Cyclic guanosine monophosphate improves salt tolerance in Solanum lycopersicum.

The cyclic nucleotide cyclic guanosine monophosphate (cGMP) is a powerful cell signaling molecule involved in biotic and abiotic stress perception and signal transduction. In the model plant Arabidopsis thaliana, salt and osmotic stress rapidly induce increase in cGMP which plays role by modulating the activity of monovalent cation transporters, possibly by direct binding to these proteins and by altering the expression of many abiotic stress responsive genes. In a recent study, a membrane permeable analogue of cGMP (8-bromo-cGMP) was found to have a promotive effect on soluble sugar, flavonoids and lignin content, and membrane integrity in Solanum lycopersicum seedlings under salt stress. However, it remains to be elucidated how salt stress affects the endogenous cGMP level in S. lycopersicum and if Br-cGMP-induced improvement in salt tolerance in S. lycopersicum involves altered cation fluxes. The current study was conducted to answer these questions. A rapid increase (within 30 s) in endogenous cGMP level was determined in S. lycopersicum roots after treatment with 100 mM NaCl. Addition of membrane permeable Br-cGMP in growth medium remarkably ameliorated the inhibitory effects of NaCl on seedlings' growth parameters, chlorophyll content and net photosynthesis rate. In salt stressed plants, Br-cGMP significantly decreased Na+ content by reducing its influx and increasing efflux while it improved plants K+ content by reducing its efflux and enhancing influx. Furthermore, supplementation with Br-cGMP improved plant's proline content and total antioxidant capacity, resulting in markedly decreased electrolyte leakage under salt stress. Br-cGMP increased the expression of Na+/H+ antiporter genes in roots and shoots of S. lycopersicum growing under salt stress, potentially enhancing plant's ability to sequester Na+ into the vacuole. The findings of this study provide insights into the mechanism of cGMP-induced salt stress tolerance in S. lycopersicum.

Higher cadmium and zinc accumulation in parsley (Petroselinum crispum) roots activates its antioxidants defense system.

Parsley (Petroselinum crispum) is herb with many biological and medicinal benefits for humans. However, growth on zinc (Zn) and cadmium (Cd) contaminated sites might get severely affected due to over accumulation of heavy metals (HM) in different plant tissues. Antioxidants play a crucial role in minimizing the negative effects of HM. The present study investigates the effects of Zn and Cd stress on P. crispum morphological parameters, enzymatic/non-enzymatic antioxidant profiling and metal accumulation in shoot/root. Plants were exposed to different concentrations of Zn (50, 100, 150 and 200 µM) and Cd (10, 20, 40 and 80 µM) along with control (no stress), in soil-less Hoagland's solution. The results showed that Zn and Cd substantially decrease the growth parameters with increased contents of malondialdehyde (MDA), hydrogen peroxide (H2O2), and electrolyte leakage (EL). Non-enzymatic antioxidant activities, like total phenolic contents (TPC) and ferric reducing antioxidant power (FRAP), were induced high in leaves only upon Cd stress and contrarily decreased upon Zn stress. Total flavonoid contents (TFC) were decreased under Zn and Cd stress. Enzymatic antioxidant activities like superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were also strongly induced upon Cd stress. At the same time, SOD and guaiacol peroxidase (GPX) activity was induced significantly upon Zn stress. Cd uptake and accumulation was notably high in roots as compared to shoots, which suggests P. crispum have a reduced ability to translocate Cd towards aboveground parts (leaves). Additionally, strong induction of antioxidants by P. crispum under Cd stress might indicate the capacity to effectively re-modulate its physiological response. However, further investigations regarding other HMs and experiments at the molecular level are still needed.

Characterization of alkaline metalloprotease isolated from halophilic bacterium Bacillus cereus and its applications in various industrial processes.

