Driving forces of the complex formation between highly charged disordered proteins.

Highly disordered complexes between oppositely charged intrinsically disordered proteins present a new paradigm of biomolecular interactions. Here, we investigate the driving forces of such interactions for the example of the highly positively charged linker histone H1 and its highly negatively charged chaperone, prothymosin α (ProTα). Temperature-dependent single-molecule Förster resonance energy transfer (FRET) experiments and isothermal titration calorimetry reveal ProTα-H1 binding to be enthalpically unfavorable, and salt-dependent affinity measurements suggest counterion release entropy to be an important thermodynamic driving force. Using single-molecule FRET, we also identify ternary complexes between ProTα and H1 in addition to the heterodimer at equilibrium and show how they contribute to the thermodynamics observed in ensemble experiments. Finally, we explain the observed thermodynamics quantitatively with a mean-field polyelectrolyte theory that treats counterion release explicitly. ProTα-H1 complex formation resembles the interactions between synthetic polyelectrolytes, and the underlying principles are likely to be of broad relevance for interactions between charged biomolecules in general.

Polymer complexation: Partially ionizable asymmetric polyelectrolytes.

Theories of bulk coacervation of oppositely charged polyelectrolytes (PE) obscure single molecule level thermodynamic details, considered significant for coacervate equilibrium, whereas simulations account for only pairwise Coulomb interaction. Also, studies of effects of asymmetry on PE complexation are rare compared to symmetric PEs. We develop a theoretical model, accounting for all entropic and enthalpic contributions at the molecular level, and the mutual segmental screened Coulomb and excluded volume interactions between two asymmetric PEs, by constructing a Hamiltonian following Edwards and Muthukumar. Assuming maximal ion-pairing in the complex, the system free energy comprising configurational entropy of the polyions and free-ion entropy of the small ions is minimized. The effective charge and size of the complex, larger than sub-Gaussian globules as for symmetric chains, increase with asymmetry in polyion length and charge density. The thermodynamic drive for complexation is found to increase with ionizability of symmetric polyions and with a decrease in asymmetry in length for equally ionizable polyions. The crossover Coulomb strength demarcating the ion-pair enthalpy-driven (low strength) and counterion release entropy-driven (high strength) is marginally dependent on the charge density, because so is the degree of counterion condensation, and strongly dependent on the dielectric environment and salt. The key results match the trends in simulations. The framework may provide a direct way to calculate thermodynamic dependencies of complexation on experimental parameters such as electrostatic strength and salt, thus to better analyze and predict observed phenomena for different sets of polymer pairs.

Polyelectrolyte complexation of two oppositely charged symmetric polymers: A minimal theory.

Interplay of Coulomb interaction energy, free ion entropy, and conformational elasticity is a fascinating aspect in polyelectrolytes (PEs). We develop a theory for complexation of two oppositely charged PEs, a process known to be the precursor to the formation of complex coacervates in PE solutions, to explore the underlying thermodynamics of complex formation, at low salts. The theory considers general degrees of solvent polarity and dielectricity within an implicit solvent model, incorporating a varying Coulomb strength. Explicit calculation of the free energy of complexation and its components indicates that the entropy of free counterions and salt ions and the Coulomb enthalpy of bound ion-pairs dictate the equilibrium of PE complexation. This helps decouple the self-consistent dependency of charge and size of the uncomplexed parts of the polyions, derive an analytical expression for charge, and evaluate the free energy components as functions of chain overlap. Complexation is observed to be driven by enthalpy gain at low Coulomb strengths, driven by entropy gain of released counterions but opposed by enthalpy loss due to reduction of ion-pairs at moderate Coulomb strengths, and progressively less favorable due to enthalpy loss at even higher Coulomb strengths. The total free energy of the system is found to decrease linearly with an overlap of chains. Thermodynamic predictions from our model are in good quantitative agreement with simulations in literature.

Three-dimensional graphene for electrochemical detection of Cadmium in Klebsiella michiganensis to study the influence of Cadmium uptake in rice plant.

