A root-specific wall-associated kinase gene, HvWAK1, regulates root growth and is highly divergent in barley and other cereals

Wall-associated receptor-like kinases (WAKs) are important candidates for directly linking the extracellular matrix with intracellular compartments and are involved in developmental processes and stress response. WAK gene family has been identified in plants such as Arabidopsis and rice. Here, we present a detailed analysis of the WAK1 gene from barley cv. Golden Promise, mapped to chromosome 5H. Three BAC clones corresponding to the WAK fragment were sequenced and the full-length WAK1 gene was characterized. The gene has three exons and two short introns with a coding region of 2,178 bp encoding a protein of 725 amino acids. A regulatory region was analyzed in ?1,000 bp sequence upstream to start codon. Using conserved domains database and SMART, various conserved domains such as GUB WAK Bind, epidermal growth factor CA, and protein kinase C as well as other regions like signal peptides, active sites, and transmembrane domains were identified. The gene organization of HvWAK1 was compared with wheat (TaWAK1) and Arabidopsis (AtWAK1), suggesting that the WAK1 gene organization has remained highly conserved. Nonetheless, WAK1 was found to be highly divergent when compared with sequences available from barley cv. Haruna Nijo (50 %), rice (46 %), wheat (21 %), Arabidopsis (25 %), and maize (19 %). This divergence may have facilitated a better adaptation to surrounding environments due to its role in communication between the extracellular matrix, cell, and outer environment. Semiquantitative RT-PCR-based expression analysis indicates HvWAK1 expression is specific to roots. Significant differences in root growth between GP wild type and GP-Ds mutant seedlings were observed under control and salt stress conditions.     

Isolation and characterization of a lead (Pb) tolerant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain HF5 for decolorization of reactive red-120 and other azo dyes

Presence of heavy metals including lead (Pb) in the textile effluents is a crucial factor affecting the growth and potential of the dye decolorizing bacterial strains. This work was planned to isolate and characterize a bacterial strain exhibiting the potential to decolorize a range of azo dyes as well as the resistance to Pb. In this study, several Pb tolerant bacteria were isolated from effluents of textile industry. These bacterial isolates were screened for their potential of decolorizing the reactive red-120 (RR120) azo dye with presence of Pb (50 mg L^?1). The most efficient isolate was further characterized for its potential to resist Pb and decolorize different azo dyes under varying cultural and incubation conditions. Out of the total 82 tested bacterial isolates, 30 bacteria were found to have varying potentials to resist the presence of lead (Pb) and carry out decolorization of an azo dye reactive red-120 (RR120) in the medium amended with Pb (50 mg L^?1). The most efficient selected bacterium, Pseudomonas aeruginosa strain HF5, was found to show a good potential not only to grow in the presence of considerable concentration of Pb but also to decolorize RR120 and other azo dyes in the media amended with Pb. The strain HF5 completely (>?90%) decolorized RR120 in mineral salt medium amended with 100 mg L^?1 of Pb and 20 g L^?1 NaCl. This strain also considerably (>?50%) decolorized RR120 up to the presence of 2000 mg L^?1 of Pb and 50 g L^?1 of NaCl but with reduced rate. The optimal decolorization of RR120 by HF5 was achieved when the pH of the Pb amended (100 mg L^?1) mineral salt media was adjusted at 7.5 and 8.5. Interestingly, this strain also showed the tolerance to a range of metal ions with varying MIC values. The Pseudomonas aeruginosa strain HF5 harboring the unique potentials to grow and decolorize the azo dyes in the presence of Pb is envisaged as a potential bioresource for devising the remediation strategies for treatment of colored textile wastewaters loaded with Pb and other heavy metal ions.     

