Actinobase: Database on molecular diversity, phylogeny and biocatalytic potential of salt tolerant alkaliphilic actinomycetes

Actinobase is a relational database of molecular diversity, phylogeny and biocatalytic potential of haloalkaliphilic actinomycetes. The main objective of this data base is to provide easy access to range of information, data storage, comparison and analysis apart from reduced data redundancy, data entry, storage, retrieval costs and improve data security. Information related to habitat, cell morphology, Gram reaction, biochemical characterization and molecular features would allow researchers in understanding identification and stress adaptation of the existing and new candidates belonging to salt tolerant alkaliphilic actinomycetes. The PHP front end helps to add nucleotides and protein sequence of reported entries which directly help researchers to obtain the required details. Analysis of the genus wise status of the salt tolerant alkaliphilic actinomycetes indicated 6 different genera among the 40 classified entries of the salt tolerant alkaliphilic actinomycetes. The results represented wide spread occurrence of salt tolerant alkaliphilic actinomycetes belonging to diverse taxonomic positions. Entries and information related to actinomycetes in the database are publicly accessible at http://www.actinobase.in. On clustalW/X multiple sequence alignment of the alkaline protease gene sequences, different clusters emerged among the groups. The narrow search and limit options of the constructed database provided comparable information. The user friendly access to PHP front end facilitates would facilitate addition of sequences of reported entries. AVAILABILITY: The database is available for free at http://www.actinobase.in.     

Organic solvent tolerance of an alkaline protease from salt-tolerant alkaliphilic <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> strain Mit-1

A salt-tolerant alkaliphilic actinomycete, Mit-1 was isolated from Mithapur, coastal region of Gujarat, India. The strain was identified as Streptomyces clavuligerus and based on 16S rRNA gene sequence (EU146061) homology; it was related to Streptomyces sp. (AY641538.1). The organism could grow with up to 15% salt and pH 11, optimally at 5% and pH 9. It was able to tolerate and secrete alkaline protease in the presence of a number of organic solvents including xylene, ethanol, acetone, butanol, benzene and chloroform. Besides, it could also utilize these solvents as the sole source of carbon with significant enzyme production. However, the organism produced spongy cell mass with all solvents and an orange brown soluble pigment was evident with benzene and xylene. Further, the enzyme secretion increased by 50-fold in the presence of butanol. With acetone and ethanol; the enzyme was highly active at 60–80°C and displayed optimum activity at 70°C. The protease was significantly stable and catalyzed the reaction in the presence of xylene, acetone and butanol. However, ethanol and benzene affected the catalysis of the enzyme adversely. Crude enzyme preparation was more stable at 37°C in solvents as compared to partially purified and purified enzymes. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes are explored and information on their enzymatic potential is still scares. To the best of our knowledge this is the first report on organic solvent tolerant protease from salt-tolerant alkaliphilic actinomycetes.     

Novel oxidatively stable subtilisin-like serine proteases from alkaliphilic Bacillus spp.: enzymatic properties, sequences, and evolutionary relationships

The genes for five subtilisin-like serine proteases from alkaliphilic strains of Bacillus exhibiting resistance to oxidative inactivation were cloned and sequenced. The deduced amino acid sequences of the enzymes were highly homologous (greater than 88% identity). They were composed of 638 or 639 amino acids, including a possible approximately 200-amino acid prepro-peptide, and unique stretches of approximately 160 amino acids were found in the C-terminal regions. The molecular masses of mature enzymes (433 or 434 amino acids) were approximately 45 kDa for all. Amino acid sequence comparison and phylogenetic analysis indicated that these enzymes are far removed from other known subtilisins in the line of molecular evolution. We propose that these novel proteases be categorized as a new class of subtilisins, named oxidatively stable, alkaline protease.     

嗜碱放线菌生理学特性的初步研究*

对从新疆、青海采集的盐碱土样中分离获得的29株嗜碱放线菌(AlkAlipbilic actinomyeetes)及1株模式菌株Nocardiopsis prasina的pH实验,不同碱性物质KOH、K2CO3、NaOH、Na2CO3对生长的影响,及对NaCl、KCl耐受性进行了研究。结果发现,少数嗜碱放线菌对Na^ 有一定的依赖性,对K^ 碱性物质敏感;一些嗜碱放线菌对CO3^2-敏感,NaCl、KCl对一些嗜碱放线菌的生长有抑制作用;4种碱性物质对Nocardiopsis菌株生长无影响,且它们可耐受高浓度NaCl、KCl。因此提出高碱性环境中是否存在K^ 、CO3^2-专一性依赖的嗜碱放线菌的推测。     

Haloalkaliphilic actinomycetes in soils of Mongolian desert steppes

The abundance of actinomycetes isolated from the soils of Mongolian desert steppes varies from several thousand to hundreds of thousands of CFU/g soil, depending on soil type and isolation medium. Eight actinomycete genera have been found in these soils: Streptomyces, Micromonospora, Saccharopolyspora, Actinomadura, Microtetraspora, Thermomonospora, Nocardia, and Dactylosporangium. The streptomycete complexes of brown desert-steppe and gray-brown desert alkaline soils include halophilic, alkaliphilic, and haloalkaliphilic species that grow most successfully on the media with a salt concentration of 5% and pH 8–9.     