Cadmium (Cd), as a hazardous pollutant present in the environment as well as within biological samples, needs to be detected and remediated at the same time. Although many types of Cd detection techniques have been developed globally, there is no evidence to analyse Cd2+ ion electrochemically using graphene-based electrode for bioaccumulation of Cd in bacteria and plants. The present study describes the fabrication and characterization of a three-dimensional reduced graphene oxide-based electrode to detect bioaccumulation of Cd within the bacterial cell and rice tissues applying differential pulse voltammetry (DPV) technique. In addition, X-ray fluorescence (XRF) and X-ray diffraction (XRD) studies were performed as supporting tools for this study in the selected Cd resistant plant growth promoting rhizobacterial (PGPR) strain, Klebsiella michiganensis MCC3089. This strain was characterized based on its plant growth promoting (PGP) traits and exhibited bioaccumulation of Cd both under high and low Cd concentrations, of which the latter is more environmentally significant. The Cd-sequestration ability of this strain was found to reduce Cd uptake within rice seedlings.

Enhancement of growth and salt tolerance of rice seedlings by ACC deaminase-producing Burkholderia sp. MTCC 12259.

Increasing soil salinity is often associated with accelerated ethylene production in plants, leading to overall growth reduction. The salt-tolerant 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing PGPR may alleviate salt stress by reducing the production of stress ethylene. In this study, a salt-tolerant ACC deaminase-producing strain named P50 was isolated from a coastal rice field in Odisha, India, which enhanced the growth of rice seedlings under salt stress. The P50 strain was identified as Burkholderia sp. based on phenotypic characteristics, MALDI-TOF MS data for ribosomal proteins and 16S rDNA sequence-based homology. Various PGP traits of strain P50 were characterized, among which the ACC deaminase activity was optimized at different physical conditions and confirmed by enzyme assay, as well as FTIR. The IAA, EPS and proline production of this strain were estimated under increasing NaCl concentrations essential for plant growth promotion under salt stress. Finally, the P50 strain was utilized in a gnotobiotic assay using rice seedlings (cv. Swarnamasuri) under saline stress. Seedlings treated with the P50 strain showed improvement in various morphological and biochemical characteristics, ROS scavenging antioxidant enzymatic activities, and reduced amounts of stress ethylene compared to non-inoculated strains under salinity. In addition, isolation of the ACC deaminase mutant of this strain was not found to reduce stress ethylene, confirming that the P50 strain was associated with a reduction in stress ethylene. Strain P50 was also found to colonize the root surfaces of rice seedlings associated with the plant-microbe interaction process. Thus, as an effective salt-tolerant PGPR, strain P50 can be utilized in salt-affected agricultural fields to improve plant growth in a sustainable manner.

The role of arsenic resistant Bacillus aryabhattai MCC3374 in promotion of rice seedlings growth and alleviation of arsenic phytotoxicity.

The biological agents have been utilized as an affordable alternative to conventional costly metal remediation technologies for last few years. The present investigation introduces arsenic (As) resistant plant growth promoting rhizobacteria (PGPR) isolated from the As-contaminated agricultural field of West Bengal, India that alleviates arsenic-induced toxicity and exhibited many plant growth promoting traits (PGP). The isolated strain designated as AS6 has identified as Bacillus aryabhattai based on phenotypic characteristics, physio-biochemical tests, MALDI-TOFMS bio-typing, FAME analysis and 16S rDNA sequence homology. The strain found to exhibit five times more resistance to arsenate than arsenite with minimum inhibitory concentrations (MIC) being 100 mM and 20 mM respectively. The result showed that accumulation of As was evidenced by SEM- EDAX, TEM-EDAX studies. The intracellular accumulation of arsenic was also confirmed as in bacterial biomass by AAS, FTIR, XRD and XRF analyses. The increased rate of As (V) reduction by this strain found to be exploited for the remediation of arsenic in the contaminated agricultural field. The strain also found to exhibit important PGP traits viz., ACC deaminase activity (2022 nmol α-ketobutyrate/mg protein/h), IAA production (166 µg/ml), N2 fixation (0.32 µgN fixed/h/mg proteins) and siderophore production (72%) etc. Positive influenced of AS6 strain on rice seedlings growth promotion under As stress was observed considering the several morphological, biochemical parameters including antioxidants activities as compared with an uninoculated set. Thus this strain might be exploited for stress amelioration and plant growth enhancement of rice cultivar under arsenic spiked agricultural soil.

