Molecular Cloning, In Silico Analysis, and Characterization of a Novel Cellulose Microfibril Swelling Gene Isolated from Bacillus sp. Strain AY8.

A novel cellulose microfibril swelling (Cms) gene of Bacillus sp. AY8 was successfully cloned and sequenced using a set of primers designed based on the conserved region of the gene from the genomic database. The molecular cloning of the Cms gene revealed that the gene consisted of 679 bp sequences encoding 225 amino acids. Further in silico analysis unveiled that the Cms gene contained the NlpC/P60 conserved region that exhibited a homology of 98% with the NlpC/P60 family proteins found in both the strains, Burkholderialata sp. and Burkholderia vietnamiensis. The recombinant Cms enzyme had a significant impact on the reduction of crystallinity indices (CrI) of various substrates including a 3%, a 3.97%, a 4.66%, and a substantial 14.07% for filter paper, defatted cotton fiber, avicel, and alpha cellulose, respectively. Additionally, notable changes in the spectral features were observed among the substrates treated with recombinant Cms enzymes compared to the untreated control. Specifically, there was a decrease in band intensities within the spectral regions of 3000-3450 cm-1, 2900 cm-1, 1429 cm-1, and 1371 cm-1 for the treated filter paper, cotton fiber, avicel, and alpha cellulose, respectively. Furthermore, the recombinant Cms enzyme exhibited a maximum cellulose swelling activity at a pH of 7.0 along with a temperature of 40 °C. The molecular docking data revealed that ligand molecules, such as cellobiose, dextrin, maltose 1-phosphate, and feruloyated xyloglucan, effectively bonded to the active site of the Cms enzyme. The molecular dynamics simulations of the Cms enzyme displayed stable interactions with cellobiose and dextrin molecules up to 100 ns. It is noteworthy to mention that the conserved region of the Cms enzyme did not match with those of the bioadditives like expansins and swollenin proteins. This study is the initial report of a bacterial cellulose microfibril swellase enzyme, which could potentially serve as an additive to enhance biofuel production by releasing fermentable sugars from cellulose.

Characterization of lignin and hemicellulose degrading bacteria isolated from cow rumen and forest soil: Unveiling a novel enzymatic model for rice straw deconstruction.

Application of greener pretreatment technology using robust ligninolytic bacteria for short duration to deconstruct rice straw and enhance bioethanol production is currently lacking. The objective of this study is to characterize three bacterial strains isolated from the milieux of cow rumen and forest soil and explore their capabilities of breaking down lignocellulose - an essential process in bioethanol production. Using biochemical and genomic analyses these strains were identified as Bacillus sp. HSTU-bmb18, Bacillus sp. HSTU-bmb19, and Citrobacter sp. HSTU-bmb20. Genomic analysis of the strains unveiled validated model hemicellulases, multicopper oxidases, and pectate lyases. These enzymes exhibited interactions with distinct lignocellulose substrates, further affirmed by their stability in molecular dynamic simulations. A comprehensive expression of ligninolytic pathways, including ß-ketoadipate, phenyl acetate, and benzoate, was observed within the HSTU-bmb20 genome. The strains secreted approximately 75-82 U/mL of cellulase, xylase, pectinase, and lignin peroxidase. FT-IR analysis of the bacterial treated rice straw fibers revealed that the intensity of lignin-related peaks decreased, while cellulose-related peaks sharpened. The values of crystallinity index for the untreated control and the treated rice straw with either HSTU-bmb18, or HSTU-bmb19, or HSTU-bmb20 were recorded to be 34.48, 28.49, 29.36, 31.75, respectively, which are much higher than that of 13.53 noted for those treated with the bacterial consortium. The ratio of fermentable cellulose in rice straw increased by 1.25-, 1.79-, 1.93- and 2.17-fold following treatments with HSTU-bmb18, HSTU-bmb20, HSTU-bmb19, and a mixed consortium of these three strains, respectively. These aggregative results suggested a novel model for rice straw deconstruction utilizing hydrolytic enzymes of the consortium, revealing superior efficacy compared to individual strains, and advancing cost-effective, affordable, and sustainable green technology.

