The oxidation state of active site thiols determines activity of saccharopine dehydrogenase at low pH

Saccharopine dehydrogenase catalyzes the NAD-dependent conversion of saccharopine to generate l-lysine and α-ketoglutarate. A disulfide bond between cysteine 205 and cysteine 249, in the vicinity of the dinucleotide-binding site, is observed in structures of the apoenzyme, while a dithiol is observed in a structure with AMP bound, suggesting preferential binding of the dinucleotide to reduced enzyme. Mutation of C205 to S gave increased values of V/E_t and V/KE_t at pH 7 compared to wild type. Primary deuterium and solvent deuterium kinetic isotope effects suggest the catalytic pathway, which includes the hydride transfer and hydrolysis steps, contributes more to rate limitation in C205S, but the rates of the two steps relative to one another remain the same. There is a large increase in the rate constants V₁/E_t and V₁/K_NADEt at pH values below 7 compared to WT. Data indicate the low pH increase in activity results from a decreased sensitivity of the C205S mutant enzyme to the protonation state of an enzyme group with a pK_a of about 7, likely responsible for a pH-dependent conformational change. Reduction of WT and C205S mutant enzymes with TCEP gives equal activities at pH 6, consistent with the increased activity observed for the C205S mutant enzyme.     

Algal diversity in flowing waters at an acidic mine drainage “barrens” in central Pennsylvania,USA

Microscopic investigations were undertaken to decipher the diversity in the lotic algal communities from acidic waters (pH 2.4–3.2) flowing overland in sheets and channels at an acid mine drainage (AMD) barrens near Kylertown, PA, USA. Microscopic observations, supplemented with taxonomic keys, aided in identification of the dominant algae, and measurement of carbon from adjacent soils was undertaken. The unicellular protist Euglena sp. was most abundant in slower flowing waters (i.e., pool near point of emergence and surficial flow sheets), while Ulothrix sp. was most abundant in faster flowing water from the central stream channel. A diverse range of unicellular microalgae such as Chlorella, Cylindrocystis, Botryococcus, and Navicula and several filamentous forms identified as Microspora, Cladophora, and Binuclearia were also recorded. The observed high algal diversity may be related to the long duration of AMD flow at this site which has led to the development of adapted algal communities. The comparatively higher carbon content in soil materials adjacent to slower flowing water sampling locations provides evidence for the important role of algae as primary producers in this extreme environment.     

Development of cyanobacterium-based biofilms and their in vitro evaluation for agriculturally useful traits

The ability of cyanobacteria to be useful as matrices for agriculturally important bacteria was evaluated. Biofilms were generated with the selected strain Anabaena torulosa after co-culturing with Azotobacter chroococcum, Pseudomonas striata, Serratia marcescens, and Mesorhizobium ciceri. The biochemical attributes were compared with individual bacterial and cyanobacterial cultures. The biofilms were characterized in terms of proteins, chlorophyll, IAA production, acetylene-reducing activity, phosphate solubilization, and antagonism towards selected phytopathogenic fungi. An enhancement in the population counts was recorded in A. torulosa–S. marcescens and A. torulosa–P. striata biofilms. The A. torulosa–A. chroococcum and A. torulosa–M. ciceri biofilms were also able to utilize new saccharides as compared to the individual cultures. Such novel biofilms with agriculturally useful traits can provide additional advantages including the broader spectrum of activity and the presence or formation of biologically active compounds; they also suggest the way to effective inoculants for sustainable and environment friendly agriculture.     

