Alkaliphilic and thermostable lipase production by leaf litter fungus Leptosphaerulina trifolii A SMR-2011.

Fungi that inhabit fire-prone forests have to be adapted to harsh conditions and fungi affiliated to Ascomycota recovered from foliar litter samples were used for bioprospecting of molecules such as enzymes. Agni's fungi isolated from leaf litter, whose spores are capable of tolerating 110 oC were screened for thermostable lipases. One of the isolates, Leptosphaerulina trifolii A SMR-2011 exhibited high positive lipase activity than other isolates while screening through agar plate assay using Tween 20 in the medium. Maximum lipase activity (173.2 U/mg) of L. trifolii was observed at six days of inoculation and decreased thereafter. Among different oils used, the maximum lipase activity was attained by soybean oil (940.1 U/mg) followed by sunflower oil (917.1 U/mg), and then by mustard oil (884.8 U/mg), showing its specificity towards unsaturated fatty acids. Among the various organic nitrogen sources tested, soybean meal showed maximum lipase activity (985.4 U/mg). The partially purified enzyme was active over a wide range of pH from 8 to 12 with a pH optimum of 11.0 (728.1 U/mg) and a temperature range of 60-80 oC with an optimal temperature of 70 oC (779.1 U/mg). The results showed that lipase produced by L. trifolii is alkali stable and retained 85% of its activity at pH 11.0. This enzyme also showed high thermal stability retaining more than 50% of activity when incubated at 60 oC to 90 °C for 2 h. The ions Ca2+ and Mn2+ induced the lipase activity, while Cu2+ and Zn2+ ions lowered the activity compared to control. These results suggests that the leaf litter fungus L. trifolii serves as a potential source for the production of alkali-tolerant and thermostable lipase.

Comparative transcriptome analysis of ectomycorrhizal fungus Pisolithus albus in response to individual and combined stress of copper and cadmium.

An ectomycorrhizal fungus Pisolithus albus establishes the natural symbiosis with plant roots on extreme heavy metal (HM)-rich soil and enables their survival in toxic metal concentrations. Understanding P. albus key genes and pathways behind strong metal tolerance is crucial for its successful application in the rehabilitation of metal-contaminated barren lands. Therefore, this study aimed to analyze the whole transcriptome profile of P. albus under individual and combined metal stress of copper (Cu) and cadmium (Cd). At 480 µM Cu and 16 µM Cd toxic concentrations, P. albus has shown growth and survival and accumulated high metal (1.46 µg Cu and 1.13 µg Cd per mg of dry mycelia). The study found a stronger response of P. albus to single-metal stress in high concentration as compared to multi-metal stress in relatively lower concentration. Hence, the intensity of fungal response to HM stress is mainly determined by the metal concentration involved in stress. We have found a total of 11 pathways significantly associated with HM stress, among which amino acid, lipid, and carbohydrate metabolisms were highly affected. The functional enrichment of differentially expressed genes has shown the induced biosynthesis of arginine, melanin, metal chelating agents, membrane phospholipids, fatty acids, folate, pantothenate, ergothioneine, and other antioxidant agents; upregulation of zinc ion uptake, potassium transporters, and lysine degradation; and reduction of phosphatidylcholine degradation, incorrect protein folding, iron uptake, and potassium efflux as the top efficient tolerance mechanisms of P. albus against HM stress. The current study would contribute to understanding fungal HM tolerance and its further utilization in the bioremediation of metal-contaminated abandoned lands. The validation of RNA-sequencing analysis with RT-qPCR of selected genes showed the high credibility of the presented data.

Characterization and genome-wide sequence analysis of an ectomycorrhizal fungus Pisolithus albus, a potential source for reclamation of degraded lands.

Pisolithus albus is a ubiquitous ectomycorrhizal fungus that establishes symbiosis with a wide range of woody plants around the globe. The symbiotic association of this fungus plays a crucial role in the nutrient cycling of their host plants and enables them to thrive in adverse environmental conditions. Based on its ecological importance and lack of genomic studies, whole-genome sequencing was carried out to analyze P. albus sequences through an Illumina HiSeq X system. The functional annotations were performed against various databases to explore genomic patterns and traits possibly attributing to its specialization. Comparative genomics of P. albus with phylogenetically related Pisolithus microcarpus and Pisolithus tinctorius (only available genomes of Pisolithus at NCBI till now) led to the identification of their unique and shared basic functional and stress adaptation capabilities. The de novo assembled genome of 56.15 Mb with 91.8% BUSCO completeness is predicted to encode 23,035 genes. The study is aimed to generate solid genomic data resources for P. albus, forming the theoretical basis for future transcriptomic, proteomic and metabolomic studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03483-5.

