Antagonistic Activity of Pseudomonas fluorescens Strain X1 Against Different Fusaria and it's In Vivo Analysis Against Fusarium udum Infected Pigeon Pea.

A plant growth-promoting rhizobacterial strain, Pseudomonas fluorescens X1 isolated from the garden soil was employed for antagonistic activity against different species of fusaria. Strain X1 inhibited four different fusaria (Fusarium moniliforme, Fusarium oxysporum, Fusarium semitectum and Fusarium udum) in dual culture plate assay, and in broth culture using cell-free culture filtrate. Scanning electron microscopic (SEM) analysis revealed deformation and shrinkage in mycelia of fusaria after treatment with strain X1. Confocal micrographs showed degeneration of nuclei inside the cells of fusaria for the same effect. Strain X1 exhibited maximum antifungal activity, when it was grown in nutrient broth yeast (NBY) medium amended with 1 mM NH4MoO4 and 1% glucose. The antifungal extracts eluted from thin-layer chromatography (TLC) followed by high performance liquid chromatography (HPLC) showed two fractions active against different fusaria. Liquid chromatography-mass spectrometry (LCMS) analysis of the two fractions 1 and 2 corresponded to molecular ions at m/z 177.16 and m/z 177.09, respectively. Infra-red (IR) analysis showed five similar absorption bands in both the fractions analysed. In vivo analysis of strain X1 alone and along with fungicide inhibited the growth of F. udum and improved the biomass and growth of pigeon pea. These results indicated that strain X1 could be possibly used as a biocontrol agent to inhibit the growth of soil-borne diseases of different fusaria including F. udum that causes wilting in pigeon pea.

Bioremediation of toxic heavy metals (THMs) contaminated sites: concepts, applications and challenges.

Heavy metal contamination is a global issue, where the prevalent contaminants are arsenic (As), cadmium (Cd), chromium (Cr)(VI), mercury (Hg), and lead (Pb). More often, they are collectively known as "most problematic heavy metals" and "toxic heavy metals" (THMs). Their treatment through a variety of biological processes is one of the prime interests in remediation studies, where heavy metal-microbe interaction approaches receive high interest for their cost effective and ecofriendly solutions. In this review, we provide an up to date information on different microbial processes (bioremediation) for the removal of THMs. For the same, emphasis is put on oxidation-reduction, biomineralization, bioprecipitation, bioleaching, biosurfactant technology, biovolatilization, biosorption, bioaccumulation, and microbe-assisted phytoremediation with their selective advantages and disadvantages. Further, the literature briefly discusses about the various setups of cleaning processes of THMs in environment under ex situ and in situ applications. Lately, the study sheds light on the manipulation of microorganisms through genetic engineering and nanotechnology for their advanced treatment approaches.

The relative impact of toxic heavy metals (THMs) (arsenic (As), cadmium (Cd), chromium (Cr)(VI), mercury (Hg), and lead (Pb)) on the total environment: an overview.

Certain five heavy metals viz. arsenic (As), cadmium (Cd), chromium (Cr)(VI), mercury (Hg), and lead (Pb) are non-threshold toxins and can exert toxic effects at very low concentrations. These heavy metals are known as most problematic heavy metals and as toxic heavy metals (THMs). Several industrial activities and some natural processes are responsible for their high contamination in the environment. In recent years, high concentrations of heavy metals in different natural systems including atmosphere, pedosphere, hydrosphere, and biosphere have become a global issue. These THMs have severe deteriorating effects on various microorganisms, plants, and animals. Human exposure to the THMs may evoke serious health injuries and impairments in the body, and even certain extremities can cause death. In all these perspectives, this review provides a comprehensive account of the relative impact of the THMs As, Cd, Cr(VI), Hg, and Pb on our total environment.

Biosorption of heavy metals by a lead (Pb) resistant bacterium, Staphylococcus hominis strain AMB-2.

In the present study, a lead (Pb)-resistant bacterium, Staphylococcus hominis strain AMB-2 was isolated from Mandoli industrial area, Delhi and selected for heavy metal biosorption considering multiple heavy metal resistance. In the batch experiment, both living and dead biomasses of strain AMB-2 showed biosorption of Pb and cadmium (Cd) in single and binary systems as analyzed through Inductively coupled plasma-optical emission spectrometry. Living biomass exhibited more biosorption of metals than dead biomass in both single and binary systems. However, in the binary system, metals competed for the attachment sites on the bacterial surface, where Pb got more preference over Cd for the same. The underlying mechanism for the biosorption was attachment of the metal ions through functional groups onto the surface of the biomass as revealed by scanning electron microscope-energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction. Conclusively, this study displayed an effective biotreatment of Pb and Cd from aqueous medium using a low-cost biosorbent prepared from S. hominis strain AMB-2 considering biosafety of microorganisms and an eco-friendly approach.

