Advances and Trends in Biotechnology and Genetics Vol. 1

Single Subunit RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Peramachi Palanivelu — 2019-07-31

Aim: To analyze various single subunit DNA dependent RNA polymerases and identify conserved motifs, active site regions among them and propose a plausible mechanism of action for these polymerases using the T7 RNA polymerase as a model system.
Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis and X-ray crystallographic data were analyzed.
Place and Duration of Study: Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625 021, India, from 2010 to 2013.
Methodology: The advanced version of Clustal Omega was used for protein sequence analysis of various SSU DNA dependent RNA polymerases from viruses, mitochondria and chloroplasts. Along with the conserved motifs identified by the bioinformatics analysis and with the data obtained by X-ray crystallographic, biochemical and site-directed mutagenesis (SDM) were also used to confirm the possible amino acids involved in the active sites and catalysis of these RNA polymerases.
Results: Multiple sequence analyses of various single subunit (SSU) DNA dependent RNA polymerases from different sources showed only a few highly conserved motifs among them, except chloroplast RNA polymerases where a large number of highly conserved motifs were found. Possible catalytic regions in all these polymerases consist of a highly conserved amino acid K and a ‘gatekeeper’ YG pair. In addition to, these polymerases also use an invariant R at the -4 position from the YG pair and an invariant S/T, adjacent to the YG pair. Furthermore, two highly conserved Ds are implicated in the metal-binding site and thus might participate in the catalytic process. The YG pair appears to be specific for DNA templates as it is not reported in RNA dependent RNA polymerases.
Conclusion: The highly conserved amino acid K, the ‘gatekeeper’ YG pair and an invariant R which are reported in all DNA polymerases, are also found in these DNA dependent RNA polymerases. Therefore, these RNA polymerases might be using the same catalytic mechanism as DNA polymerases. The catalytic amino acid K could act as the proton abstractor and generate the necessary nucleophile at the 3’-OH and the YG pair, R and the S/T might involve in the template binding and selection of nucleoside triphosphates (NTPs) for polymerization reactions. The two highly conserved Ds could act as the ‘NTP charge shielder’ and orient the alpha phosphate of incoming NTPs for the reaction at the 3’-OH growing end.

Electrochemical Evaluation of Interactions from Cratylia mollis Seed Lectin with Inhibitor Carbohydrate

Sandra Rodrigues de Souza — 2019-07-31

Aims: To evaluate the charge transference and the electrochemical potential of Cratylia mollis seed lectin, Cramoll 1,4, adsorbed on Nafion beads after the interaction of glucose ligand.

Study Design: Based on detection of electrochemical currents and potentials of Cramoll 1,4 by electrochemical techniques.

Place and Duration of Study: Department of Chemistry, Catholic University of Pernambuco, between April 2014 and November 2015.

Methodology: Cyclic Voltammetry (CV) was performed in an electrochemical cell containing three electrodes (a calomel electrode, a platinum wire counter electrode and a platinum electrode), connected to a potentiostat to obtain electrochemical currents related to the charge transference. An electrochemical cell containing a calomel electrode and a platinum electrode coupled to a multimeter was used to register the potentials. A saline solution was used as support to control the charge distribution inside of the cell. Cramoll 1,4-glucose interaction was evaluated in the concentration of 100, 200 and 300 mM of glucose.

Results: CV measurements showed significant charge transference after Cramoll 1,4-glucose interaction. Cathodic and anodic peaks paired near 100 mV were detected in the range 100-300 mM glucose, achieving a maximum current response of 1300 μA, approximately. Positive electrochemical potentials of Cramoll 1,4 adsorbed to Nafion-beads was achieved showing a linear behaviour with the increase of glucose concentration at 300 mM.

Conclusion: The system is useful for characterization of lectin-carbohydrate interactions and as a glucose sensor to estimate the activity of lectins. The use of cyclic voltammetry allowed the detection of a redox potential of 100 mV to Cramoll 1,4/Nafion-beads through the interaction with different glucose concentrations and constitutes another approach to determine Cramoll 1,4 activity. Electrochemical potential measurements generate a linear response for glucose concentrations, being an alternative for glucose sensing. Charge transference of lectins is a great phenomenon to evaluate lectin-carbohydrate interactions in biological systems.