Use of plant products and copepods for control of the dengue vector, <Emphasis Type="Italic">Aedes aegypti</Emphasis>

The efficacy of plant extracts (neem tree, Azadirachta indica A. Juss.; Meliaceae) and copepods [Mesocyclops aspericornis (Daday)] for the control of the dengue vector Aedes aegypti L. was tested in the laboratory. Neem Seed Kernel Extract (NSKE) at 25, 50, 100, 200 and 400 ppm caused significant mortality of Ae. aegypti larvae. Lethal concentrations (LC₅₀ and LC₉₀) were worked out. The LC₅₀ and LC₉₀ values for I to IV larval instars were 111.98, 138.34, 158.93, 185.22 ppm and for pupae was 146.13 ppm, respectively. The LC₉₀ value of I instar was 372.95 ppm, II instar was 422.77 ppm, III instar was 440.63 ppm, IV instar was 456.96 ppm, and pupae was 476.92 ppm, respectively. A study was conducted to test the whether the predatory efficiency of copepods on first instars changed in the presence of NSKE. The percentage of predatory efficiency of copepod was 6.80% in treatments without NSKE and the percentage of predatory efficiency increased up to 8.40% when copepods were combined with NSKE. This increase in predation efficiency may caused by detrimental effects of the neem active principle compound (Azadirachtin) on the mosquito larvae. Our results suggest that the combined application of copepods and neem extract to control Aedes populations is feasible. Repeated application of neem does not cause changes in copepod populations, because neem is highly degradable in the environment.     

Gene and QTL detection in a three-way barley cross under selection by a mixed model with kinship information using SNPs

Quantitative trait locus (QTL) detection is commonly performed by analysis of designed segregating populations derived from two inbred parental lines, where absence of selection, mutation and genetic drift is assumed. Even for designed populations, selection cannot always be avoided, with as consequence varying correlation between genotypes instead of uniform correlation. Akin to linkage disequilibrium mapping, ignoring this type of genetic relatedness will increase the rate of false-positives. In this paper, we advocate using mixed models including genetic relatedness, or ‘kinship’ information for QTL detection in populations where selection forces operated. We demonstrate our case with a three-way barley cross, designed to segregate for dwarfing, vernalization and spike morphology genes, in which selection occurred. The population of 161 inbred lines was screened with 1,536 single nucleotide polymorphisms (SNPs), and used for gene and QTL detection. The coefficient of coancestry matrix was estimated based on the SNPs and imposed to structure the distribution of random genotypic effects. The model incorporating kinship, coancestry, information was consistently superior to the one without kinship (according to the Akaike information criterion). We show, for three traits, that ignoring the coancestry information results in an unrealistically high number of marker–trait associations, without providing clear conclusions about QTL locations. We used a number of widely recognized dwarfing and vernalization genes known to segregate in the studied population as landmarks or references to assess the agreement of the mapping results with a priori candidate gene expectations. Additional QTLs to the major genes were detected for all traits as well.     

Characterization of the Biocidal Spectrum of Extracellular Filtrates of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

The effect of dichloromethane (DCM) and ethyl acetate (EA) extract of a cyanobacterium, Microcystis aeruginosa was evaluated against unicellular cyanobacteria and the phytopathogenic fungus Rhizoctonia solani. Fractionation of the filtrate showed the presence of five spots of different Rf values on silica gel coated plates indicating the presence of a number of compounds in the extract. A marked reduction in growth (52%) of the fungus was recorded on the plates supplemented with cyanobacterial extract, indicating the involvement of anti-fungal metabolite(s). The extract did not show any negative influence on seed germination and growth of seedlings of wheat, rice and mung, emphasizing the suitability of the compound for use in agriculture.     

BBProF: An Asynchronous Application Server for Rapid Identification of Proteins Associated with Bacterial Bioleaching