Biodecolorization of textile azo dyes by isolated yeast from activated sludge: Issatchenkia orientalis JKS6

An ascomycetous yeast strain isolated from activated sludge could decolorize Reactive Black 5 azo dye at 200 mg l^?1 up to 90 % within 12–18 h under agitated condition. Yeast decolorization ability was investigated at different RB5 concentrations and, at higher dye concentration, 500 mg l^?1, the decolorization was found to be 98 % after 36 h incubation time. Extensive decolorization (95–99 %) was obtained in presence of five other azo dyes, Reactive Orange 16, Reactive Red 198, Direct Blue 71, Direct Yellow 12, and Direct Black 22, by isolated yeast. HPLC analysis, UV–vis spectra and colorless biomass obtained after complete decolorization showed that the decolorization occured through a biodegradation mechanism. Decolorization was occurred during the exponential growth phase which is associated to primary metabolism. Laccase production by the yeast cells was not detected. The isolated yeast was characterized according to phenotypical and molecular procedures and was closely related (99 % identity) to Issatchenkia orientalis.     

An omp gene enhances cell tolerance of Cu(II) in Sinorhizobium meliloti CCNWSX0020

The main aim of this work was to study molecular characterization of a DNA fragment conferring resistance to Cu(II) in Sinorhizobium meliloti CCNWSX0020. The strain CCNWSX0020, resistant to 1.4 mmol l^?1 Cu(II) in tryptone-yeast extract medium was isolated from Medicago lupulina growing in mine tailings of Fengxian County, China. The availability of the complete genome sequence of S. meliloti CCNWSX0020 provides an opportunity for investigating genes that play significant roles in Cu(II) resistance. A copper resistance gene, with a length of 1,445 bp, encoding 481 amino acids, designated omp, was identified by cDNA-amplified fragment length polymorphism from S. meliloti CCNWSX0020. The expression of omp gene strongly increased in the presence of Cu(II). The omp-defective mutants display sensitivities to Cu(II) compared with their wild types. The Cu(II)-sensitive phenotype of the mutant was complemented by a 1.5-kb DNA fragment containing omp gene. BLAST analysis revealed that this gene encoded a hypothetical outer membrane protein with 75 % similarity to outer membrane efflux protein in Rhizobium leguminosarum bv. viciae 3841. These studies suggested that the omp product was involved in the Cu(II) tolerance of S. meliloti CCNWSX0020.     

Degradation and detoxification of azo dyes by a salt-tolerant yeast <Emphasis Type="Italic">Cyberlindnera samutprakarnensis</Emphasis> S4 under high-salt conditions

A new yeast strain which was capable of degrading various azo dyes under high-salt conditions was identified in this study. The results showed that the yeast named S4 was identified as Cyberlindnera samutprakarnensis through 26S rDNA sequence analysis and could decolorize more than 97% of Acid Red B (ARB) within 18 h under the optimal conditions. The acute toxicity of ARB sharply decreased after degradation. NADH–DCIP reductase and lignin peroxidase were determined as the key reductase and oxidase of the yeast S4, respectively. Furthermore, it was proposed that ARB was degraded by strain S4 successively through reduction of azo bonds, hydroxylation, deamination, desulfonation and finally to the TCA cycle.     

Decolorization of azo dyes by marine Shewanella strains under saline conditions

Azo dye decolorization was studied with Shewanella strains under saline conditions. Growing cells of Shewanella algae and Shewanella marisflavi isolated from marine environments demonstrated better azo dye decolorization capacities than the other three strains from non-saline sources. Cell suspensions of S. algae and S. marisflavi could decolorize single or mixed azo dyes with different structures. Decolorization kinetics were described with Michaelis–Menton equation, which indicated better decolorization performance of S. algae over S. marisflavi. Lactate and formate were identified as efficient electron donors for amaranth decolorization by the two strains. S. algae and S. marisflavi could decolorize amaranth at up to 100 g?L^?1 NaCl or Na₂SO₄. However, extremely low concentration of NaNO₃ exerted strong inhibition on decolorization. Both strains could remove the color and COD of textile effluent during sequential anaerobic–aerobic incubation. Lower concentrations of NaCl (20–30 g?L^?1) stimulated the activities of azoreductase, laccase, and NADH-DCIP reductase. The decolorization intermediates were identified by high-performance liquid chromatography and Fourier transform infrared spectroscopy. Decolorization metabolites of amaranth were less toxic than original dye. These findings improved our knowledge of azo-dye-decolorizing Shewanella species and provided efficient candidates for the treatment of dye-polluted saline wastewaters.     