Role of Ectomycorrhizal Symbiosis Behind the Host Plants Ameliorated Tolerance Against Heavy Metal Stress.

Soil heavy metal (HM) pollution, which arises from natural and anthropogenic sources, is a prime threat to the environment due to its accumulative property and non-biodegradability. Ectomycorrhizal (ECM) symbiosis is highly efficient in conferring enhanced metal tolerance to their host plants, enabling their regeneration on metal-contaminated lands for bioremediation programs. Numerous reports are available regarding ECM fungal potential to colonize metal-contaminated lands and various defense mechanisms of ECM fungi and plants against HM stress separately. To utilize ECM-plant symbiosis successfully for bioremediation of metal-contaminated lands, understanding the fundamental regulatory mechanisms through which ECM symbiosis develops an enhanced metal tolerance in their host plants has prime importance. As this field is highly understudied, the present review emphasizes how plant's various defense systems and their nutrient dynamics with soil are affected by ECM fungal symbiosis under metal stress, ultimately leading to their host plants ameliorated tolerance and growth. Overall, we conclude that ECM symbiosis improves the plant growth and tolerance against metal stress by (i) preventing their roots direct exposure to toxic soil HMs, (ii) improving plant antioxidant activity and intracellular metal sequestration potential, and (iii) altering plant nutrient uptake from the soil in such a way to enhance their tolerance against metal stress. In some cases, ECM symbiosis promotes HM accumulation in metal stressed plants simultaneous to improved growth under the HM dilution effect.

Protection from metal toxicity by Hsp40-like protein isolated from contaminated soil using functional metagenomic approach.

Pollution in the environment due to accumulation of potentially toxic metals results in deterioration of soil and water quality, thus impacting health of all living organisms including microbes. In the present investigation, a functional metagenomics approach was adopted to mine functional genes involved in metal tolerance from potentially toxic metal contaminated site. Eukaryotic cDNA library (1.0-4.0 kb) was screened for the genes providing tolerance to cadmium (Cd) toxicity through a functional complementation assay using Cd-sensitive Saccharomyces cerevisiae mutant ycf1Δ. Out of the 98 clones able to recover growth on Cd-supplemented selective medium, one clone designated as PLCc43 showed more tolerance to Cd along with some other clones. Sequence analysis revealed that cDNA PLCc43 encodes a 284 amino acid protein harbouring four characteristic zinc finger motif repeats (CXXCXGXG) and showing partial homology with heat shock protein (Hsp40) of Acanthamoeba castellanii. qPCR analysis revealed the induction of PLCc43 in the presence of Cd, which was further supported by accumulation of Cd in ycf1Δ/PLCc43 mutant. Cu-sensitive (cup1Δ), Zn-sensitive (zrc1Δ) and Co-sensitive (cot1Δ) yeast mutant strains were rescued from sensitivity when transformed with cDNA PLCc43 indicating its ability to confer tolerance to various potentially toxic metals. Oxidative stress tolerance potential of PLCc43 was also confirmed in the presence of H2O2. Present study results suggest that PLCc43 originating from a functional eukaryotic gene of soil community play an important role in detoxification of potentially toxic metals and may be used as biomarker in various contaminated sites.

Endolichenic fungal diversity associated with some lichens of the Western Ghats.

A total of 389 strains of fungi belonging to 38 species were isolated from 10 lichen species of the Western Ghats, southern India. All the lichens screened, irrespective of their growth forms or location, harboured endolichenic fungi. Most of the fungi belonged to the Ascomycotina. Chaetomium sp. 1, Hypoxylon investiens, Nemania bipapillata, Nodulisporium sp., Paecilomyces sp., and Sporormiella intermedia were the most common ones and were present in 5 or more lichen species. Of the 28 sporulating fungi, 7 belonged to Xylariales, and their total colonization frequency in all the lichens was also high. This observation further substantiates the high ecological amplitude of Xylariales, possibly due to overlapping ecological niches and their ability to inhibit co-occurring endolichenic fungi.

Hetero-Polysaccharides Secreted from Dunaliella salina Exhibit Immunomodulatory Activity Against Peripheral Blood Mononuclear Cells and RAW 264.7 Macrophages.