Assessment of heavy metal contamination and Hg-resistant bacteria in surface water from different regions of Delhi, India.

The present study aims to monitor the surface water quality of different regions in Delhi (India). With many physical and chemical properties, all samples had a high load of pollution in which Najafgarh drain (Nd) exhibited maximum and laboratory tap water (Ltw) minimum contamination. Water samples contained notable amounts of heavy metals including Cr, Cd, As, Cu, Pb and Hg. A total of 88 Hg-resistant bacteria were isolated from all the regions except Ltw. Among all the samples, the density of Hg-resistant bacteria was highest in sample of Nd and their morphotype heterogeneity was highest in sample collected from river Yamuna nearby Kashmiri gate (Kg). Different strains showed different patterns of resistance to different heavy metals and antibiotics. Multiple antibiotic resistance (MAR) indices were high in two samples, the highest reported in a sample taken from river Yamuna nearby Majnu ka tila (Mkt) (0.34). The 12.5% and 24.45% isolates showed ß- and α-hemolytic natures, respectively that might be of pathogenic concern. In this account, high concentrations of heavy metals and their resistant bacteria in surface water have severely damaged the quality of water and their resources and produced high risk to the associated life forms.

Inducible cellulase production from an organic solvent tolerant Bacillus sp. SV1 and evolutionary divergence of endoglucanase in different species of the genus Bacillus

Abstract Bacteria are important sources of cellulases with various industrial and biotechnological applications. In view of this, a non-hemolytic bacterial strain, tolerant to various environmental pollutants (heavy metals and organic solvents), showing high cellulolytic index (7.89) was isolated from cattle shed soil and identified as Bacillus sp. SV1 (99.27% pairwise similarity with Bacillus korlensis). Extracellular cellulases showed the presence of endoglucanase, total cellulase and β-glucosidase activities. Cellulase production was induced in presence of cellulose (3.3 times CMCase, 2.9 times FPase and 2.1 times β-glucosidase), and enhanced (115.1% CMCase) by low-cost corn steep solids. An in silico investigation of endoglucanase (EC 3.2.1.4) protein sequences of three Bacillus spp. as query, revealed their similarities with members of nine bacterial phyla and to Eukaryota (represented by Arthropoda and Nematoda), and also highlighted of a convergent and divergent evolution from other enzymes of different substrate [(1,3)-linked beta-d-glucans, xylan and chitosan] specificities. Characteristic conserved signature indels were observed among members of Actinobacteria (7 aa insert) and Firmicutes (9 aa insert) that served as a potential tool in support of their relatedness in phylogenetic trees.

Inducible cellulase production from an organic solvent tolerant Bacillus sp. SV1 and evolutionary divergence of endoglucanase in different species of the genus Bacillus.

Bacteria are important sources of cellulases with various industrial and biotechnological applications. In view of this, a non-hemolytic bacterial strain, tolerant to various environmental pollutants (heavy metals and organic solvents), showing high cellulolytic index (7.89) was isolated from cattle shed soil and identified as Bacillus sp. SV1 (99.27% pairwise similarity with Bacillus korlensis). Extracellular cellulases showed the presence of endoglucanase, total cellulase and ß-glucosidase activities. Cellulase production was induced in presence of cellulose (3.3 times CMCase, 2.9 times FPase and 2.1 times ß-glucosidase), and enhanced (115.1% CMCase) by low-cost corn steep solids. An in silico investigation of endoglucanase (EC 3.2.1.4) protein sequences of three Bacillus spp. as query, revealed their similarities with members of nine bacterial phyla and to Eukaryota (represented by Arthropoda and Nematoda), and also highlighted of a convergent and divergent evolution from other enzymes of different substrate [(1,3)-linked beta-d-glucans, xylan and chitosan] specificities. Characteristic conserved signature indels were observed among members of Actinobacteria (7 aa insert) and Firmicutes (9 aa insert) that served as a potential tool in support of their relatedness in phylogenetic trees.