Cloning, Expression, and Characterization of Iron Superoxide Dismutase in Sonneratia alba, a Highly Salt Tolerant Mangrove Tree

In this study, a novel iron superoxide dismutase (FeSOD) gene from Sonneratia alba was cloned and then expressed in Escherichia coli Rosetta-gami, designated as SaFeSOD. The DNA sequence of SaFeSOD contained a 786-bp open reading frame which encodes a 261 amino-acid protein of 30.0 kDa. The 651-bp fragment coding for putative mature SaFeSOD was amplified and inserted into pET15b for expression. This recombinant SaFeSOD was subsequently isolated by Ni-trap column protein purification system. The apparent molecular mass of the purified enzyme was 25 kDa on SDS-PAGE. In comparison with FeSODs from other plant species, all iron-binding sites (His 27, His 80, Asp 164 and His 168) of SaFeSOD were conserved. SaFeSOD was found to have good pH stability in the pH range of 3.5–9.5 at 25 °C after 1 h incubation and was relatively stable and showed 78 % activity when incubated in 50 °C for 1 h. Quantitative real-time PCR experiments demonstrated that SaFeSOD was expressed in leaf, stem, flower, fruit and root tissues with the highest expression in leaf tissues.     

Degradation of Sulphonated Azo Dye Red HE7B by Bacillus sp. and Elucidation of Degradative Pathways

Bacteria capable of degrading the sulfonated azo dye Red HE7B were isolated from textile mill effluent contaminated soil. The most efficient isolate was identified as Bacillus sp. Azo1 and the isolate could successfully decolorize up to 89 % of the dye. The decolorized cultural extract analyzed by HPLC confirmed degradation. Enzymatic analysis showed twofold and fourfold increase in the activity of azoreductase and laccase enzymes, respectively, indicating involvement of both reductive and oxidative enzymes in biodegradation of Red HE7B. Degraded products which were identified by GC/MS analysis included various metabolites like 8-nitroso 1-naphthol, 2-diazonium naphthalene. Mono azo dye intermediate was initially generated from the parent molecule. This mono azo dye was further degraded by the organism, into additional products, depending on the site of cleavage of R–N=N–R molecule. Based on the degradation products identified, three different pathways have been proposed. The mechanism of degradation in two of these pathways is different from that of the previously reported pathway for azo dye degradation. This is the first report of a microbial isolate following multiple pathways for azo dye degradation. Azo dye Red HE7B was observed to be phytotoxic, leading to decrease in root development, shoot length and seedling fresh weight. However, after biotreatment the resulting degradation products were non-phytotoxic.     

Structural characterization of a hypothetical protein: a potential agent involved in trimethylamine metabolism in Catenulispora acidiphila

Catenulispora acidiphila is a newly identified lineage of actinomycetes that produces antimicrobial activities and represents a promising source of novel antibiotics and secondary metabolites. Among the discovered protein coding genes, 68 % were assigned a putative function, while the remaining 32 % are genes encoding “hypothetical” proteins. Caci_0382 is one of the “hypothetical” proteins that has very few homologs. Sequence analysis shows that the protein belongs to the NTF2-like protein family. The structure of Caci_0382 demonstrates that it shares the same fold and has a similar active site as limonene-1,2-epoxide hydrolase, which suggests that it may have a related function. Using a fluorescence thermal shift assay, we identified stabilizing compounds that suggest potential natural ligands of Caci_0382. Using this information, we determined the crystal structure in complex with trimethylamine to provide a better understanding of the function of this uncharacterized protein.     