Characterization of Growth-Promoting Activities of Consortia of Chlorpyrifos Mineralizing Endophytic Bacteria Naturally Harboring in Rice Plants-A Potential Bio-Stimulant to Develop a Safe and Sustainable Agriculture.

Eighteen pesticide-degrading endophytic bacteria were isolated from the roots, stems, and leaves of healthy rice plants and identified through 16S rRNA gene sequencing. Furthermore, biochemical properties, including enzyme production, dye degradation, anti-bacterial activities, plant-growth-promoting traits, including N-fixation, P-solubilization, auxin production, and ACC-deaminase activities of these naturally occurring endophytic bacteria along with their four consortia, were characterized. Enterobacter cloacae HSTU-ABk39 and Enterobacter sp. HSTU-ABk36 displayed inhibition zones of 41.5 ± 1.5 mm, and 29 ± 09 mm against multidrug-resistant human pathogenic bacteria Staphylococcus aureus and Staphylococcus epidermidis, respectively. FT-IR analysis revealed that all eighteen isolates were able to degrade chlorpyrifos pesticide. Our study confirms that pesticide-degrading endophytic bacteria from rice plants play a key role in enhancing plant growth. Notably, rice plants grown in pots containing reduced urea (30%) mixed with either endophytic bacterial consortium-1, consortium-2, consortium-3, or consortia-4 demonstrated an increase of 17.3%, 38.6%, 18.2%, and 39.1% yields, respectively, compared to the control plants grown in pots containing 100% fertilizer. GC-MS/MS analysis confirmed that consortia treatment caused the degradation of chlorpyrifos into different non-toxic metabolites, including 2-Hydroxy-3,5,6 trichloropyridine, Diethyl methane phosphonate, Phorate sulfoxide, and Carbonochloridic. Thus, these isolates could be deployed as bio-stimulants to improve crop production by creating a sustainable biological system.

Enhanced rice plant (BRRI-28) growth at lower doses of urea caused by diazinon mineralizing endophytic bacterial consortia and explorations of relevant regulatory genes in a Klebsiella sp. strain HSTU-F2D4R.

Endophytic biostimulant with pesticide bioremediation activities may reduce agrochemicals application in rice cultivation. The present study evaluates diazinon-degrading endophytic bacteria, isolated from rice plants grown in the fields with pesticide amalgamation, leading to increased productivity in high-yielding rice plants. These endophytes showed capabilities of decomposing diazinon, confirmed by FT-IR spectra analysis. Growth promoting activities of these endophytes can be attributed to their abilities to produce an increased level of IAA content and to demonstrate high level ACC-deaminase activities. Furthermore, these endophytes demonstrated enhanced level of extracellular cellulase, xylanase, amylase, protease and lignin degrading activities. Five genera including Enterobacter, Pantoea, Shigella, Acinetobacter, and Serratia, are represented only by the leaves, while four genera such as Enterobacter, Escherichia, Kosakonia, and Pseudomonas are represented only by the shoots. Five genera including, Klebsiella, Enterobacter, Pseudomonas, Burkholderia, and Bacillus are represented only by the roots of rice plants. All these strains demonstrated cell wall hydrolytic enzyme activities, except pectinase. All treatments, either individual strains or consortia of strains, enhanced rice plant growth at germination, seedling, vegetative and reproductive stages. Among four (I-IV) consortia, consortium-III generated the maximum rice yield under 70% lower doses of urea compared to that of control (treated with only fertilizer). The decoded genome of Klebsiella sp. HSTU-F2D4R revealed nif-cluster, chemotaxis, phosphates, biofilm formation, and organophosphorus insecticide-degrading genes. Sufficient insecticide-degrading proteins belonging to strain HSTU-F2D4R had interacted with diazinon, confirmed in molecular docking and formed potential catalytic triads, suggesting the strains have bioremediation potential with biofertilizer applications in rice cultivation.

