S. cerevisiae genome sequence, we discuss the ways in which the S. cerevisiae sequence and annotation have changed, consider the multiple sources of experimental and comparative data on which these changes are based, and describe our methods for evaluating, incorporating and documenting these new data.     

FTIR micro‐reflectance absorption spectroscopic analysis of chemisorbed reaction films for tribological applications

Chemisorbed reaction films (CRFs) were prepared by using iron (Fe) particles (100–200 mess size) and thio (sulfur) derivatives of ethyl octadecenoate and methyl 12‐hydroxy octadecenoate in the light viscosity paraffin liquid medium. The reaction was conducted in a simulated condition of tribo‐chemical situation. CRFs were obtained in solid amorphous phase. The CRFs were examined for elemental composition and layer analysis using C‐H‐N‐O‐S analyzer and Fourier transform infrared spectroscopy micro‐reflectance absorption spectroscopic technique. Further, the CRFs were isolated into organic solvent soluble fractions using polar solvents of increasing polar strength. Their elemental analyses were studied, and chemical constitutions were known. Friction coefficient and wear scar diameter were evaluated by high frequency reciprocating rig, PLINT TE‐77 machine. Thermal stability was studied using thermogravimetric analysis technique in nitrogen environment. These studies inferred that CRFs appeared varying in its composition, luster, phase, chemical structure and thermal stability. Nonetheless, these were also found anomalous in elemental distribution throughout the layer structure of the CRFs. Copyright © 2015 John Wiley & Sons, Ltd.     

Friction and wear characteristics of organic thiophosphoro derivatives — a critical appraisal

The friction- and wear-reducing characteristics of a number of oil soluble sulphurised and phosphosulphurised derivatives for automotive and industrial applications have been reviewed. A critical appraisal of the suggested causes and mechanism of friction reduction and antiwear characteristics has been made. A review of the investigations carried out so far indicates that the creation of in situ films of simple inorganic salts, such as molybdenum disulphide, iron sulphide or phosphates of low shear strength and lamellar structures, does not appear to be the primary cause of low friction and wear. The authors have, therefore, undertaken a programme to synthesise and study the chemistry and tribochemical reactions in relation to friction and wear characteristics of hydrocarbon-soluble thiophosphoro derivatives of alkyl phenol, alcohols and fatty esters, in order to establish relationships between the reactivity, chemical nature and crystal structure of films formed on rubbing surfaces, and antifriction and antiwear characteristics. Molybdenum salts of various phosphorothio derivatives of pentadecylphenol and lauryl oleate of specific structures have been synthesised, and the friction and wear properties of their blends in mineral oil base stock have been studied. It can be clearly inferred from the results that type of bonding between sulphur, phosphorus and molybdenum, and reactivity of these derivatives with rubbing surfaces, determine their friction-reducing and antiwear characteristics. Their reactivity with iron and the nature of films formed are under investigation.     

First and second law analyses of a typical solar air dryer system: a case study

This communication presents an experimental study based on energy and exergy analyses of a typical solar air dryer. On the basis of the experimental results, the first and the second law efficiencies are calculated with respect to the available solar radiation. It is found that the second law efficiency is much less than the first law efficiency irrespective of the mass flow rate of the circulating fluid. It is also found that both efficiencies are an increasing function with respect to the mass flow rate of the working fluid. For a particular day, it is also found that first and second law efficiencies fluctuate according to radiation. The results obtained in this article are found to be consistent with those obtained by earlier authors as given in the literature.