Computational elucidation of phylogenetic, structural and functional characteristics of Pseudomonas Lipases.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) catalyzes tri-, di-, and monoacyl glycerol of fat into glycerol and fatty acids. It has important roles in the digestion of lipids in living organisms and industrially as laundry detergents along with proteases. The microbial lipases are more stable, active and economically feasible compared to plant and animal sources. Hence, much attention was given to the maximum production of the enzyme from the microbial sources. The phylogenetic analysis revealed that the amino acid sequence of lipase protein and their corresponding cDNA of Pseudomonas aeruginosa clustered with Pseudomonas stutzeri among different species of Pseudomonas, while P. aeruginosa PA1 clustered with P. aeruginosa SJTD-1 among different strains of P. aeruginosa. The lipase of P. aeruginosa PA1 was a monomeric, acidic and thermostable protein having a molecular weight ranging in between 32.72 to 34.89 kDa. The protein was abundant with random coils and alpha helices in its secondary structure. The tertiary model showed 96.310 score as an overall quality factor. Hence, this in silico study gives some useful information about the lipase protein without performing crystal structure assessment by X-ray Crystallography or NMR study in wet lab experiments which could be helpful for isolation and characterization of the enzyme in vitro.

Characterization of Cd-resistant Klebsiella michiganensis MCC3089 and its potential for rice seedling growth promotion under Cd stress.

Application of heavy metal resistant plant growth promoting rhizobacteria has an important role as they help to evade metal-induced toxicity in plants on one hand and enhance plant growth on the other. The present study is therefore focused on the characterization of a cadmium resistant bacterial strain isolated from heavy metal contaminated rhizospheric soil designated as S8. This S8 strain was selected in terms of cadmium resistance and plant growth promoting traits. Moreover, it also showed resistance to lead and arsenic to a considerable extent. The selected strain S8 was identified as Klebsiella michiganensis by modern approaches of bacterial taxonomy. The plant growth promoting traits exhibited by the strain include 1-aminocyclopropane-1-carboxylic acid deaminase activity (58.33 ng α-keto butyrate/mg protein/h), Indole-3-acetic acid production (671 µg/ml), phosphate solubilization (71.98 ppm), nitrogen fixation (3.72 µg of nitrogen fixed/h/mg protein) etc. Besides, the strain also exhibited high cadmium removal efficiency (73-97%) from the medium and intracellular accumulation as well. Its efficiency to alleviate cadmium-induced toxicity was determined against a rice cultivar in terms of morphological and biochemical changes. Enhanced growth and reduced oxidative stress were detected in presence of the bacterium. On the basis of these results, it can be concluded that K. michiganensis strain S8 is cadmium accumulating plant growth promoting rhizobacterium that can be applied in cadmium contaminated agricultural soil to achieve better productivity of rice.

Bioaccumulation of cadmium by Enterobacter sp. and enhancement of rice seedling growth under cadmium stress.