Phytoremediation of arsenic from the contaminated soil using transgenic tobacco plants expressing ACR2 gene of Arabidopsis thaliana.

We have cloned, characterized and transformed the AtACR2 gene (arsenic reductase 2) of Arabidopsis thaliana into the genome of tobacco (Nicotiana tabacum, var Sumsun). Our results revealed that the transgenic tobacco plants are more tolerant to arsenic than the wild type ones. These plants can grow on culture medium containing 200µM arsenate, whereas the wild type can barely survive under this condition. Furthermore, when exposed to 100µM arsenate for 35days the amount of arsenic accumulated in the shoots of transgenic plants was significantly lower (28µg/g d wt.) than that found in the shoots of non-transgenic controls (40µg/g d wt.). However, the arsenic content in the roots of transgenic plants was significantly higher (2400µg/g d. wt.) than that (2100µg/g d. wt.) observed in roots of wild type plants. We have demonstrated that Arabidopsis thaliana AtACR2 gene is a potential candidate for genetic engineering of plants to develop new crop cultivars that can be grown on arsenic contaminated fields to reduce arsenic content of the soil and can become a source of food containing no arsenic or exhibiting substantially reduced amount of this metalloid.

Comparative genome analysis of Lysinibacillus B1-CDA, a bacterium that accumulates arsenics.

Previously, we reported an arsenic resistant bacterium Lysinibacillus sphaericus B1-CDA, isolated from an arsenic contaminated lands. Here, we have investigated its genetic composition and evolutionary history by using massively parallel sequencing and comparative analysis with other known Lysinibacillus genomes. Assembly of the sequencing reads revealed a genome of ~4.5 Mb in size encompassing ~80% of the chromosomal DNA. We found that the set of ordered contigs contains abundant regions of similarity with other Lysinibacillus genomes and clearly identifiable genome rearrangements. Furthermore, all genes of B1-CDA that were predicted be involved in its resistance to arsenic and/or other heavy metals were annotated. The presence of arsenic responsive genes was verified by PCR in vitro conditions. The findings of this study highlight the significance of this bacterium in removing arsenics and other toxic metals from the contaminated sources. The genetic mechanisms of the isolate could be used to cope with arsenic toxicity.

Bioremediation of hexavalent chromium (VI) by a soil-borne bacterium, Enterobacter cloacae B2-DHA.

Chromium and chromium containing compounds are discharged into the nature as waste from anthropogenic activities, such as industries, agriculture, forest farming, mining and metallurgy. Continued disposal of these compounds to the environment leads to development of various lethal diseases in both humans and animals. In this paper, we report a soil borne bacterium, B2-DHA that can be used as a vehicle to effectively remove chromium from the contaminated sources. B2-DHA is resistant to chromium with a MIC value of 1000 µg mL(-1) potassium chromate. The bacterium has been identified as a Gram negative, Enterobacter cloacae based on biochemical characteristics and 16S rRNA gene analysis. TOF-SIMS and ICP-MS analyses confirmed intracellular accumulation of chromium and thus its removal from the contaminated liquid medium. Chromium accumulation in cells was 320 µg/g of cells dry biomass after 120-h exposure, and thus it reduced the chromium concentration in the liquid medium by as much as 81%. Environmental scanning electron micrograph revealed the effect of metals on cellular morphology of the isolates. Altogether, our results indicate that B2-DHA has the potential to reduce chromium significantly to safe levels from the contaminated environments and suggest the potential use of this bacterium in reducing human exposure to chromium, hence avoiding poisoning.

Data in support of the comparative genome analysis of Lysinibacillus B1-CDA, a bacterium that accumulates arsenics.