Molecular characterization and expression of type-I interferon gene in Labeo rohita

Genes coding for type-I interferon (I-IFN) has been cloned from Labeo rohita, a commercially important and widely cultured fish in India and South East Asia. In the present study, full-length gene of I-IFN was amplified and sequenced. The sequence analysis revealed that I-IFN consists of 1,786 bp genomic sequence with four introns and five exons and an ORF of 546 bp encoding for a putative protein of 181 amino acids. The mature protein has a molecular weight of 18.97 kDa and consists of 158 amino acids and a signal peptide of 23 amino acids at the N terminus. The sequence carries I-IFN signature motif, one glycosylation site, two conserved cystine amino acids and other conserved amino acids. The sequence showed highest similarity to that of Cyprinus carpio (84 %). In silico analysis of the rohu I-IFN protein was done using various bioinformatic tools. The constitutive expression of I-IFN gene was found to be more in spleen compared to gill and kidney in real time PCR assay. Expression of I-IFN increased about 20-fold in cultured kidney cell 2 h after induction with poly I:C and showed maximum expression at 8 h post-induction.     

Properties of NAD (P) H Azoreductase from Alkaliphilic Red Bacteria Aquiflexum sp. DL6

Azoreductase plays a key role in bioremediation and biotransformation of azo dyes. It initializes the reduction of azo bond in azo dye metabolism under aerobic or anaerobic conditions. In the present study, we isolated an alkaliphilic red-colored Aquiflexum sp. DL6 bacterial strain and identified by 16S rRNA method. We report nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-dependent azoreductase purified from Aquiflexum sp. DL6 by a combination of ammonium sulfate precipitation and chromatography methods. The azoreductase was purified up to 30-fold with 37 % recovery. The molecular weight was found to be 80 kDa. The optimum activity was observed at pH 7.4 and temperature 60 °C with amaranth azo dye as a substrate. The thermal stability of azoreductase was up to 80 °C. The azoreductase has shown a wide range of substrate specificity, including azo dyes and nitro aromatic compounds. Metal ions have no significant inhibitory action on azoreductase activity. The apparent K _m and V _max values for amaranth azo dye were 1.11 mM and 30.77 U/mg protein respectively. This NAD (P) H azoreductase represents the first azoreductase to be characterized from alkaliphilic bacteria.     

Neuropeptide Y reduces the expression of <Emphasis Type="Italic">PLCB2</Emphasis>, <Emphasis Type="Italic">PLCD1</Emphasis> and selected <Emphasis Type="Italic">PLC</Emphasis> genes in cultured human endothelial cells

The ability of mycobacteria to grow and invade target tissues is the key component in the process of Mycobacterium bovis infection. Therefore, analysis of the proteins responsible for cell invasion will assist clinicians in combating bovine tuberculosis. The Mb1514 gene of M. bovis encodes a hypothetical invasion protein (designated here as MbINV protein), whose function has not yet been directly identified. In this study, the Mb1514 gene from M. bovis was cloned, and expressed in E. coli. The recombinant MbINV protein (a single band of approximately 28 kDa) was purified for biological analysis. Our data demonstrated that recombinant MbINV protein significantly inhibited the viability of RAW264.7 macrophages in a dose-dependent manner (P < 0.05), and induced cell necrosis, indicating that the protein is toxic. MbINV protein infection significantly enhanced the mRNA expression levels of TNF-α, IL-1β, and NOS2 (P < 0.01), suggesting that MbINV protein may be one of the virulence factors which directly interact with macrophages and modulate the host immune response to M. bovis. An invasion inhibition assay showed that MbINV-inhibited M. bovis invasion of RAW264.7 cells in a concentration-dependant manner, demonstrating it is an invasion protein.     