Microbial proteases are one of the most demanding enzymes for various industries with diverse applications in food, pharmaceutics, and textile industries to name the few. An extracellular alkaline metalloprotease was produced and purified from moderate halophilic bacterial strain, Bacillus cereus TS2, with some unique characteristics required for various industrial applications. The protease was produced in basal medium supplemented with casein and was partially purified by ion exchange chromatography followed by ammonium sulphate precipitation. The alkaline metalloprotease has molecular weight of 35 kDa with specific activity of 535.4 µM/min/mg. It can work at wide range of pH from 3 to 12, while showing optimum activity at pH 10. Similarly, the alkaline metalloprotease is stable till the temperature of 80 °C and works at wide range of temperature from 20 to 90 °C with optimum activity at 60 °C. The turnover rate increases in the presence of NaCl and Co+2 with k cat/KM of 1.42 × 103 and 1.27 × 103 s-1.M-1 respectively, while without NaCl and Co+2 it has a value of 7.58× 102. The alkaline metalloprotease was relatively resistant to thermal and solvent mediated denaturation. Applications revealed that the metalloprotease was efficient to remove hair from goat skin, remove blood stains and degrade milk, thus can be a potential candidate for leather, detergent, and food industry.

Essential Oil from Tagetes minuta Has Antiquorum Sensing and Antibiofilm Potential against Pseudomonas aeruginosa Strain PAO1.

Biofilms are complex communities of microorganisms that are enclosed in a matrix that shows increased resistance to antimicrobial and immunological encounters. Mostly, the traditional methods to control biofilm are exhausted; therefore, the aim is to evaluate the potential of essential oil (EO) from Tagetes minuta to encounter biofilm and other related virulence factors. The EO of T. minuta was extracted through steam-distillation, analyzed on gas chromatography-mass spectrometry, and the biofilm inhibition assays were performed with various concentrations of EO. Mainly the EO from T. minuta contains cis-ß-ocimene (29.1%), trans-tagetenone (23.1%), and cis-tagetenone (17.7%). The virulence factors were monitored while applying different concentrations of EO and it was recorded that the EO from T. minuta significantly inhibited the virulence factors linked with quorum sensing (QS), such as pyocyanin production, protease production, and swarming motility. Biofilm formation is one of the most important virulence factors associated with the QS pathway and was inhibited up to 79% in the presence of EO. Antibacterial activity against the PAO1 of EO was not so promising particularly and it has high MIC (325 µg/mL) and MBC (5000 µg/mL). EO is quite efficient to inhibit biofilm in a very small concentration of 20 µg/mL, which confirms that the biofilm inhibition by EO is not by killing bacterial cells but by inhibiting the QS pathway. The study on PAO1 constructs carrying various QS reported genes confirmed that the EO interferes with the QS pathway that ultimately controls various virulence factors caused by PAO1.

Biodegradation of monocrotophos by Brucella intermedia Msd2 isolated from cotton plant.

Widespread and inadequate use of Monocrotophos has led to several environmental issues. Biodegradation is an ecofriendly method used for detoxification of toxic monocrotophos. In the present study, Msd2 bacterial strain was isolated from the cotton plant growing in contaminated sites of Sahiwal, Pakistan. Msd2 is capable of utilizing the monocrotophos (MCP) organophosphate pesticide as its sole carbon source for growth. Msd2 was identified as Brucella intermedia on the basis of morphology, biochemical characterization and 16S rRNA sequencing. B. intermedia showed tolerance of MCP up to 100 ppm. The presence of opd candidate gene for pesticide degradation, gives credence to B. intermedia as an effective bacterium to degrade MCP. Screening of the B. intermedia strain Msd2 for plant growth promoting activities revealed its ability to produce ammonia, exopolysaccharides, catalase, amylase and ACC-deaminase, and phosphorus, zinc and potassium solubilization. The optimization of the growth parameters (temperatures, shaking rpm, and pH level) of the MCP-degrading isolate was carried out in minimal salt broth supplemented with MCP. The optimal pH, temperature, and rpm for Msd2 growth were observed as pH 6, 35 °C, and 120 rpm, respectively. Based on optimization results, batch degradation experiment was performed. Biodegradation of MCP by B. intermedia was monitored using HPLC and recorded 78% degradation of MCP at 100 ppm concentration within 7 days of incubation. Degradation of MCP by Msd2 followed the first order reaction kinetics. Plant growth promoting and multi-stress tolerance ability of Msd2 was confirmed by molecular analysis. It is concluded that Brucella intermedia strain Msd2 could be beneficial as potential biological agent for an effective bioremediation for polluted environments.

Decolorization of azo dyes by a novel aerobic bacterial strain Bacillus cereus strain ROC.