This study is a part of our long term project on bioremediation of toxic metals and other pollutants for protection of human health and the environment from severe contamination. The information and results presented in this data article are based on both in vitro and in silico experiments. in vitro experiments were used to investigate the presence of arsenic responsive genes in a bacterial strain B1-CDA that is highly resistant to arsenics. However, in silico studies were used to annotate the function of the metal responsive genes. By using this combined study consisting of in vitro and in silico experiments we have identified and characterized specific genes from B1-CDA that can be used as a potential tool for removal of arsenics as well as other heavy metals from the contaminated environment.

Isolation and characterization of a Lysinibacillus strain B1-CDA showing potential for bioremediation of arsenics from contaminated water.

The main objective of this study was to identify and isolate arsenic resistant bacteria that can be used for removing arsenic from the contaminated environment. Here we report a soil borne bacterium, B1-CDA that can serve this purpose. B1-CDA was isolated from the soil of a cultivated land in Chuadanga district located in the southwest region of Bangladesh. The morphological, biochemical and 16S rRNA analysis suggested that the isolate belongs to Lysinibacillus sphaericus. The minimum inhibitory concentration (MIC) value of the isolate is 500 mM (As) as arsenate. TOF-SIMS and ICP-MS analysis confirmed intracellular accumulation and removal of arsenics. Arsenic accumulation in cells amounted to 5.0 mg g(-1) of the cells dry biomass and thus reduced the arsenic concentration in the contaminated liquid medium by as much as 50%. These results indicate that B1-CDA has the potential for remediation of arsenic from the contaminated water. We believe the benefits of implementing this bacterium to efficiently reduce arsenic exposure will not only help to remove one aspect of human arsenic poisoning but will also benefit livestock and native animal species. Therefore, the outcome of this research will be highly significant for people in the affected area and also for human populations in other countries that have credible health concerns as a consequence of arsenic-contaminated water.

In silico and in vivo studies of molecular structures and mechanisms of AtPCS1 protein involved in binding arsenite and/or cadmium in plant cells.

This paper reports a continuation of our previous research on the phytochelatin synthase1 (PCS1) gene involved in binding and sequestration of heavy metals or metalloids in plant cells. Construction of a 3D structure of the Arabidopsis thaliana PCS1 protein and prediction of gene function by employing iterative implementation of the threading assembly refinement (I-TASSER) revealed that PC ligands (3GC-gamma-glutamylcysteine) and Gln50, Pro53, Ala54, Tyr55, Cys56, Ile102, Gly161, His162, Phe163, Asp204 and Arg211 residues are essential for formation of chelating complex with cadmium (Cd²âº) or arsenite (AsIII). This finding suggests that the PCS1 protein might be involved in the production of the enzyme phytochelatin synthase, which might in turn bind, localize, store or sequester heavy metals in plant cells. For validation of the in silico results, we included a T-DNA tagged mutant of Arabidopsis thaliana, SAIL_650_C12, (mutation in AtPCS1 gene) in our investigation. Furthermore, using reverse transcriptase PCR we confirmed that the mutant does not express the AtPCS1 gene. Mutant plants of SAIL_650_C12 were exposed to various amounts of cadmium (Cd²âº) and arsenite (AsIII) and the accumulation of these toxic metals in the plant cells was quantified spectrophotometrically. The levels of Cd²âº and AsIII accumulation in the mutant were approximately 2.8 and 1.6 times higher, respectively, than that observed in the wild-type controlled plants. We confirmed that the results obtained in in silico analyses complement those obtained in in vivo experiments.

In silico and in vivo studies of an Arabidopsis thaliana gene, ACR2, putatively involved in arsenic accumulation in plants.