Characterization of a salt resistant bacterial strain <Emphasis Type="Italic">Proteus</Emphasis> sp. NA6 capable of decolorizing reactive dyes in presence of multi-metal stress

Microbial biotechnologies for the decolorization of textile wastewaters have attracted worldwide attention because of their economic suitability and easiness in handling. However, the presence of high amounts of salts and metal ions in textile wastewaters adversely affects the decolorization efficiency of the microbial bioresources. In this regard, the present study was conducted to isolate salt tolerant bacterial strains which might have the potential to decolorize azo dyes even in the presence of multi-metal ion mixtures. Out of the tested 48 bacteria that were isolated from an effluent drain, the strain NA6 was found relatively more efficient in decolorizing the reactive yellow-2 (RY2) dye in the presence of 50 g L^?1 NaCl. Based on the similarity of its 16S rRNA gene sequence and its position in a phylogenetic tree, this strain was designated as Proteus sp. NA6. The strain NA6 showed efficient decolorization (>90 %) of RY2 at pH 7.5 in the presence of 50 g L^?1 NaCl under static incubation at 30 °C. This strain also had the potential to efficiently decolorize other structurally related azo dyes in the presence of 50 g L^?1 NaCl. Moreover, Proteus sp. NA6 was found to resist the presence of different metal ions (Co⁺², Cr⁺⁶, Zn⁺², Pb⁺², Cu⁺², Cd⁺²) and was capable of decolorizing reactive dyes in the presence of different levels of the mixtures of these metal ions along with 50 g L^?1 NaCl. Based on the findings of this study, it can be suggested that Proteus sp. NA6 might serve as a potential bioresource for the biotechnologies involving bioremediation of textile wastewaters containing the metal ions and salts.     

Depolymerization and decolorization of kraft lignin by bacterium Comamonas sp. B-9

There is no commercial or industrial-scale process for the remediation of black liquor using microorganisms to date. One of the most important causes is that most microorganisms are not able to use lignin as their principal metabolic carbon or energy source. The bacterial strain Comamonas sp. B-9 has shown remarkable ability to degrade kraft lignin and decolorize black liquor using lignin as its principal metabolic carbon and energy source. This report looks at the depolymerization and decolorization of kraft lignin by Comamonas sp. B-9. The degradation, decolorization, and total carbon removal reached 45, 54, and 47.3 %, respectively, after 7 days treatment. Comamonas sp. B-9 was capable of depolymerizing kraft lignin effectively as analyzed by gel permeation chromatography and decolorization via degrading benzene ring structures as shown using Fourier transform infrared spectroscopy analysis.     

Neurexophilin 1 Gene Polymorphism in Chickens and Its Variation Among Species

Neurexophilin 1 (nxph1) has been considered a potential candidate marker for sperm storage in chicken sperm storage tubules. In this work, one mutation of chicken nxph1 was detected. We analyzed 18 nxph1 gene sequences from 18 species. The coding sequence length of the zebra fish nxph1 gene is 819 bp; that of the other species is 816 bp. Amino acid alignment analysis revealed that the gene product is a conserved protein, especially in mammals. The sequences of mammals are highly conserved. We found 202 conserved amino acids (70–271), and there were only eight mutations in the remaining 69 amino acids. That level of conservation could be due to the nxph1 gene having been subjected to substantial constraints or strong purifying selection during millions of years of evolution.     

A bacterial laccase from marine microbial metagenome exhibiting chloride tolerance and dye decolorization ability

Laccases are blue multicopper oxidases with potential applications in environmental and industrial biotechnology. In this study, a new bacterial laccase gene of 1.32 kb was obtained from a marine microbial metagenome of the South China Sea by using a sequence screening strategy. The protein (named as Lac15) of 439 amino acids encoded by the gene contains three conserved Cu²⁺-binding domains, but shares less than 40% of sequence identities with all of the bacterial multicopper oxidases characterized. Lac15, recombinantly expressed in Escherichia coli, showed high activity towards syringaldazine at pH 6.5–9.0 with an optimum pH of 7.5 and with the highest activity occurring at 45 °C. Lac15 was stable at pH ranging from 5.5 to 9.0 and at temperatures from 15 to 45 °C. Distinguished from fungal laccases, the activity of Lac15 was enhanced twofold by chloride at concentrations lower than 700 mM, and kept the original level even at 1,000 mM chloride. Furthermore, Lac15 showed an ability to decolorize several industrial dyes of reactive azo class under alkalescent conditions. The properties of alkalescence-dependent activity, high chloride tolerance, and dye decolorization ability make the new laccase Lac15 an alternative for specific industrial applications.     