Bacteria-mediated plant growth promotion and bioremediation of heavy metal containing soil is a widely accepted eco-friendly method. The present study is aimed to screen out cadmium resistant bacterial strain from metal contaminated rice rhizosphere and evaluate its effects on the growth of rice seedlings under cadmium stress. Among four different isolates (designated as S1, S2, S3 and S5), the S2 isolate was screened on the basis of different PGP traits and multi heavy metal resistance (minimum inhibitory concentration for cadmium, lead and arsenic were 3500, 2500 and 1050 µg/ml respectively). The selected S2 strain has ability to produce ACC deaminase (236.11 ng α-keto-butyrate/mg protein/h), IAA (726 µg/ml), solubilize phosphate (73.56 ppm) and fix nitrogen (4.4 µg of nitrogen fixed/h/mg protein). The selected strain was identified as Enterobacter sp. on the basis of phenotypic characterization, MALDI-TOF MS analysis of ribosomal proteins, FAME analysis and 16 S rDNA sequence homology. The high cadmium removal efficiency (> 95%) of this strain from the growth medium was measured by Atomic Absorption Spectrophotometer and it was due to intracellular cadmium accumulation evidenced by SEM-EDX-TEM-EDX study. SEM analysis also revealed no distortion of surface morphology of this strain even grown in the presence of high cadmium concentration (3000 µg/ml). Inoculation of this strain with rice seedlings significantly enhanced various morphological, biochemical characters of seedling growth compared with un-inoculated seedlings under Cd stress. The strain also exhibited alleviation of cadmium-induced oxidative stress, reduction of stress ethylene and decreased the accumulation of cadmium in seedlings as well that conferred cadmium tolerance to the plant. Thus the S2 strain could be considered as a potent heavy metal resistant PGPR applicable in heavy metal contaminated agricultural soil for bioremediation and plant growth promotion as well. MAIN FINDING: A cadmium resistant plant growth promoting Enterobacter sp. was isolated that accumulated cadmium evidenced by SEM-TEM-EDX study. It reduced Cd uptake and enhanced growth in rice seedlings.

Alleviation of phytotoxic effects of cadmium on rice seedlings by cadmium resistant PGPR strain Enterobacter aerogenes MCC 3092.

Heavy metal resistant PGPR mediated bioremediation, phytostimulation and stress alleviation is an eco-friendly method for sustainable agriculture in the metal contaminated soil. The isolation of such PGPR is highly demanding to reduce heavy metals in contaminated cultivated fields for agricultural benefit. The present study was successful to isolate a potent multi-heavy metal resistant PGPR strain, identified as Enterobacter aerogenes strain K6 based on MALDI-TOF MS, FAME analysis and 16S rDNA sequence homology, from rice rhizosphere contaminated with a variety of heavy metals/metalloid near industrial area. The strain exhibited high degree of resistance to Cd2+, Pb2+ and As3+ upto 4000 µg/mL, 3800 µg/mL and 1500 µg/mL respectively. Intracellular Cd accumulation of this strain was evidenced by AAS-SEM-TEM-EDX-XRF studies. Moreover, it showed several important PGP traits like IAA production, nitrogen fixation, phosphate solubilization, ACC deaminase activity even under high Cd stress (upto 3000 µg/mL). The combined effect of Cd resistance and PGP activities of this strain was manifested to the significant (p < 0.05) growth promotion of rice seedling under Cd stress by reducing oxidative stress (through antioxidants), stress ethylene and Cd uptake in seedlings. Thus K6 strain conferred Cd-tolerance in rice seedlings and could be applied as PGPR in contaminated fields.

A halotolerant Enterobacter sp. displaying ACC deaminase activity promotes rice seedling growth under salt stress.

Agricultural productivity is proven to be hampered by the synthesis of reactive oxygen species (ROS) and production of stress-induced ethylene under salinity stress. One-aminocyclopropane-1-carboxylic acid (ACC) is the direct precursor of ethylene synthesized by plants. Bacteria possessing ACC deaminase activity can use ACC as a nitrogen source preventing ethylene production. Several salt-tolerant bacterial strains displaying ACC deaminase activity were isolated from rice fields, and their plant growth-promoting (PGP) properties were determined. Among them, strain P23, identified as an Enterobacter sp. based on phenotypic characteristics, matrix-assisted laser desorption ionization-time of flight mass spectrometry data and the 16S rDNA sequence, was selected as the best-performing isolate for several PGP traits, including phosphate solubilization, IAA production, siderophore production, HCN production, etc. Enterobacter sp. P23 was shown to promote rice seedling growth under salt stress, and this effect was correlated with a decrease in antioxidant enzymes and stress-induced ethylene. Isolation of an acdS mutant strain enabled concluding that the reduction in stress-induced ethylene content after inoculation of strain P23 was linked to ACC deaminase activity.