Recovery of metal value, especially from low-grade ores and overburden minerals using acidophilic bacteria through the process of bioleaching is an environmentally benign and commercially scalable biotechnology. In recent years, while the “OMICS” landscape has been witnessing extensive application of computational tools to understand and interpret global biological sequence data, a dedicated bioinformatic server for analysis of bacterial information in the context of its bioleaching ability is not available. We have developed an on-line Bacterial Bioleaching Protein Finder (BBProF) System, which rapidly identifies novel proteins involved in a bacterial bioleaching process and also performs phylogenetic analysis of 16S rRNA genes. BBProF uses the features of Asynchronous Java Script and XML (AJAX) to provide an efficient and fast user experience with minimal requirement of network bandwidth. In the input module the server accepts any bacterial or archaeal complete genome sequence in RAW format and provides a list of proteins involved in the microbial leaching process. BBProF web server is integrated with the European Bioinformatics Institute (EBI) web services such as BLAST for homology search and InterProScan for functional characterization of output protein sequences. Studying evolutionary relationship of bacterial strains of interest using Muscle and ClustalW2 phylogeny web services from EBI is another key feature of our server, where 16S rRNA gene sequences are considered as input through a JQUERY interface along with the sequences present in the BBProF database library. Complete genome sequences of 24 bioleaching microorganism characterized by genomic and physiological study in the laboratory and their respective 16S rRNA gene sequences were stored in the database of the BBProF library. To our knowledge BBProF is the first integrated bioinformatic web server that demonstrates its utility in identifying potential bioleaching bacteria. We hope that the server facilitate ongoing comparative genomic studies on of bioleaching microorganisms and also assist in identification and design of novel microbial consortia that are optimally efficient bioleaching agents.     

Prevalence of genetic variants associated with cardiovascular disease risk and drug response in the Southern Indian population of Kerala

BACKGROUND AND AIM:

This study reports the prevalence of five clinically significant variants associated with increased risk of cardiovascular disorders, and variable responses of individuals to commonly prescribed cardiovascular drugs in a South Indian population from the state of Kerala.

MATERIALS AND METHODS:

Genomic DNA isolated from 100 out-patient samples from Kerala were sequenced to examine the frequency of clinically relevant polymorphisms in the genes MYBPC3 (cardiomyopathy), SLCO1B1 (statin-induced myopathy), CYP2C9, VKORC1 (response to warfarin) and CYP2C19 (response to clopidogrel).

RESULTS:

Our analyses revealed the frequency of a 25 bp deletion variant of MYBPC3 associated with risk of cardiomyopathy was 7%, and the SLCO1B1 “C” allele associated with risk for statin-induced myopathy was 15% in this sample group. Among the other variants associated with dose-induced toxicity of warfarin, VKORC1 (c.1639G>A), was detected at 22%, while CYP2C9*3 and CYP2C9*2 alleles were present at a frequency of 15% and 3% respectively. Significantly, the tested sample population showed high prevalence (66%) of CYP2C19*2 variant, which determines response to clopidogrel therapy.

CONCLUSIONS:

We have identified that certain variants associated with cardiovascular disease and related drug response in the five genes, especially those in VKORC1, CYP2C19 and MYBPC3, are highly prevalent in the Kerala population, with almost 2 times higher prevalence of CYP2C19*2 variant compared with other regions in the country. Since the variants chosen in this study have relevance in disease phenotype and/or drug response, and are detected at a higher frequency, this study is likely to encourage clinicians to perform genetic testing before prescribing therapy.     

Inhibition of pathogenic bacterial biofilm by biosurfactant produced by Lysinibacillus fusiformis S9

A biosurfactant producing microbe isolated from a river bank was identified as Lysinibacillus fusiformis S9. It was identified with help of biochemical tests and 16S rRNA gene phylogenetic analysis. The biosurfactant S9BS produced was purified and characterized as glycolipid. The biosurfactant showed remarkable inhibition of biofilm formation by pathogenic bacteria like Escherichia coli and Streptococcus mutans. It was interesting to note that at concentration of 40 μg ml^?1 the biosurfactant did not show any bactericidal activity but restricted the biofilm formation completely. L. fusiformis is reported for the first time to produce a glycolipid type of biosurfactant capable of inhibiting biofilm formation by pathogenic bacteria. The biosurfactant inhibited bacterial attachment and biofilm formation equally well on hydrophilic as well as hydrophobic surfaces like glass and catheter tubing. This property is significant in many biomedical applications where the molecule should help in preventing biofouling of surfaces without being toxic to biotic system.