Identification of single nucleotide polymorphisms in the agouti signaling protein (ASIP) gene in some goat breeds in tropical and temperate climates

The agouti-signaling protein (ASIP) plays a major role in mammalian pigmentation as an antagonist to melanocortin-1 receptor gene to stimulate pheomelanin synthesis, a major pigment conferring mammalian coat color. We sequenced a 352 bp fragment of ASIP gene spanning part of exon 2 and part of intron 2 in 215 animals representing six goat breeds from Nigeria and the United States: West African Dwarf, predominantly black; Red Sokoto, mostly red; and Sahel, mostly white from Nigeria; black and white Alpine, brown and white Spanish and white Saanen from the US. Twenty haplotypes from nine mutations representing three intronic, one silent and five missense (p.S19R, p.N35K, p.L36V, p.M42L and p.L45W) mutations were identified in Nigerian goats. Approximately 89 % of Nigerian goats carry haplotype 1 (TGCCATCCG) which seems to be the wild type configuration of mutations in this region of the gene. Although we found no association between these polymorphisms in the ASIP gene and coat color in Nigerian goats, in-silico functional analysis predicts putative deleterious functional impact of the p.L45W mutation on the basic amino-terminal domain of ASIP. In the American goats, two intronic mutations, g.293G>A and g.327C>A, were identified in the Alpine breed, although the g.293G>A mutation is common to American and Nigerian goat populations. All Sannen and Sahel goats in this study belong to haplotypes 1 of both populations which seem to be the wild-type composite ASIP haplotype. Overall, there was no clear association of this portion of the ASIP gene interrogated in this study with coat color variation. Therefore, additional genomic analyses of promoter sequence, the entire coding and non-coding regions of the ASIP gene will be required to obtain a definite conclusion.     

Decolorization of textile azo dye and Congo red by an isolated strain of the dissimilatory manganese-reducing bacterium Shewanella xiamenensis BC01

Shewanella xiamenensis BC01 (SXM) was isolated from sediment collected off Xiamen, China and was identified based on the phylogenetic tree of 16S rRNA sequences and the gyrB gene. This strain showed high activity in the decolorization of textile azo dyes, especially methyl orange, reactive red 198, and recalcitrant dye Congo red, decolorizing at rates of 96.2, 93.0, and 87.5 %, respectively. SXM had the best performance for the specific decolorization rate (SDR) of azo dyes compared to Proteus hauseri ZMd44 and Aeromonas hydrophila NIU01 strains and had an SDR similar to Shewanella oneidensis MR-1 in Congo red decolorization. Luria-Bertani medium was the optimal culture medium for SXM, as it reached a density of 4.69 g-DCW L^?1 at 16 h. A mediator (manganese) significantly enhanced the biodegradation and flocculation of Congo red. Further analysis with UV–VIS, Fourier Transform Infrared spectroscopy, and Gas chromatography–mass spectrometry demonstrated that Congo red was cleaved at the azo bond, producing 4,4′-diamino-1,1′-biphenyl and 1,2′-diamino naphthalene 4-sulfonic acid. Finally, SEM results revealed that nanowires exist between the bacteria, indicating that SXM degradation of the azo dyes was coupled with electron transfer through the nanowires. The purpose of this work is to explore the utilization of a novel, dissimilatory manganese-reducing bacterium in the treatment of wastewater containing azo dyes.