Previously, our in silico analyses identified four candidate genes that might be involved in uptake and/or accumulation of arsenics in plants: arsenate reductase 2 (ACR2), phytochelatin synthase 1 (PCS1) and two multi-drug resistant proteins (MRP1 and MRP2) [Lund et al. (2010) J Biol Syst 18:223-224]. We also postulated that one of these four genes, ACR2, seems to play a central role in this process. To investigate further, we have constructed a 3D structure of the Arabidopsis thaliana ACR2 protein using the iterative implementation of the threading assembly refinement (I-TASSER) server. These analyses revealed that, for catalytic metabolism of arsenate, the arsenate binding-loop (AB-loop) and residues Phe-53, Phe-54, Cys-134, Cys-136, Cys-141, Cys-145, and Lys-135 are essential for reducing arsenate to arsenic intermediates (arsenylated enzyme-substrate intermediates) and arsenite in plants. Thus, functional predictions suggest that the ACR2 protein is involved in the conversion of arsenate to arsenite in plant cells. To validate the in silico results, we exposed a transfer-DNA (T-DNA)-tagged mutant of A. thaliana (mutation in the ACR2 gene) to various amounts of arsenic. Reverse transcriptase PCR revealed that the mutant exhibits significantly reduced expression of the ACR2 gene. Spectrophotometric analyses revealed that the amount of accumulated arsenic compounds in this mutant was approximately six times higher than that observed in control plants. The results obtained from in silico analyses are in complete agreement with those obtained in laboratory experiments.

A new opecoelid species (Trematoda: Opecoelidae) from the threespine stickleback Gasterosteus aculeatus L. in California.

Plagioporus kolipinskii n. sp. (Trematoda: Opecoelidae) is described from the intestine of the threespine stickleback, Gasterosteus aculeatus L., from Lobos Creek, a freshwater stream in Presidio, San Francisco County, California. Plagioporus kolipinskii is morphologically somewhat similar to 4 (P. serotinus , P. angusticollis , P. macrouterinus , and P. shawi []) of the 12 currently recognized North American species of the genus, but can be readily distinguished from all 4 in possessing a much larger acetabulum and a larger ovary relative to the testes. In addition, the new species can be distinguished from P. angusticollis by a smaller cirrus sac; from P. macrouterinus by the elongated shape of its body and reduced extent of its uterus (the uterus of P. macrouterinus extends posteriorly to the intersection of the testes); from P. shawi by a much-shorter cirrus sac (which reaches the ovary in P. shawi), an unlobed ovary (as opposed to a quadrilobed ovary in P. shawi), and fewer eggs that are also larger relative to body size.

Paclobutrazol affects the resistance of black spruce to high light and thermal stress.

Detached needles from 20-week-old black spruce (Picea mariana (Mill.) B.S.P.) seedlings root-drenched with 60 mg of paclobutrazol were exposed to two temperatures (22 and 50 degrees C) and two light treatments (100 and 1900 &mgr;mol m(-2) s(-1) PAR) in a factorial combination for 4 h in vitro. Mean dry weights of individual needles from paclobutrazol-treated plants were approximately 1.9 times heavier than that of needles from untreated controls at 22 degrees C, but no differences were observed following incubation at 50 degrees C. Numbers of cells per needle remained constant in all treatments. Chlorophyll and carotenoid contents per needle were higher in seedlings treated with paclobutrazol than in untreated control seedlings, and the differences were most pronounced in the high temperature plus high light treatment. In low light at 50 degrees C, quantum efficiency of photosystem II was 45% higher in needles of paclobutrazol-treated seedlings than in needles of untreated control seedlings, but quantum efficiency of needles from treated seedlings declined when needles were exposed to high light at either temperature. Peroxidase and superoxide dismutase activities were up-regulated by paclobutrazol, whereas catalase activities were depressed and no significant differences were observed between treated and control needles at 50 degrees C in either light treatment. Paclobutrazol treatment did not moderate the depressive effects of high temperature on total soluble protein or on the activity of ribulose-1,5-bisphosphate carboxylase. In contrast, high activities of phosphoenolpyruvate carboxylase were maintained in paclobutrazol-treated needles under all stress conditions, whereas large losses in activity were recorded in untreated needles at 50 degrees C. Collectively, these observations suggest that paclobutrazol treatment may convey resistance to excessive light and high temperatures by increasing the potential of conifers to limit damage caused by oxidative stress.