Evolutionary Relationships and Taxa-Specific Conserved Signature Indels Among Cellulases of Archaea, Bacteria, and Eukarya.

The cellulases from different cellulolytic organisms have evolutionary relationships, which range from single-celled prokaryotes to the complex eukaryotes of the living world. This in silico analysis revealed the presence of a conserved cellulase domain along with evolutionary relationships among cellulases from several species of Archaea, Bacteria, and Eukarya. The amino acid sequences of cellulases from Archaea and Bacteria showed closer identity with their domain or phylum members that provided insights into convergent and divergent evolution of cellulases from other enzymes with different substrate specificities. Evolutionary relatedness was also observed in phylogenetic trees among a number of cellulase sequences of diverse taxa. In cellulases, propensity for alanine, glycine, leucine, serine, and threonine was high, but low for cysteine, histidine, and methionine. Catalytic aspartic acid had a higher propensity than glutamic acid, and both were involved in regular expression patterns. Characteristic group and multigroup-specific conserved signature indels located in the catalytic domains of cellulases were observed that further clarified evolutionary relationships. These indels can be distinctive molecular tools for understanding phylogeny and identification of unknown cellulolytic species of common evolutionary descent in different environments.

Production, Optimization, and Characterization of Organic Solvent Tolerant Cellulases from a Lignocellulosic Waste-Degrading Actinobacterium, Promicromonospora sp. VP111.

High costs of natural cellulose utilization and cellulase production are an industrial challenge. In view of this, an isolated soil actinobacterium identified as Promicromonospora sp. VP111 showed potential for production of major cellulases (CMCase, FPase, and ß-glucosidase) utilizing untreated agricultural lignocellulosic wastes. Extensive disintegration of microcrystalline cellulose and adherence on it during fermentation divulged true cellulolytic efficiency of the strain. Conventional optimization resulted in increased cellulase yield in a cost-effective medium, and the central composite design (CCD) analysis revealed cellulase production to be limited by cellulose and ammonium sulfate. Cellulase activities were enhanced by Co(+2) (1 mM) and retained up to 60 °C and pH 9.0, indicating thermo-alkaline tolerance. Cellulases showed stability in organic solvents (25 % v/v) with log P ow ≥ 1.24. Untreated wheat straw during submerged fermentation was particularly degraded and yielded about twofold higher levels of cellulases than with commercial cellulose (Na-CMC and avicel) which is especially economical. Thus, this is the first detailed report on cellulases from an efficient strain of Promicromonospora that was non-hemolytic, alkali-halotolerant, antibiotic (erythromycin, kanamycin, rifampicin, cefaclor, ceftazidime) resistant, multiple heavy metal (Mo(+6) = W(+6) > Pb(+2) > Mn(+2) > Cr(+3) > Sn(+2)), and organic solvent (n-hexane, isooctane) tolerant, which is industrially and environmentally valuable.

Myroides indicus sp. nov., isolated from garden soil.

A novel aerobic, non-motile, rod-shaped, catalase- and oxidase-positive bacterial strain, designated UKS3T, was isolated from garden soil, and subjected to polyphasic taxonomic analysis. Strain UKS3T formed whitish, viscous colonies on nutrient agar and was Gram-staining negative. Phylogenetic analysis, based on 16S rRNA gene sequence, showed that maximum pairwise similarity occurs with representatives of the genus Myroides. The most closely related species include Myroides marinus JS-08T (92.7 % sequence similarity), Myroides phaeus MY15T (92.7 %), Myroides odoratus DSM 2801T (91.5 %) and Myroides odoratimimus CCUG 39352T (91.4 %). Strain UKS3T contained menaquinone-6 (MK-6) as the major respiratory quinone and iso-C15 : 0 (40.2 %), anteiso-C15 : 0 (9.4 %) and iso-C17 : 0 3-OH (8.5 %) as major fatty acids. Phosphatidylethanolamine, phospholipids and three aminolipids were the major polar lipids. The DNA G+C content of strain UKS3T was 36.8 ± 2.0 mol%. On the basis of phenotypic, chemotaxonomic and molecular analysis, strain UKS3T represents a novel species of the genus Myroides, for which the name Myroides indicus sp. nov., is proposed. The type strain is UKS3T ( = DSM 28213T = NCIM 5555T ).

Enhanced production of steviol glycosides in mycorrhizal plants: a concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes.