Influence of Microorganisms on Corrosion Induction and Protection

Rawia Mansour — 2019-07-31

The microbial corrosion or the microbiologically induced corrosion (MIC) is defined as the deterioration of metals as a result of metabolic activities of microorganisms. MIC leads to important economical losses in many industries and services. Anaerobic Sulfate-reducing bacteria (SRB) is a serious type of MIC commonly found in the oil and gas industry sector and are responsible for most highly corrosion damages to offshore steel structures. The SRB present in the crude oil uses the sulfate as an electron acceptor generating corrosive hydrogen sulfide (H2S) as one of their products. Other types of MIC are the aerobic iron and manganese bacteria mainly responsible about the accelerated pitting attacks of stainless steel. Most MIC takes the form of pits that form below the colonies of living organic matter and mineral and the deposits of biological origin. The most affected devices by MIC are stainless and carbon steel tanks, pipelines, heat exchangers, fuel storage tanks. On the other hand, many beneficial bacteria, (such as Bacillus lichiniformis) have the ability to protect metal surfaces from corrosion via different mechanisms including biofilms and formation of sticky protective layer of γ-poly-glutamate or antibiotics on metal surfaces. The aim of the present work is to present a spotlight on the history and the role of microorganisms in corrosion induction and prevention. This chapter includes corrosion inhibition mechanisms employing beneficial microorganisms with special reference to microbial biofilms to avoid the dramatic economic loss due to corrosion. On the contrary, different types of harmful microorganisms included in corrosion are also discussed including iron and sulfur reducing bacteria.

Actinobacteria: A Renewable Source of Bioactive Molecules with Medical, Industrial and Pharmacological Importance

Rosilma de O. Araujo-Melo — 2019-07-31

The Actinobacteria receive much attention, since they produce a wide variety of metabolites, including antibiotics, antitumor agents, antioxidant molecules and enzyme inhibitors. These bacteria can be found in various habitats, including soil, ocean, extreme environments, mangrove, lichen, plants, and animals. The classification of Actinobacteria based upon the morphological observation, physiological and biochemical characteristics is not enough to differentiate the genera of this phylum. Following, a complementary identification is performed based on the distribution of specific constituents of the cellular wall, such as diaminopimelic acid and carbohydrates. With the advent of molecular biology, the identification of genera and species became more reliable. The screening of microbial natural products has become an important route to discover new bioactive compounds in order to develop new therapeutic agents. Actinobacteria remains one of the leading producers of biopharmaceuticals; endophytic Actinobacteria also yield secondary metabolites with wide range of biological activity. This review focuses on gathering relevant information on identification, classification, chemical diversity of Actinobacteria, as well as reveals some biotechnological applications of these bacteria. Actinobacteria are microorganisms widely distributed in nature, inhabiting mainly soil, and plants. These prokaryotes are broadly responsible for the production of various metabolites commercially available, such as antibiotics, antifungal compounds, enzymes, and chemotherapeutic agents. Endophytic Actinobacteria also produce active substances, and have important functions in the development of plants with agro-industrial interest, as well as in research efforts against multidrug-resistant bacteria, in order to find new effective compounds that can be used in clinical routine. This review provides data with a focus on spreading the importance of these microorganisms, as well as turns the attention to the fact that more studies are necessary for application of these Actinobacteria as innovative biotechnological tools.