Synthetic dyes are widely used as colorant compounds in various industries for different purposes. Among all the dyestuffs, azo dyes constitute the largest and the most used class of dyes. These dyes and their intermediate products are common contaminants of ground water and soil in developing countries. Biological methods have been found to be promising for the treatment and degradation of these compounds. In the present study, we focused on the biological removal of azo dyes (Reactive orange 16 and Reactive black 5) under aerobic conditions using an indigenous bacterial strain isolated from contaminated industrial areas. The bacterial isolate was identified as Bacillus cereus strain ROC. Degradation experiments under agitation with both free and immobilized cells indicates that this strain degrades both azo- dyes in 5 days. The immobilized cells were more proficient than their free cell counterparts. The toxicity of the biotransformation products formed after decolorization were assessed by conducting bacteriotoxic and phytotoxic assays. All the toxicity assays indicate that the dyes' degraded products were non-toxic in nature, as compared to the dyes themselves. The kinetics of the azo dyes' degradation was also studied at various initial concentration ranges from 50 mg/L to 250 mg/L by growth independent kinetic models. Zero-order kinetics were fit to the experimental data, producing values of least squares regression (R2) greater than 0.98, which indicates that the bacterial strain degrades both dyes by co-metabolism rather than utilizing them as sole energy source. These results indicate that the Bacillus cereus ROC strain has great potential to degrade dye-contaminated water and soil.

Dimethoate residues in Pakistan and mitigation strategies through microbial degradation: a review.

Organophosphate pesticides (OPs) are used extensively for crop protection worldwide due to their high water solubility and relatively low persistence in the environment compared to other pesticides, such as organochlorines. Dimethoate is a broad-spectrum insecticide that belongs to the thio-organophosphate group of OPs. It is applied to cash crops, animal farms, and houses. It has been used in Pakistan since the 1960s, either alone or in a mixture with other OPs or pyrethroids. However, the uncontrolled use of this pesticide has resulted in residual accumulation in water, soil, and tissues of plants via the food chain, causing toxic effects. This review article has compiled and analyzed data reported in the literature between 1998 and 2021 regarding dimethoate residues and their microbial bioremediation. Different microorganisms such as bacteria, fungi, and algae have shown potential for bioremediation. However, an extensive role of bacteria has been observed compared to other microorganisms. Twenty bacterial, three fungal, and one algal genus with potential for the remediation of dimethoate have been assessed. Active bacterial biodegraders belong to four classes (i) alpha-proteobacteria, (ii) gamma-proteobacteria, (iii) beta-proteobacteria, and (iv) actinobacteria and flavobacteria. Microorganisms, especially bacterial species, are a sustainable technology for dimethoate bioremediation from environmental samples. Yet, new microbial species or consortia should be explored.

H-NS family protein MvaU downregulates phenazine-1-carboxylic acid (PCA) biosynthesis via binding to an AT-rich region within the promoter of the phz2 gene cluster in the rhizobacterium Pseudomonas strain PA1201.

Histone-like nucleoid-structuring (H-NS) proteins are key regulators in gene expression silencing and in nucleoid compaction. The H-NS family member proteins MvaU in Pseudomonas aeruginosa are thought to bind the same AT-rich regions of chromosomes and function to coordinate the control of a common set of genes. Here, we explored the molecular mechanism by which MvaU controls PCA biosynthesis in P. aeruginosa PA1201. We present evidence suggesting that MvaU is self-regulated. Deletion of mvaU significantly increased PCA production, and PCA production sharply decreased when mvaU was over-expressed. MvaU transcriptionally repressed phz2 cluster expression and consequently reduced PCA biosynthesis. ß-galactosidase assays confirmed that base pairing near the -35 box is required when MvaU regulates PCA production in PA1201. Electrophoretic mobility shift assays (EMSA) and additional point mutation analysis demonstrated that MvaU directly bound to an AT-rich motif within the promoter of the phz2 cluster. Chromatin immunoprecipitation (ChIP) analysis also indicated that MvaU directly bound to the P5 region of the phz2 cluster promoter. MvaU repression of PCA biosynthesis was independent of QscR and OxyR in PA1201 and neither PCA or H2O2 were the environmental signals that induced mvaU expression. These findings detail a new MvaU-dependent regulatory pathway of PCA biosynthesis in PA1201 and provide a foundation to increase PCA fermentation titer by genetic engineering.

