Inactivation of ntcA gene revealed differential proteome expression and induction of some hypothetical proteins in the cyanobacterium Anabaena sp. PCC 7120 and its derivative ntcA mutant under different levels of calcium

Abstract

Calcium is an important macronutrient for both prokaryotes and eukaryotes. It acts as an important second messenger mediating rapid response to environmental conditions. The present investigation deals with proteome profiling of Anabaena 7120 and its derivative ntcA mutant in response to varied calcium doses (0, 1 and 10?mM CaCl₂). Concentration of 1?mM CaCl₂ salt was the optimum concentration whereas 10?mM CaCl₂ was the inhibitory concentration for both the wild type and mutant strains. The results showed highly significant alteration in terms of protein abundance and differential response related to key processes of photosynthesis, energy and metabolism, nitrogen metabolism, oxidative and antioxidative defence, transport and signalling and fatty acid metabolism. In the wild type proteins related to photosynthesis and nitrogen metabolism showed upregulation at 1?mM CaCl₂ concentration while antioxidative defence related proteins were down-regulated. In the mutant however, proteins related to photosynthesis and nitrogen metabolism exhibited severe down-regulation. Some hypothetical proteins were also realized during proteome analysis. Overall, our results suggested that NtcA have a potential role in regulation of calcium ion dependent key processes underlying in various metabolic activities of the cyanobacterium Anabaena 7120.     

Unravelling the emerging threats of microplastics to agroecosystems

Reviews in Environmental Science and Bio/Technology - In the past few decades, pollution from microplastics has emerged as an important issue on a global scale. These plastic particles are mainly...     

Effect of Al and heavy metals on enzymes of nitrogen metabolism of fast and slow growing rhizobia under explanta conditions

Most of the legume crops are affected by metal stress present in the soil mainly due to contaminated agrochemicals and sewage sludge. The effect of aluminium, and heavy metals copper, iron and molybdenum on growth and activity of enzymes of fast and slow growing rhizobial sps. was studied. Sinorhizobium meliloti RMP₅ was found to be more tolerant to metal stress than Bradyrhizobium BMP₁. Both the strains were extremely sensitive to Al than other metals. Al was much more deleterious for the enzymatic activities (nitrate reduction, nitrite reduction, nitrogenase and uptake hydrogenase) of strain RMP₅ and BMP₁. Cu showed inhibitory effect on growth and enzyme activities of Bradyrhizobium strain at all concentrations. However, in S. meliloti RMP₅ all the tested enzymatic activities increased up to the concentration of 0.1 mM Cu. Fe enhanced the growth and enzyme activities of S. meliloti RMP₅ and Bradyrhizobium BMP₁ up to 100 mM concentration. Mo enhanced all the tested enzymatic activities of S. meliloti RMP₅ up to 1 mM. Nitrate and nitrite reduction activities of Bradyrhizobium BMP₁ increased up to 1 mM concentration. However, nitrogenase and hydrogenase activities of Bradyrhizobium BMP₁ got enhanced only up to 0.5 mM Mo. Both Fe and Mo are the key components of the enzyme nitrogenase and nitrate reductase and enhanced the growth and enzyme activities of both the sps. The study of physiology of nitrogen fixing ability of both fast and slow growing rhizobial strains reported that the supplementation of Mo and Fe in soils along with the biological formulations will enhance the process of symbiotic nitrogen fixation.     

The possible role of 10398A and 16189C mtDNA variants in providing susceptibility toT2DM in two North Indian populations: a replicative study

The role of mitochondria in causing diseases is becoming evident as more and more studies are focusing on this organelle of the cell. This is largely attributed to its reactive oxygen species (ROS) production property. In the context of diabetes, ROS is suggested to trigger different forms of insulin resistance involving different mechanisms. The suggestive role of a mtDNA variant G10398A in increasing ROS production and the impaired response to oxidative stress due to T16189C variant is worth addressing as genetic susceptibility factors in type 2 diabetes mellitus (T2DM). A case control study on 312 T2DM cases and ethnically matched 466 controls involving two North Indian populations, referred as cohort 1 and cohort 2 (in a replicative study), was undertaken to test such a genetic association. A statistically significant association was observed for 10398A allele in both the cohorts [cohort1 (OR = 2.67 95% CI 1.77–4.00); cohort2 (OR = 1.76 95%CI 1.12–2.77)]. The analysis of G10398A/T16189C haplotypic combinations revealed that 10398A/16189C haplotype provides a risk in both the cohorts. To sum up the study suggests that 10398A and 16189C alleles provide susceptiblity to T2DM independently as well as together.     

