Understanding alternative fluxes/effluxes through comparative metabolic pathway analysis of phylum actinobacteria using a simplified approach

Actinobacteria are known for their diverse metabolism and physiology. Some are dreadful human pathogens whereas some constitute the natural flora for human gut. Therefore, the understanding of metabolic pathways is a key feature for targeting the pathogenic bacteria without disturbing the symbiotic ones. A big challenge faced today is multiple drug resistance by Mycobacterium and other pathogens that utilize alternative fluxes/effluxes. With the availability of genome sequence, it is now feasible to conduct the comparative in silico analysis. Here we present a simplified approach to compare metabolic pathways so that the species specific enzyme may be traced and engineered for future therapeutics.     

Contribution of K99 and D319 to substrate binding and catalysis in the saccharopine dehydrogenase reaction

Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to l-lysine and α-ketoglutarate. Lysine 99 is within hydrogen-bond distance to the α-carboxylate of the lysine substrate and D319 is in the vicinity of the carboxamide side chain of NADH. Both are conserved and may be important to the overall reaction. Replacing K99 with M gives decreases of 110-, 80- and 20-fold in the V(2)/K(m) values for lysine, α-ketoglutarate and NADH, respectively. Deuterium isotope effects on V and V/K(Lys) increase, while the solvent deuterium isotope effects decrease compared to the C205S mutant enzyme. Data for K99M suggest a decreased affinity for all reactants and a change in the partition ratio of the imine intermediate to favor hydrolysis. A change in the bound conformation of the imine and/or the distance of the imine carbon to C4 of the nicotinamide ring of NADH is also suggested. Changing D319 to A decreases V(2)/K(NADH) by 33-fold. Primary deuterium and solvent deuterium isotope effects decrease compared to C205S suggesting a non-isotope sensitive step has become slower. NADH binds to enzyme first, and sets the site for binding of lysine and α-ketoglutarate. The slower step is likely the conformational change generated upon binding of NADH.     

Down-regulation of hepatic G-6-Pase expression in hyperglycemic rats: Intervention with biogenic gold nanoconjugate

Chronic diabetes extensively complicates the glucose metabolism to onset and progress the complication. Concurrently, several contemporary medicines, especially organo-metallic formulations, are emerging to treat hyperglycemia. The current study aims to emphasize the gold nanoparticles (GNPs) potential for glucose metabolism regulation in Streptozotocin (STZ) induced diabetes. Quantitative real-time polymerase chain reaction (RT-PCR) was carried out to detect the mRNA expression of Glucose transporters 2 (GLUT2), Glucokinase (GK) and Glucose 6 Phosphatase (G-6-Pase). The study shows remarkable results such as the prognostic effect of GNPs in reinforcing the repression of enzyme complex G-6-Pase about 13.3-fold when compared to diabetes control. Also, molecular docking studies showed significant inhibition of G-6-Pase by the terpenoid ligands with alpha and beta amyrin from leaf extract of Couroupita guianensis. Thus the study explored the novel mechanism of G-6-Pase downregulated by GNPs intervention that majorly contributes to the regulation of circulatory glucose homeostasis during diabetes.     

Rapid identification of homozygosity and site of wild relative introgressions in wheat through chromosome‐specific KASP genotyping assays

For future food security, it is important that wheat, one of the most widely consumed crops in the world, can survive the threat of abiotic and biotic stresses. New genetic variation is currently being introduced into wheat through introgressions from its wild relatives. For trait discovery, it is necessary that each introgression is homozygous and hence stable. Breeding programmes rely on efficient genotyping platforms for marker‐assisted selection (MAS). Recently, single nucleotide polymorphism (SNP)‐based markers have been made available on high‐throughput Axiom^® SNP genotyping arrays. However, these arrays are inflexible in their design and sample numbers, making their use unsuitable for long‐term MAS. SNPs can potentially be converted into Kompetitive allele‐specific PCR (KASP?) assays that are comparatively cost‐effective and efficient for low‐density genotyping of introgression lines. However, due to the polyploid nature of wheat, KASP assays for homoeologous SNPs can have difficulty in distinguishing between heterozygous and homozygous hybrid lines in a backcross population. To identify co‐dominant SNPs, that can differentiate between heterozygotes and homozygotes, we PCR‐amplified and sequenced genomic DNA from potential single‐copy regions of the wheat genome and compared them to orthologous copies from different wild relatives. A panel of 620 chromosome‐specific KASP assays have been developed that allow rapid detection of wild relative segments and provide information on their homozygosity and site of introgression in the wheat genome. A set of 90 chromosome‐nonspecific assays was also produced that can be used for genotyping introgression lines. These multipurpose KASP assays represent a powerful tool for wheat breeders worldwide.     