Stevia rebaudiana (Bertoni) produces steviol glycosides (SGs)--stevioside (stev) and rebaudioside-A (reb-A) that are valued as low calorie sweeteners. Inoculation with arbuscular mycorrhizal fungi (AMF) augments SGs production, though the effect of this interaction on SGs biosynthesis has not been studied at molecular level. In this study transcription profiles of eleven key genes grouped under three stages of the SGs biosynthesis pathway were compared. The transcript analysis showed upregulation of genes encoding 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes viz.,1-deoxy-D-xylulose 5-phospate synthase (DXS), 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR) and 2-C-methyl-D-erytrithol 2,4-cyclodiphosphate synthase (MDS) in mycorrhizal (M) plants. Zn and Mn are imperative for the expression of MDS and their enhanced uptake in M plants could be responsible for the increased transcription of MDS. Furthermore, in the second stage of SGs biosynthesis pathway, mycorrhization enhanced the transcription of copalyl diphosphate synthase (CPPS) and kaurenoic acid hydroxylase (KAH). Their expression is decisive for SGs biosynthesis as CPPS regulates flow of metabolites towards synthesis of kaurenoid precursors and KAH directs these towards steviol synthesis instead of gibberellins. In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. While higher transcription of all the three characterized UGTs in M plants explains augmented production of SGs; higher transcript levels of UGT76G1, specifically improved reb-A to stev ratio implying increased sweetness. The work signifies that AM symbiosis upregulates the transcription of all eleven SGs biosynthesis genes as a result of improved nutrition and enhanced sugar concentration due to increased photosynthesis in M plants.

Arbuscular mycorrhiza increase artemisinin accumulation in Artemisia annua by higher expression of key biosynthesis genes via enhanced jasmonic acid levels.

It is becoming increasingly evident that the formation of arbuscular mycorrhiza (AM) enhances secondary metabolite production in shoots. Despite mounting evidence, relatively little is known about the underlying mechanisms. This study suggests that increase in artemisinin concentration in Artemisia annua colonized by Rhizophagus intraradices is due to altered trichome density as well as transcriptional patterns that are mediated via enhanced jasmonic acid (JA) levels. Mycorrhizal (M) plants had higher JA levels in leaf tissue that may be due to induction of an allene oxidase synthase gene (AOS), encoding one of the key enzymes for JA production. Non-mycorrhizal (NM) plants were exogenously supplied with a range of methyl jasmonic acid concentrations. When leaves of NM and M plants with similar levels of endogenous JA were compared, these matched closely in terms of shoot trichome density, artemisinin concentration, and transcript profile of artemisinin biosynthesis genes. Mycorrhization increased artemisinin levels by increasing glandular trichome density and transcriptional activation of artemisinin biosynthesis genes. Transcriptional analysis of some rate-limiting enzymes of mevalonate and methyl erythritol phosphate (MEP) pathways revealed that AM increases isoprenoids by induction of the MEP pathway. A decline in artemisinin concentration in shoots of NM and M plants treated with ibuprofen (an inhibitor of JA biosynthesis) further confirmed the implication of JA in the mechanism of artemisinin production.

In silico approach to study adaptive divergence in nucleotide composition of the 16S rRNA gene among bacteria thriving under different temperature regimes.

Bacteria exist in a wide range of habitats ranging from psychrophilic through mesophilic to thermophilic. These different habitats have distinct environmental restriction for their existence. These microorganisms evolve themselves to survive in a specific habitat through the phenotypic and genotypic changes. In the bacterial domain, in silico analysis of 16S rRNA gene sequences using Mega 5.2 software by computing nucleotide composition, and evaluating their significance by statistical analysis using analysis of variance through Statistical Package for the Social Sciences (SPSS) version 16.0, revealed the habitat-specific bias in the occurrence of four types of nucleosides (A, T, C, and G) in the 16S rRNA gene. This hypothesis is also supported by Duncan's multiple range significance test at p=0.05 and also by the clustering of bacterial species of the same habitat group in the neighbor-joining tree of 150 different bacterial species of different psychrophilic, mesophilic, and thermophilic habitats (50 from each). The results on the probability of substitution (transition and transversion) in 16S rRNA gene sequences suggest that there is a habitat-specific selection pressure that possibly happens at the level of replication and repair process that results in a decreasing frequency of occurrence of adenine and thymine in the order psychrophilic>mesophilic>thermophilic species, and in an increasing frequency of occurrence of cytosine and guanine in the order psychrophilic

An in silico [correction of insilico] approach to bioremediation: laccase as a case study.