Effects of selective protein kinase c isozymes in prostaglandin2alpha-induced Ca2+ signaling and luteinizing hormone-induced progesterone accumulation in the mid-phase bovine corpus luteum

A single-cell approach for measuring the concentration of cytoplasmic calcium ions ([Ca(2+)](i)) and a protein kinase C-epsilon (PKCepsilon)-specific inhibitor were used to investigate the developmental role of PKCepsilon in the prostaglandin F(2alpha)(PGF(2alpha))-induced rise in [Ca(2+)](i) and the induced decline in progesterone accumulation in cultures of cells isolated from the bovine corpus luteum. PGF(2alpha) increased [Ca(2+)](i) in Day 4 large luteal cells (LLCs), but the response was significantly lower than in Day 10 LLCs (4.3 +/- 0.6, n = 116 vs. 21.3 +/- 2.3, n = 110). Similarly, the fold increase in the PGF(2alpha)-induced rise in [Ca(2+)](i) in Day 4 small luteal cells (SLCs) was lower than in Day 10 SLCs (1.6 +/- 0.2, n = 198 vs. 2.7 +/- 0.1, n = 95). A PKCepsilon inhibitor reduced the PGF(2alpha)-elicited calcium responses in both Day 10 LLCs and SLCs to 3.5 +/- 0.3 (n = 217) and 1.3 +/- 0.1 (n = 205), respectively. PGF(2alpha) inhibited LH-stimulated progesterone (P(4)) accumulation only in the incubation medium of Day 10 luteal cells. Both conventional and PKCepsilon-specific inhibitors reversed the ability of PGF(2alpha) to decrease LH-stimulated P(4) accumulation, and the PKCepsilon inhibitor was more effective at this than the conventional PKC inhibitor. In conclusion, the evidence indicates that PKCepsilon, an isozyme expressed in corpora lutea with acquired PGF(2alpha) luteolytic capacity, has a regulatory role in the PGF(2alpha)-induced Ca(2+) signaling in luteal steroidogenic cells, and that this in turn may have consequences (at least in part) on the ability of PGF(2alpha) to inhibit LH-stimulated P(4) synthesis at this developmental stage.     

Acetoxy drug: protein transacetylase of buffalo liver-characterization and mass spectrometry of the acetylated protein product

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K(m) (1667 microM) and V(max) (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys(51), Lys(82), Lys(123), Lsy(181), Lys(191) and Lys(210)) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.     

The Interactive Effect of SIRT1 Promoter Region Polymorphism on Type 2 Diabetes Susceptibility in the North Indian Population

Our previous studies have implicated genes mainly involved in the activity of pancreatic β cells in type 2 diabetes (T2D) susceptibility in the North Indian population. Recent literature on the role of SIRT1 as a potential master switch modulating insulin secretion and regulating gene expression in pancreatic β cells has warranted an evaluation of SIRT1 promoter region polymorphisms in the North Indian population, which is the main focus of the present study. 1542 samples (692 T2D patients and 850 controls) were sequenced for the 1.46 kb region upstream the translation start site of the SIRT1 gene. We performed a functional characterization of the SIRT1 promoter region polymorphisms using luciferase assay and observed a single-nucleotide polymorphism (SNP), rs12778366, in association with SIRT1 expression. We propose that TT, the high-expressing genotype of SNP rs12778366 in the SIRT1 promoter region and present in >80% of the North Indian population, was favored under conditions of feast-famine cycles in evolution, which has turned out to be a cause of concern in the present sedentary lifestyle under ad libitum conditions. Case-control association analysis did not implicate rs12778366 in T2DM per se in the studied population. However, our earlier reported risk genotype combinations of mt-ND3, PGC1α, and UCP2-866, when compared with the protective genotype combinations, in the background of the high-expressing TT genotype of SIRT1 SNP rs12778366, showed a very high additive risk [corrected odd ratio (OR) = 8.91; p = 6.5×10⁻¹¹]. The risk level was considerably low in the genotype backgrounds of TX (OR = 6.68; p = 2.71×10⁻¹²) and CX (OR = 3.74; p = 4.0×10⁻³). In addition, we screened other reported T2D-associated polymorphisms: PIK3R1 rs3730089, IRS1 rs1801278, and PPP1R3 rs1799999, which did not show any significant association in North Indian population. The present paper emphasizes the importance of gene interactions in the biological pathways of T2D, a complex lifestyle disease.     