Characterization of cadmium-resistant Klebsiella pneumoniae MCC 3091 promoted rice seedling growth by alleviating phytotoxicity of cadmium.

Cadmium (Cd) phytotoxicity in agricultural land is a major global concern now-a-days resulting in very poor yield. Plant growth-promoting rhizobacteria (PGPR)-mediated bioremediation is one of the convenient strategies for detoxification of Cd from the soil and for plant growth promotion under Cd stress. The selected strain K5 was identified as Klebsiella pneumoniae based on MALDI-TOF MS ribosomal protein and 16S rDNA sequence-based homology. The strain possessed several PGP traits viz. IAA production (3413 µg/mL), phosphate solubilization (80.25 ppm), ACC deaminase activity (40 ng α-ketobutyrate/mg protein/h), N2 fixation ability (1.84 µg N2 fixed/h), etc. and has the highest Cd resistance (4000 µg/mL) among Cd-resistant PGPR so far reported. This strain efficiently accumulated Cd and remained viable under Cd stress as confirmed by AAS-TEM-EDX analysis and viability test, respectively. The significant (p < 0.05) positive effect of the strain was reflected in various plant growth parameters like increased seed germination (50 to 90%), root length (5-fold), shoot length (about 2-fold), root fresh weight (> 2-fold), and shoot fresh weight (1.23-fold) under Cd stress compared with uninoculated set. Moreover, the positive impact of this strain on antioxidant enzyme activity (CAT, MDA, SOD) and several other biochemical parameters (proline, α-amylase, protease, total sugar, total protein, chlorophyll content) were also measured that favors plant growth promotion under Cd stress. Besides, the K5 strain also decreased stress-ethylene level under Cd stress and reduction of Cd accumulation in seedling (> 1.5-fold) was conducive to alleviate Cd phytotoxicity. Hence, K. pneumoniae strain K5 can be used as a phytostimulating and Cd-bioremediating biofertilizer for sustainable agriculture in heavy metal-contaminated sites.

In silico structural and functional analysis of Mesorhizobium ACC deaminase.

Nodulation is one of the very important processes of legume plants as it is the initiating event of fixing nitrogen. Although ethylene has essential role in normal plant metabolism but it has also negative impact on plants particularly in nodule formation in legume plants. It is also produced due to a variety of biotic or abiotic stresses. 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase is a rhizobial enzyme which cleaves ACC (immediate precursor of ethylene) into α-ketobutyrate and ammonia. As a result, the level of ethylene from the plant cells is decreased and the negative impact of ethylene on nodule formation is reduced. ACC deaminase is widely studied in several plant growth promoting rhizobacterial (PGPR) strains including many legume nodulating bacteria like Mesorhizobium sp. It is an important symbiotic nitrogen fixer belonging to the class - alphaproteobacteria under the order Rhizobiales. ACC deaminase has positive role in Legume-rhizobium symbiosis. Rhizobial ACC deaminase has the potentiality to reduce the adverse effects of ethylene, thereby triggering the nodulation process. The present study describes an in silico comparative structural (secondary structure prediction, homology modeling) and functional analysis of ACC deaminase from Mesorhizobium spp. to explore physico-chemical properties using a number of bio-computational tools. M. loti was selected as a representative species of Mesorhizobium genera for 3D modelling of ACC deaminase protein. Correlation by the phylogenetic relatedness on the basis of both ACC deaminase enzymes and respective acdS genes of different strains of Mesorhizobium has also studied.

An in silico structural, functional and phylogenetic analysis with three dimensional protein modeling of alkaline phosphatase enzyme of Pseudomonas aeruginosa.