Laccase (E.C. 1.10.3.2) is one of the well-studied enzymes used for bioremediation of xenobiotics such as phenols, anilines, etc. Its broad substrate specificity offers a wide opportunity for screening pollutants in order to predict potential targets for degradation. Present study utilizes protein-ligand docking as a tool to achieve the said. For virtual screening, a set of pollutants were selected from five different industries from EPA. X-ray crystal structures of laccase enzymes were taken from the Brookhaven Protein Data Bank (PDB). Two-dimensional structures of pollutants were downloaded from the NCBI Pubchem, which were further converted into three-dimensional structures using CORINA. Protein-ligand docking was carried out using GOLD. Nearly 30 and 17% of the selected datasets showed the best average GOLD fitness score for fungal and bacterial laccase enzyme respectively, suggesting thereby that laccase might be able to oxidize these pollutants. Moreover, in few cases like anthracene, phenanthrene, etc., there is experimental data to support this hypothesis. Similar kind of work would be helpful to find putative pollutants for other biodegradative enzymes.

Mg2+ decreases arrhenius energies of activation for high temperature catalysis of phosphatases in Thermoactinomyces vulgaris.

The nonspecific acid and alkaline phosphatases of Thermoactinomyces vulgaris were found to be optimally active at 65 degrees C and 70 degrees C, respectively, indicating the thermophilic nature of these enzymes in this obligate thermophile. Mg(2+), when added in the assay mixture (in the form of MgCl(2)), increased the specific activities of these enzymes without affecting their respective temperature optima. This divalent cation decreased the Arrhenius energies of activation (E ( A )) of both acid and alkaline phosphatases, as substantiated by Mg(2+)-dependent decrease in the slopes of their Arrhenius plots, which were found to be linear. Thus, Mg(2+)-dependent stimulation of high temperature catalysis of T. vulgaris phosphatases appeared to be accomplished by the decrease in their E ( A )values by this divalent cation, and such unique feature of these enzymes might be associated with their evolutionary adaptation in this thermophilic actinomycete to support its growth at elevated temperatures. The catalytic role of Mg(2+ )in enhancing the phosphatase activities was specified by the fact that this metal ion was able to recover the enzyme activities inhibited by dialysis and EDTA.

Ca2+ dependence and inhibitory effects of trifluoperazine on plasma membrane ATPase of Thermoactinomyces vulgaris.

Ca2+ enhanced the plasma membrane Ca(2+)-ATPase (PMCA) specific activities in wild-type strain 1227 and mutant strains 1278, 1286, and 1261 of Thermoactinomyces vulgaris. The Ca(2+)-ATPase specific activities showed marked increase with increasing concentrations of Ca2+ added in the form of CaCl2 in the culture medium and reached the optimum values at 0.6 mM in strains 1227, 1278, and 1286 and at 0.7 mM in strain 1261 of T. vulgaris. Trifluoperazine, a specific blocker of calmodulin, when added in vivo at concentrations of 2 microM and 8 microM along with the respective optimal concentrations of Ca2+, decreased the PMCA-specific activities to a low level in a dose-dependent manner. The results of the present investigation suggest the presence of a Ca(2+)-dependent protein activator (CaDPA) in the microenvironment constituting this enzyme; and such Ca(2+)-modulated protein has been assigned to play an important role in the enhancement of PMCA levels in this aerobic, spore-forming, thermophilic actinomycete.

Ca2+ -dependence and inhibition of transformation by trifluoperazine and chlorpromazine in Thermoactinomyces vulgaris.

Ca(2+) enhanced the transformation frequency of Thermoactinomyces vulgaris (stock no. 1278) of an auxotrophic strain by the chromosomal DNA isolated from a prototrophic strain (stock no. 1227). The number of transformants showed a marked increase with increasing concentration of CaCl(2) upto 0.05 mM; and above this concentration, the transformation frequency decreased significantly. Antipsychotic drugs that are potent calmodulin inhibitors, like trifluoperazine and chlorpromazine, when applied in the concentration range of 0.01-0.04 mM along with optimal CaCl(2) concentration to the cultures of the recipient cells, resulted in a significant inhibition in the frequency of Ca(2+)-stimulated transformation. The results of present investigation suggest the involvement of a Ca(2+)-dependent protein activator in the development of Ca(2+)-mediated competence, which could have played an important role in the enhancement of genetic transformation in this aerobic spore forming thermophilic actinomycete.