Diverse mechanisms adopted by fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> PGC2 during the inhibition of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> and <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Rhizoctonia solani and Phytophthora capsici are two of the most destructive phytopathogens occurring worldwide and are only partly being managed by traditional control strategies. Fluorescent Pseudomonas isolates PGC1 and PGC2 were checked for the antifungal potential against R. solani and P. capsici. Both the isolates were screened for the ability to produce a range of antifungal compounds. The results of this study indicated the role of chitinase and β-1,3-glucanase in the inhibition of R. solani, however, antifungal metabolites of a non-enzymatic nature were responsible for inhibition of P. capsici. The study confirmed that multiple and diverse mechanisms are adopted by the same antagonist to suppress different phytopathogens, as evidenced in case of R. solani and P. capsici.     

Molecular characterization of ‘Clover proliferation’ phytoplasma subgroup-D (16SrVI-D) associated with vegetables crops in India

Nine vegetable plants species exhibiting phytoplasma suspected symptoms of white/purple leaf, little leaf, flat stem, witches’ broom, phyllody and leaf yellowing were observed in experimental fields at Indian Agricultural Research Institute, New Delhi from December 2015 to July 2016. Total DNA extracted from the three healthy and three symptomatic leaves of all the nine vegetables were subjected to PCR assays using phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. No amplifications of DNA were observed in first round PCR assays with primer pair P1/P7 from any of the symptomatic samples. However, phytoplasma DNA specific fragments of ~ 1.3 kb were amplified from Apium graveolens L. (two isolates), Brassica oleracea vr. capitata L. (one isolate) and Solanum melongena L. (one isolate) by using 3Far/3Rev primer pair and 1.2 kb fragment was amplified from Lactuca sativa L. (one isolate) by using R16F2n/R16R2 primer pair. No DNA amplification was seen in other symptomatic vegetable samples of tomato, carrot, cucurbit, bitter gourd and Amaranthus species utilizing either P1/P7 primer pair followed by 3Far/3Rev or R16F2n/R16R2 primer pairs. Out of three leafhopper species collected from the symptomatic vegetable fields, only Hishimonus phycitis was found positive for association of phytoplasma. No DNA amplifications were observed in healthy plant samples and insects collected from non-symptomatic fields. Comparative sequence comparison analyses of 16S rDNA of positive found vegetable phytoplasma strains revealed 100% sequence identities among each other and with phytoplasma strains of ‘clover proliferation’ (16SrVI) group. Phytoplasma sequences, virtual RFLPs and phylogenetic analyses of 16S rDNA sequence comparison confirmed the identification of 16SrVI subgroup D strain of phytoplasmas in four vegetables and one leafhopper (HP) species. Further virtual RFLP analysis of 16S rDNA sequence of the vegetables phytoplasma strains confirmed their taxonomic classification with strains of ‘clover proliferation’ subgroup D. Since, H. phycitis feeding on symptomatic vegetable species in the study was also tested positive for the 16SrVI phytoplasma subgroup-D as of vegetables; it may act as potent natural reservoir of 16SrVI-D subgroup of phytoplasmas infecting vegetable and other important agricultural crops.