A cyanobacterial photorespiratory bypass model to enhance photosynthesis by rerouting photorespiratory pathway in C3 plants.

Plants employ photosynthesis to produce sugars for supporting their growth. During photosynthesis, an enzyme Ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco) combines its substrate Ribulose 1,5 bisphosphate (RuBP) with CO2 to produce phosphoglycerate (PGA). Alongside, Rubisco also takes up O2 and produce 2-phosphoglycolate (2-PG), a toxic compound broken down into PGA through photorespiration. Photorespiration is not only a resource-demanding process but also results in CO2 loss which affects photosynthetic efficiency in C3 plants. Here, we propose to circumvent photorespiration by adopting the cyanobacterial glycolate decarboxylation pathway into C3 plants. For that, we have integrated the cyanobacterial glycolate decarboxylation pathway into a kinetic model of C3 photosynthetic pathway to evaluate its impact on photosynthesis and photorespiration. Our results show that the cyanobacterial glycolate decarboxylation bypass model exhibits a 10% increase in net photosynthetic rate (A) in comparison with C3 model. Moreover, an increased supply of intercellular CO2 (Ci) from the bypass resulted in a 54.8% increase in PGA while reducing photorespiratory intermediates including glycolate (- 49%) and serine (- 32%). The bypass model, at default conditions, also elucidated a decline in phosphate-based metabolites including RuBP (- 61.3%). The C3 model at elevated level of inorganic phosphate (Pi), exhibited a significant change in RuBP (+ 355%) and PGA (- 98%) which is attributable to the low availability of Ci. Whereas, at elevated Pi, the bypass model exhibited an increase of 73.1% and 33.9% in PGA and RuBP, respectively. Therefore, we deduce a synergistic effect of elevation in CO2 and Pi pool on photosynthesis. We also evaluated the integrative action of CO2, Pi, and Rubisco carboxylation activity (Vcmax) on A and observed that their simultaneous increase raised A by 26%, in the bypass model. Taken together, the study potentiates engineering of cyanobacterial decarboxylation pathway in C3 plants to bypass photorespiration thereby increasing the overall efficiency of photosynthesis.

Anti-biofilm activity of plant derived extracts against infectious pathogen-Pseudomonas aeruginosa PAO1.

BACKGROUND: Biofilm forming ability of Pseudomonas aeruginosa make them vulnerable, because it makes them recalcitrant against various antibiotics. Quorum sensing (QS) is cell density based signaling that helps in bacterial cell-cell communication, which regulated various virulence factors such as pigment and biofilm formation that contribute in the establishment of chronic infections. The interruption of QS is one of the effective approach to control various virulence factors. Present study was intended with the aim to authenticate antibiofilm potential in different solvents based extracts of selected medicinal plant species viz. Berginia ciliata, Clematis grata and Clematis viticella traditionally used by the inhabitants of Himalayan region of Pakistan to treat various pathogenic diseases. P. aeruginosa PAO1, an opportunistic pathogen and involves in various life-threatening infections specifically in immune deficient patients was used as a model pathogen. METHODS: Plants were extracted in various organic (ethanol, methanol, acetone, ethyl acetate, hexane, chloroform) as well as in aqueous solvents and their ability to inhibit biofilm was measured. Biofilm of PAO1 was grown in Jensen's medium while growing at 30°C and crystal violet assay was performed to assess the biofilm inhibiting activity of plant extracts. RESULTS: Solvents play a vital role in extraction of plant components and it was found that the plants in various solvents exhibit different activity against the PAO1 biofilm. Comparatively, 1% methanolic extract of B. ciliata (rhizome with skin), showed more than 80% inhibition of biofilm formation without effecting on the growth of the bacterium. Significant correlation between flavonoids content and antibiofilm activity in methanolic extract revealed the contribution of secondary metabolites in P. aeruginosa (PAO1) biofilm inhibition. CONCLUSION: Our study revealed that plants under investigation more specifically B. ciliata could be a potential candidate for drug discovery to treat P. aeruginosa PAO1, induced infectious diseases especially for its biofilm treatment.