Spermatophore consumption in a cephalopod

An individual''s gametes can represent a nourishing food source for a manipulative mate. Here, we provide evidence of ejaculate and sperm consumption in a cephalopod. Through labelling male spermatophores with ¹⁴C radiolabel, we found that female squid, Sepiadarium austrinum, consumed the spermatophores of their partners and directed the nutrients received into both somatic maintenance and egg production. We further show that in this species—where fertilization occurs externally in the female''s buccal cavity—sperm storage is short-term (less than 21 days). The combination of female spermatophore consumption and short-term external sperm storage has the potential to exert strong selection on male ejaculates and reproductive strategies.     

Deletion of the eIF2α Kinase GCN2 Fails to Rescue the Memory Decline Associated with Alzheimer’s Disease

Emerging evidence suggests that dysregulated translation through phosphorylation of eukaryotic initiation factor-2α (eIF2α) may contribute to Alzheimer’s disease (AD) and related memory impairments. However, the underlying mechanisms remain unclear. Here, we crossed knockout mice for an eIF2α kinase (GCN2: general control nonderepressible-2 kinase) with 5XFAD transgenic mice, and investigated whether GCN2 deletion affects AD-like traits in this model. As observed in AD brains, 5XFAD mice recapitulated significant elevations in the β-secretase enzyme BACE1 and the CREB repressor ATF4 concomitant with a dramatic increase of eIF2α phosphorylation. Contrary to expectation, we found that GCN2^−/− and GCN2^+/− deficiencies aggravate rather than suppress hippocampal BACE1 and ATF4 elevations in 5XFAD mice, failing to rescue memory deficits as tested by the contextual fear conditioning. The facilitation of these deleterious events resulted in exacerbated β-amyloid accumulation, plaque pathology and CREB dysfunction in 5XFAD mice with GCN2 mutations. Notably, GCN2 deletion caused overactivation of the PKR-endoplasmic reticulum-related kinase (PERK)-dependent eIF2α phosphorylation pathway in 5XFAD mice in the absence of changes in the PKR pathway. Moreover, PERK activation in response to GCN2 deficiency was specific to 5XFAD mice, since phosphorylated PERK levels were equivalent between GCN2^−/− and wild-type control mice. Our findings suggest that GCN2 may be an important eIF2α kinase under the physiological condition, whereas blocking the GCN2 pathway under exposure to significant β-amyloidosis rather aggravates eIF2α phosphorylation leading to BACE1 and ATF4 elevations in AD.     

Detection, selective isolation and characterisation of Dactylosporangium strains from diverse environmental samples

A culture-independent, nested PCR procedure based on genus-specific oligonucleotide primers detected the presence of members of the genus Dactylosporangium in 14 out of 21 diverse environmental samples. Clones generated from the 14 positive environmental samples formed distinct phyletic lines in the dactylosporangial 16S rRNA gene tree. Presumptive dactylosporangiae were isolated from 7 of these samples using a medium designed to be selective for members of the genus Dactylosporangium, namely Streptomyces Isolation Medium supplemented with gentamicin and antifungal antibiotics. One hundred and two out of 219 representative presumptive dactylosporangiae were considered as authentic members of the genus Dactylosporangium as they gave PCR amplification products using the genus-specific primers and had chemical features typical of dactylosporangiae. Representative of the Dactylosporangium isolates formed distinctive phyletic lines in the dactylosporangial 16S rRNA gene tree, contained the non-ribosomal peptide and type-I polyketide synthase genes and inhibited the growth of Bacillus subtilis, Kocuria rhizophila and Staphylococcus aureus strains. It is evident from these results that the genus Dactylosporangium is underspeciated, widely distributed in natural habitats and is a potentially rich source of novel secondary metabolites.     

Expression level of a chimeric kinase governs entry into sporulation in Bacillus subtilis

Upon starvation, Bacillus subtilis cells switch from growth to sporulation. It is believed that the N-terminal sensor domain of the cytoplasmic histidine kinase KinA is responsible for detection of the sporulation-specific signal(s) that appears to be produced only under starvation conditions. Following the sensing of the signal, KinA triggers autophosphorylation of the catalytic histidine residue in the C-terminal domain to transmit the phosphate moiety, via phosphorelay, to the master regulator for sporulation, Spo0A. However, there is no direct evidence to support the function of the sensor domain, because the specific signal(s) has never been found. To investigate the role of the N-terminal sensor domain, we replaced the endogenous three-PAS repeat in the N-terminal domain of KinA with a two-PAS repeat derived from Escherichia coli and examined the function of the resulting chimeric protein. Despite the introduction of a foreign domain, we found that the resulting chimeric protein, in a concentration-dependent manner, triggered sporulation by activating Spo0A through phosphorelay, irrespective of nutrient availability. Further, by using chemical cross-linking, we showed that the chimeric protein exists predominantly as a tetramer, mediated by the N-terminal domain, as was found for KinA. These results suggest that tetramer formation mediated by the N-terminal domain, regardless of the origin of the protein, is important and sufficient for the kinase activity catalyzed by the C-terminal domain. Taken together with our previous observations, we propose that the primary role of the N-terminal domain of KinA is to form a functional tetramer, but not for sensing an unknown signal.