Phosphorus is a primary macronutrient required for normal plant health, metabolism and survival. It is present in soil in compound insoluble form for which plant cannot uptake it directly from the soil. Some phosphate solubilizing bacteria possess some important enzymes for phosphate solubilization as well as mineralization. Alkaline (or basic) phosphatase (EC 3.1.3.1) is a type of zinc containing dimeric hydrolase enzyme responsible for removing the phosphate groups from various kinds of molecules including nucleotides, proteins, and alkaloids. Unlike acid phosphatases alkaline phosphatases are most effective in an alkaline environment. Alkaline phosphatases (ALPs) are of immense importance in various agricultural industries including dairy industries for testing successful pasteurization process. In this present study, Pseudomonas aeruginosa phosphatase was selected for a detailed computational investigation to exploit its physicochemical characteristics, structural properties including 3D model, model quality analysis, phylogenetic assessment and functional analysis using a number of available standard bioinformatics tools. The protein having average molecular weight about 51 kDa, was found thermostable and alkaline in nature belonging to metalloenzyme superfamily. Specifically, the thermostable behavior of the protein is suitable for the dairy industry. Moreover, this theoretical overview will help researchers to get an idea about the predicted protein structure and it may also help to design genetically engineered phosphate solubilizing bacteria by designing specific primers.

Granzyme B as a diagnostic marker of tuberculosis in patients with and without HIV coinfection.

Immunodiagnostic tests for tuberculosis (TB) are based on the estimation of interferon γ (IFN-γ) or IFN-γ-secreting CD4(+) T cells following ex vivo stimulation with ESAT6 and CFP-10. Sensitivity of these tests is likely to be compromised in CD4(+) T-cell-depleted situations, like HIV-TB coinfection. CD4(+) and CD8(+) T cells, isolated from 3 groups, viz., HIV-negative patients with active TB, HIV-TB coinfected patients, and healthy household contacts (HHCs) were cocultivated with autologous dendritic cells, and the cytokine response to rESAT6 stimulation was compared between groups in supernatants. While CD4(+) T-cell stimulation yielded significantly elevated levels of IFN-γ and interleukin 4 in HIV-negative TB patients, compared to HHCs, the levels of both these cytokines were nondiscriminatory between HIV-positive TB patients and HHCs. However, CD8(+) T-cell stimulation yielded significantly elevated granzyme B titers in both groups of patients, irrespective of HIV coinfection status. Hence, contrary to IFN-γ, granzyme B might be a useful diagnostic marker for Mycobacterium tuberculosis infection particularly in HIV coinfected patients.

Ethnicity-tailored novel set of ESAT-6 peptides for differentiating active and latent tuberculosis.

Differentiation between active and latent TB is a diagnostic challenge in TB-endemic regions. The commercially available IFN-γ-release assays are unsuitable for achieving this discrimination. We, therefore, screened ESAT-6 and CFP-10 proteins through population coverage analysis to identify minimal sets of peptides that can discriminate between these two forms of TB in a North Indian population. Comparing the diagnostic performance of a set of 2 ESAT-6 peptides (positions: 16-36; 59-79) to that of the QuantiFERON(®)-TB Gold IT (QFTGIT) assay, we observed significant difference in IFN-γ and TNF-α levels between patients (n = 15) and their age- and sex-matched healthy household contacts (n = 15). While the mean (±SD) IFNγ titer was 241.8 (±219.24) IU/ml for patients, the same in controls was 564.2 (±334.82) IU/ml (p = 0.039). Similarly the TNFα response was significantly higher in patients, compared to controls (796.47 ± 175.21 IU/ml vs. 481.81 ± 378.72 IU/ml; p = 0.047). IL-4 response to these peptides was non- discriminatory between the two groups. The QFTGIT Assay, however, elicited no significant difference in IFN-γ, TNF-α or IL-4 levels. Hence we conclude that IFN-γ or TNF-α response to these ESAT-6 peptides has the potential to differentiate between active and latent TB in our population.