Immobilized cells of a novel bacterium increased the degradation of N-methylated carbamates under low temperature conditions.

Carbamates are synthetic pesticides, extensively used throughout the world due to their broad specificity against various insect pests. However, their enormous and inadequate use have made them a potential threat to the environment. At low temperature, degradation of carbamates becomes difficult mainly because of low biological activity. In the present study, we isolated a bacterial strain from a low temperature climate where the N-methylated carbamates are used for crop protection. The bacterium, was identified as Pseudomonas plecoglossicida strain (TA3) by 16S rRNA analysis. Degradation experiments with both free and immobilized cells in minimal salt medium indicated that the strain TA3 utilized carbaryl, carbofuran and aldicarb as both carbon and nitrogen source. TA3 can grow well at 4 °C and demonstrated the ability to degrade three carbamates (50 µgml-1) at low temperature. The immobilized cells were found more efficient than their free cells counter parts. Immobilized cells has ability to degrade 100% of carbamates at 30 °C while 80% at 4 °C but incase of their free cells counter parts the efficiency to degrade carbamates was less which was 60% at 4 °C and 80% at 30 °C. TA3 free cellsextract also depicted high activity against all the three carbamates even at 4 °C indicating a possible enzymatic mechanism of degradation.

A newly isolated Pseudomonas sp. can degrade endosulfan via hydrolytic pathway.

Endosulfan an organochlorinated pesticide was used extensively throughout the world. Its enormous and inadequate use creates environmental as well as health problems. A bacterial strain capable to utilize endosulfan as a sole source of sulfur was isolated from pesticide contaminated soil and identified as Pseudomonas sp. on the basis of 16S rRNA. Batch experiments were conducted at various initial concentrations of endosulfan, i.e. 5, 25, 50, 75 and 100 mg/l to study its rate of degradation. After three days of incubation, 70-80% of each initial concentration was degraded by the isolated strain as compared to the control. Degradation of endosulfan increased with the time of incubation and maximum degradation was observed after 5 days of incubation. GC-MS revealed that the major metabolite was endosulfan lactone, which accumulated after 5 days of incubation. Kinetic studies at various initial concentrations also revealed that the bacterium has very promising attitude to utilize endosulfan as sole source of sulfur. It was observed that the addition of auxiliary sulfur Fe(SO4)3 in any concentration (0.05, 0.01 and 0.1%) decreased the rate of degradation of endosulfan. The ratio of µmax/ Ks was high (0.03 mg/l) when endosulfan was single sulfur source as compared to the value recorded when Fe(SO4)3 was added alongwith the endosulfan. This indicates that the newly isolated bacterium attacks sulfur moiety for its degradation.

Risk assessment of pesticide exposure on health of Pakistani tobacco farmers.

Tobacco is an important cash crop of Pakistan. Pesticides are commonly used to increase the crop yield, but their health impact has not been studied yet. The objectives of the study were to determine the frequency of pesticide poisoning and to explore the knowledge, attitudes and practices (KAP) towards safety measures among the tobacco farmers in Swabi, Pakistan. One hundred and five tobacco farmers involved in pesticide application were randomly selected from two villages of district Swabi. A structured questionnaire was used for clinical and KAP information. Plasma cholinesterase (PChE) levels were measured by Ellman's method by using GD Italy kits. All tobacco farmers were males with a mean (SD) age of 26 (9) years. The majority of the farmers reported multiple symptoms headache, dizziness, vomiting, shortness of breath, muscle weakness and skin rash correlate with the clinically significant depression of PChE levels. Out of 105 pesticide applicators, 58 (55%) had post-exposure reduction in PChE levels <20% from baseline, 35 (33%) had mild poisoning (20-40% reduction) and 12 (11%) had moderate poisoning (>40% reduction). Most of the farmers did not use any personal protective equipment during pesticide handling. Only a few used shoes (31%), masks (14%) and gloves (9%) during pesticide spray. In conclusion, the tobacco farmers had mild to moderate pesticide poisoning, which was correlated with depression in PChE levels. Moreover, most farmers had little knowledge about the safety measures, casual attitude and unsatisfactory safety practices with regard to the use of basic protective equipments during pesticide applications on the tobacco crop.