Proline Substitution of Dimer Interface β-strand Residues as a Strategy for?the Design of Functional Monomeric Proteins

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.     

A Conserved Rubredoxin Is Necessary for Photosystem II Accumulation in Diverse Oxygenic Photoautotrophs

In oxygenic photosynthesis, two photosystems work in tandem to harvest light energy and generate NADPH and ATP. Photosystem II (PSII), the protein-pigment complex that uses light energy to catalyze the splitting of water, is assembled from its component parts in a tightly regulated process that requires a number of assembly factors. The 2pac mutant of the unicellular green alga Chlamydomonas reinhardtii was isolated and found to have no detectable PSII activity, whereas other components of the photosynthetic electron transport chain, including photosystem I, were still functional. PSII activity was fully restored by complementation with the RBD1 gene, which encodes a small iron-sulfur protein known as a rubredoxin. Phylogenetic evidence supports the hypothesis that this rubredoxin and its orthologs are unique to oxygenic phototrophs and distinct from rubredoxins in Archaea and bacteria (excluding cyanobacteria). Knockouts of the rubredoxin orthologs in the cyanobacterium Synechocystis sp. PCC 6803 and the plant Arabidopsis thaliana were also found to be specifically affected in PSII accumulation. Taken together, our data suggest that this rubredoxin is necessary for normal PSII activity in a diverse set of organisms that perform oxygenic photosynthesis.     

Arsenic accumulation in Ocimum spp. and its effect on growth and oil constituents

A comparative evaluation of As accumulation and subsequent effects upon exposure to arsenite [As(III)] was performed in three species of Ocimum. Plants accumulated high amount of As (μg g^?1 dry weight; dw) (662 in O. tenuiflorum, 764 in O. basilicum and 831 in O. gratissimum at 100 μM As(III) after 10 days) with the order of accumulation being roots > stem > leaves. A significant reduction in plant height and biomass was observed. However, essential oil yield and major oil constituents, such as eugenol, methyl chevicol, and linalool, increased at lower As(III) concentrations [mostly up to 25 μM As(III)] in all three species. Positively, no detectable amount of As was found in oil of any species. The study proposes that Ocimum may be used as a phytoremediator and at the same time as a source of essential oils under proper regulation.     

Metabolite profiling can assist variability analysis in Trichoderma species

Variability in 41 isolates of Trichoderma belonging to 21 species was observed in the phenolic acid profile of their culture filtrates. The phenolic acid profiles were observed to be very stable in the culture filtrate of Trichoderma species. The similarity in phenolic acid profile was recorded and based on it the species were grouped into three distinct groups, viz. highly similar, moderately similar and least similar. Of the 21 species, seven species showed highly similar trend, whereas two and four species showed moderate and least similarity in their phenolic acid profiles, respectively. Looking into the stability of phenolic acid profile in the culture filtrate of the Trichoderma species the present tool may help in diversity analysis in Trichoderma species originating from different geographical areas.     

Endophytic bacteria from tomato and chilli,their diversity and antagonistic potential against Ralstonia solanacearum

A total of 82 endophytic bacteria of tomato and chilli was isolated from different locations of tropical Islands of Andaman and Nicobar, India. Based on in vitro screening, 16 bacterial isolates that effectively inhibited Ralstonia solanacearum (a bacterial wilt pathogen) were characterised for their diversity and identified through Microbial Identification System (Biolog). Diversity analysed through BOX-PCR showed low similarity index among the antagonistic bacteria. Based on the in vitro antagonistic activities, the selected isolates were further characterised for siderophore, indole acetic acid production, phosphate solubilisation and other extracellular enzymes; it is found that most of the isolates were positive for these properties. The production of these metabolites may be responsible for the inhibition of the pathogen R. solanacearum. The isolates BECS3, BECS6 and BECS7 showed multiple attributes and demonstrated plant growth promotion properties through tomato- and chilli-based bioassay under greenhouse conditions. These bacterial inoculations were found to result in significant increase in root, shoot and biomass of both tomato and chilli. Hence, these isolates can be further formulated and used for field application.     

Characterisation,genetic diversity and antagonistic potential of 2,4-diacetylphloroglucinol producing Pseudomonas fluorescens isolates in groundnut-based cropping systems of Andhra Pradesh,India

This study is focused on isolation and characterisation of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing Pseudomonas fluorescens isolates from different soils of groundnut-based cropping systems in Andhra Pradesh. In our studies, 21 isolates of P. fluorescens were isolated and confirmed through various biochemical tests, of which five were tested positive for 2,4-DAPGproduction with specific primers. Biocontrol potential of these isolates on groundnut stem rot pathogen (Sclerotium rolfsii) was determined through in vitro dual culture assays. The eight isolates were found effective against S. rolfsii (up to 75% inhibition) in dual culture method. All the five 2,4-DAPG-producing Plant Growth-Promoting Rhizobacteria isolates were highly antagonistic to S. rolfsii. Genetic diversity of these P. fluorescens isolates was determined by random amplification of polymorphic DNA analysis. Overall, our results suggest that the prevalence of 2,4-DAPG-producing fluorescent Pseudomonads in different crop rhizospheres of groundnut-based cropping systems.     

Use of Genetically Engineered Microorganisms (GEMs) for the Bioremediation of Contaminants

ABSTRACT

This paper presents a critical review of the literature on the application of genetically engineered microorganisms (GEMs) in bioremediation. The important aspects of using GEMs in bioremediation, such as development of novel strains with desirable properties through pathway construction and the modification of enzyme specificity and affinity, are discussed in detail. Particular attention is given to the genetic engineering of bacteria using bacterial hemoglobin (VHb) for the treatment of aromatic organic compounds under hypoxic conditions. The application of VHb technology may advance treatment of contaminated sites, where oxygen availability limits the growth of aerobic bioremediating bacteria, as well as the functioning of oxygenases required for mineralization of many organic pollutants. Despite the many advantages of GEMs, there are still concerns that their introduction into polluted sites to enhance bioremediation may have adverse environmental effects, such as gene transfer. The extent of horizontal gene transfer from GEMs in the environment, compared to that of native organisms including benefits regarding bacterial bioremediation that may occur as a result of such transfer, is discussed. Recent advances in tracking methods and containment strategies for GEMs, including several biological systems that have been developed to detect the fate of GEMs in the environment, are also summarized in this review. Critical research questions pertaining to the development and implementation of GEMs for enhanced bioremediation have been identified and posed for possible future research.