AYA and HANA: New Seedless Mandarin’s Triploid Hybrids Selected in Morocco

Handaji Najat — 2019-07-31

Seedlessness is a major objective in Morocco Citrus improvement programme. Triploidy has played an important role in the development of new seedless mandarin cultivars for fresh fruit market and has received increasing attention for the consumers. Citrus triploid hybrids can be recovered through 2x x 2x taking advantage of the unreduced (2n) gametes formation. 2x x 4x and 4x x 2x sexual hybridizations are also widely exploited. In that case, many series of diploid crosses were assessed. At maturity, the fruits have been harvested, and small embryos were extracted from undeveloped seed and cultured on the medium of Murashig and Skoog supplemented with 1mg/l gibberellic acid according. Triploids seedling selected by flow cytometry analysis were budded on Troyer citrange and planted in INRA domain with spacing 6 m X 3 m. Two new seedless selections of mandarins have been evaluated in a citrus breeding program at Morocco INRA: Hana and Aya that were compared with their female parent clementine Sidi Aissa. These promising seedless mandarins were identified by easy-peeling, juicy fruits, have a pleasant taste and are sweet like the Sidi Aissa Clementine. These clones, showing commercial interest, were described and registered in Moroccan official catalogue. Triploidy confirmed its importance in obtaining seedless hybrids. This study is significant because it is the first triploids Citrus selection work in Menzeh INRA in Morocco. We found considerable differences most of the pomological characteristics. To make an objective comparison of fruit quality, precocity, and yield of these selected genotypes, they all need to be grown using many rootstocks, soils, climates, and cultural practices. Adaptation studies will also be required for the selected mandarins triploids. Therefore, selected in the first stage of selection should be studied in the second stage of selection.

Prospection of Endophyte Microorganisms from Bauhinia monandra Leaves with Mainly Identification of Actinobacteria

Rosilma de O. Araujo-Melo — 2019-07-31

The present work aimed the prospection of microorganisms from Bauhinia monandra leaves, with the purpose to identify endophytics to obtain strains with possible biotechnological applications. B. monandra leaves, disinfected with hypochlorite solution, were macerated in phosphate buffered saline and seeded in ten culture media containing antibacterial or antifungal agents. The endophytic filamentous fungus strains detected belonged to the genera Penicillium, Curvullaria and Aspergillus. Non-filamentous endophyte bacteria were grouped in the genera Bacillus, Burkholderia, Enterobacter and strains of endophytic Actinobacteria were classified as Streptomyces and Nocardiopsis. The isolation of endophytic microorganisms with nine culture media revealed better bacteria development with L-arginine agar; inorganic salt starch agar and potato dextrose agar were superior to Actinobacteria and fungus strains, respectively. The present study revealed the predominance of the genus Penicillium in leaves of B. monandra. This work introduces the first data of identification from endophyte Actinobacteria in the leaves of B. monandra.

Use of Palm Male Inflorescence and River-Sand as Acclimatization Substrate for Plantain (Musa sp.) Cultivars

Ekwa Yawa Monono — 2019-07-31

The objective of this work was to investigate the use of Palm Male Inflorescence (PMI) and river-sand as substrate for the acclimatization of plantain. Plantlets from three plantain cultivars (Batard, Ebanga and French Clair) were obtained after 16 weeks of tissue cultures and the plantlets were subjected to routine acclimatization under screen house conditions using two different substrates mixed in different ratios (100% Sand, 100% PMI, 75% PMI, 60% PMI and 50% PMI). The experiment was arranged in a completely randomized design with ten (10) replications; each replicate consisting of one micro-pot. The different substrates used significantly influenced the performance of the cultivars. The best medium for acclimatization for French Clair was 60% PMI in terms of percentage survival of plantlets (96.88%), plantlet height (6.03 cm), diameter (0.60 cm), number of leaves (4.42 leaves), leaf area (20.23 cm²), leaf emergence rate (1.64), number of roots (7.70 roots), and root length (18.86 cm). Ebanga plantlets had the best results with 75% PMI in terms of percentage survival of plantlets (96.88%), plantlet height (6.18 cm), diameter (0.62 cm), number of leaves (4.39 leaves), leaf area (20.48 cm²), leaf emergence rate (1.76), and total fresh weight (10.05 g). Meanwhile with Batard cultivar, 50% PMI was the best substrate in terms of percentage survival of plantlets (96.88%), plantlet height (4.41 cm), diameter (0.55 cm), number of leaves (4.55 leaves), leaf area (12.96 cm²), leaf emergence rate (1.55), and number of roots (5.73 roots). The relationship between the different variables assessed shows that plant height have a very strong positive correlation with pseudostem diameter (0.94), number of leaves (0.80), leaf area (0.98), and leaf emergence rate (0.77). This study clearly show that PMI can be a viable substrate to use with sand in plantlet acclimatization; however, the different plant cultivars had optimal result at different proportions of PMI.