Several species of microalgae have been known to produce exopolysaccharides (EPS) with potential immune activity. In the present investigation, ethyl acetate fraction of crude EPS secreted by Dunaliella salina was explored for immunomodulatory activity against peripheral blood mononuclear cells (PBMC) and RAW 264.7 macrophages. Effect of EPS on cell growth and cytokines production were measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and ELISA respectively. Griess reagent was used for measuring the nitric oxide production in RAW 264.7 macrophages. FTIR analysis and mass spectroscopy were carried out for the characterization. Ethyl acetate fraction exhibited dose dependent increase in proliferative index and cytokines production (IFN-γ, TNF-α, TGF-ß). At low concentration (250 and 500 µg/mL), it showed growth inhibition and at higher concentration (1000 and 1500 µg/mL), it enhanced the cell growth. Interestingly, the pronounced increased TNF-α production was observed in ethyl acetate fraction treated PBMC cells at higher concentration (750 and 1000 µg/mL) indicating the immunostimulatory effect. In RAW cells, concentration dependent diminished cell growth (IC50 = 691 µg/mL) and nitric oxide production (IC50 = 630 µg/mL) was observed. FTIR analysis showed the presence of polysaccharides due to the detection of hydroxyl (-OH), Carbonyl (C-O) and alkyl (C-H) groups. Mass spectroscopy results revealed ethyl acetate fraction as penta-saccharide (m/z = 887.56 and 886.54) which are confirmed to be hetero-polysaccharides consisting of hexoses and pentoses along with association of ions. These results suggest that penta-saccharide (ethyl acetate fraction) isolated from D. salina may have the potential to be used for therapeutic purpose as immunomodulatory agent.

Metal induction of two metallothionein genes in the ectomycorrhizal fungus Suillus himalayensis and their role in metal tolerance.

Metallothioneins (MTs) are small proteins with highly conserved cysteine residues and are involved in metal homeostasis and metal detoxification. Two metallothionein genes ShMT1 and ShMT2 from the ectomycorrhizal fungus Suillus himalayensis were characterised for their potential role in heavy metal detoxification. The response of these MTs to the exogenous concentrations of copper and cadmium was studied by qPCR analysis. The exogenous copper but not the cadmium at the tested concentrations induced the expression of the MT genes. The functional role of ShMTs was validated by expressing the two genes through functional complementation in yeast mutant strain cup1Δ (copper-sensitive), ycf1Δ (cadmium- sensitive) and zrc1Δ (zinc-sensitive). The mutant strain successfully expressed the two genes resulting in wild-type phenotype restoration of copper, cadmium and zinc tolerance. The present study shows that the ectomycorrhizal fungus S. himalayensis encodes two metallothionein genes (ShMT1 and ShMT2) which are more inducible by copper than cadmium and could play an important role in their detoxification.

Metagenomics analysis reveals a new metallothionein family: Sequence and metal-binding features of new environmental cysteine-rich proteins.

Metallothioneins are cysteine-rich proteins, which function as (i) metal carriers in basal cell metabolism and (ii) protective metal chelators in conditions of metal excess. Metallothioneins have been characterized from different eukaryotic model and cultivable species. Presently, they are categorized in 15 families but evolutionary relationships between these metallothionein families remain unresolved. Several cysteine-rich protein encoding genes that conferred Cd-tolerance in Cd-sensitive yeast mutants have previously been isolated from soil eukaryotic metatranscriptomes. They were called CRPs for "cysteine-rich proteins". These proteins, of unknown taxonomic origins, share conserved cysteine motifs and could be considered as metallothioneins. In the present work, we analyzed these CRPs with respect to their amino acid sequence features and their metal-binding abilities towards Cd, Zn and Cu metal ions. Sequence analysis revealed that they share common features with different known metallothionein families, but also exhibit unique specific features. Noticeably, CRPs display two separate cysteine-rich domains which, when expressed separately in yeast, confer Cd-tolerance. The N-terminal domain contains some conserved atypical Cys motifs, such as one CCC and two CXCC ones. Five CRPs were expressed and purified as recombinant proteins and their metal-binding characteristics were studied. All these CRPs chelated Cd(II), Zn(II) and Cu(I), although displaying a better capacity for Zn(II) coordination. All CRPs are able to confer Cd-tolerance, and four of them confer Zn-tolerance in the Zn-sensitive zrc1Δ yeast mutant. We designated these CRPs as environmental metallothioneins belonging to a new formerly undescribed metallothionein family.