Selection and Characterization of Phosphate Solubilizing Bacteria Isolated from the Rhizosphere of Three Cultivated Legumes in Morocco

Abderrazak Rfaki — 2019-07-31

Phosphorus is the most important nutrient for the growth and development of plants. Phosphatesolubilizing bacteria (PSB) are known to influence plant growth by enhancing the availability of soluble P. This study aimed to isolate PSB from the rhizosphere of three cultivated legumes (fava bean, chickpea, and green peas) and evaluate their plant growth promotion (PGP) traits. The capacity of isolates to solubilize unavailable phosphate was quantitatively evaluated in both agar plate and broth assays using National Botanical Research Institute's phosphate (NBRIP) medium. One hundred twenty-seven (127) PSBs were isolated from fifteen fields at different locations in Meknes, and according to their phosphate solubilization activity in vitro, eight of these isolates showing the greatest solubilization activity were selected for 16S rDNA sequencing. The selected isolates were further tested for other PGP traits, such as N2 fixation activity, indole-3-acetic acid (IAA) biosynthesis, and siderophore production. P solubilization index (SI) of selected isolates varied from 2.97 to 4.5 and amount of solubilized phosphorus ranged from 50.95 to 97.49 µg mL-1. All eight isolates showed the potential to produce IAA, and only two strains T4 and T13 showed positive N2-fixation activity. Also, all selected strains were able to produce siderophores, except two (T4 and T13). The selected PSBs were tentatively identiﬁed as belonging to seven genera, Rhizobium, Paraburkholderia, Bacillus, Pantoea, Rahnella, Klebsiella and Enterobacter. This research extends the knowledge on phosphate solubilizing bacteria in the rhizosphere of three cultivated legumes from Meknes region and our selected strains may be used as efficient bio-inoculants in the field to decrease environmental pollution and promoting sustainable agriculture.

Molecular Differentiation of Five Quinoa (Chenopodium quinoa Willd.) Genotypes Using Inter-simple Sequence Repeat (ISSR) Markers

A. M. M. Al-Naggar — 2019-07-31

Knowledge of genetic diversity is one of the important tools used for genetic management of quinoa accessions for plant breeding. This research aimed to molecularly characterize five quinoa genotypes using ISSR markers to reveal genetic polymorphism and identify unique markers for each genotype. Analysis of inter-simple sequence repeats (ISSR) revealed that 10 ISSR primers produced 53 amplicons, out of them 33 were polymorphic and the average percentage of polymorphism was 61.83%. The number of amplicons per primer ranged from 3 (HB-13, HB-10, HB-8 and 17898A) to 10 (HB-15) with an average of 5.3 fragments/primer across the different quinoa genotypes. Data showed a total number of unique ISSR markers of 24; eleven of them were positive and 13 were negative. Using ISSR analysis, we were able to identify some unique bands associated with quinoa genotypes. The genetic similarity ranged from 49% (between Ollague and each of QL-3 and Chipaya) to 76% (between CICA-17 and CO-407). The results indicated that all the five quinoa genotypes differ from each other at the DNA level where the average of genetic similarity (GS) between them was about 59%. The dendrogram separated the quinoa genotypes into two clusters; the first cluster included two genotypes (QL-3 and Chipaya). The second cluster was divided into two groups; the first group included two genotypes (CICA-17 and CO-407) and the second group included only one genotype (Ollague). Our results indicated that ISSR technique is useful in the establishment of the genetic fingerprinting and estimation of genetic relationships among quinoa genotypes. Also, this technique could detect enough polymorphism in the studied quinoa genotypes to distinguish each genotype from the others. Furthermore, the use of these results in the future is important for quinoa germplasm management and improvement as well as for the selection strategies of parental lines that facilitate the prediction of crosses in order to produce hybrids with higher performance. Using ISSR analysis, we were able to identify unique bands associated with quinoa genotypes. These bands might also be used in breeding programs for differentiating among Chinopodium quinoa varieties.