Validation and high-resolution mapping of a major quantitative trait locus for alkaline salt tolerance in soybean using residual heterozygous line

Alkaline soil restricts soybean plant growth and yield. In our previous study, a major alkaline salt tolerance quantitative trait locus (QTL) was identified in soybean on chromosome 17. In this study, the residual heterozygous line (RHL46), which was selected from a population of F6 recombinant inbred lines (RILs) derived from a cross between an alkaline salt-sensitive soybean cultivar Jackson and a tolerant wild soybean accession JWS156-1, was used for validation and high-resolution mapping of the QTL. In a large segregating population (n = 1,109), which was produced by self-pollinating heterozygotes of RHL46, segregation of alkaline salt tolerance showed a continuous distribution, and the tolerant plants were predominant. Linkage mapping analysis revealed a major QTL with a large dominant effect for alkaline salt tolerance, and the highest LOD score was detected between the single sequence repeat (SSR) markers GM17-12.2 and Satt447. Furthermore, 10 fixed recombinant lines carrying chromosome fragments of different lengths in the QTL region were selected from the RHL46 progeny. Phenotype evaluation and SSR marker analysis of the recombinant lines narrowed down the QTL to a 3.33-cM interval region between the markers GM17-11.6 and Satt447 with a physical map length of approximately 771 kb. High-resolution mapping of the alkaline salt tolerance QTL will be useful not only for marker-assisted selection in soybean breeding programs but also for map-based cloning of the alkaline salt tolerance gene in order to understand alkaline salt tolerance in soybean and other plant species.     

Detection and partial characterization of the chromatin-associated proteases of yeast Saccharomyces cerevisiae

The chromatin fraction was prepared from yeast Saccharomyces cerevisiae free from cytoplasmic contamination except for a trace of mitochondria. When the yeast chromatin was incubated with histones as a substrate it showed three peaks of proteolytic activity as approximately pH 4, pH 7 and pH 11. These activities were separated from each other by differential extractions from chromatin and successive gel filtration through Sephadex G-100. Proteases were partially characterized by affinity labeling with [3H]diisopropylfluorophosphate (iPr2P-F) and by various protease inhibitors. The neutral and the alkaline proteases were serine proteases with a molecular mass of 35 kDa and 25 kDa respectively. The acidic protease showed a molecular size larger than 100 kDa on the gel filtration, and was probably an aspartyl protease because it was most strongly inhibited by pepstatin. A iPr2P-F-binding protein with a molecular mass of 66 kDa, found in chromatin, was likely to be converted to the alkaline protease of 25 kDa when chromatin was incubated at pH 10 or in 6 M urea/0.1 M phosphoric acid at the extraction. The distribution of proteolytic activities and iPr2P-F-binding proteins were compared among chromatins from different strains and from cells in different growth phases and it was found that these three proteases were present in all of them but with different proportions. Considering that rat liver chromatin contains equivalents to these proteases [Tsurugi, K. and Ogata, K. (1982) J. Biochem. (Tokyo) 92, 1369-1381], the results suggested that they play some important roles in the function of eukaryotic chromatin.     

Molecular cloning,nucleotide sequence and expression of the structural gene for a thermostable alkaline protease from Bacillus sp. no. AH-101

Summary Alkaliphilic Bacillus sp. no. AH-101 produces an extremely thermostable alkaline serine protease that has a high optimum pH (pH 12–13) and shows keratinolytic activity. The gene encoding this protease was cloned in Escherichia coli and expressed in B. subtilis. The cloned protease was identical to the AH-101 protease in its optimum pH and thermostability at high alkaline pH. An open reading frame of 1083 bases, identified as the protease gene, was preceded by a putative Shine-Dalgarno sequence (AAAGGAGG) with a spacing of 11 bases. The deduced amino acid sequence revealed a pre-pro-peptide of 93 residues followed by the mature protease comprising 268 residues. AH-101 protease showed slightly higher homology to alkaline proteases from alkaliphilic bacilli (61.2% and 65.3%) than to those from neutrophilic bacilli (54.9–56.7%). Also AH-101 protease and other proteases from alkaliphilic bacilli shared common amino acid changes and a four amino acid deletion when compared to the proteases from neutrophilic bacilli. AH-101 protease, however, was distinct among the proteases from alkaliphilic bacilli in showing the lowest homology to the others.Correspondence to: H. Takami     

Molecular cloning, nucleotide sequence, and expression of the structural gene for alkaline serine protease from alkaliphilic Bacillus sp. 221.

The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus subtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6-61.7%). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.     

Identification of a major QTL allele from wild soybean (Glycine soja Sieb. & Zucc.) for increasing alkaline salt tolerance in soybean

Salt-affected soils are generally classified into two main categories, sodic (alkaline) and saline. Our previous studies showed that the wild soybean accession JWS156-1 (Glycine soja) from the Kinki area of Japan was tolerant to NaCl salt, and the quantitative trait locus (QTL) for NaCl salt tolerance was located on soybean linkage group N (chromosome 3). Further investigation revealed that the wild soybean accession JWS156-1 also had a higher tolerance to alkaline salt stress. In the present study, an F₆ recombinant inbred line mapping population (n = 112) and an F₂ population (n = 149) derived from crosses between a cultivated soybean cultivar Jackson and JWS156-1 were used to identify QTL for alkaline salt tolerance in soybean. Evaluation of soybean alkaline salt tolerance was carried out based on salt tolerance rating (STR) and leaf chlorophyll content (SPAD value) after treatment with 180 mM NaHCO₃ for about 3 weeks under greenhouse conditions. In both populations, a significant QTL for alkaline salt tolerance was detected on the molecular linkage group D2 (chromosome 17), which accounted for 50.2 and 13.0% of the total variation for STR in the F₆ and the F₂ populations, respectively. The wild soybean contributed to the tolerance allele in the progenies. Our results suggest that QTL for alkaline salt tolerance is different from the QTL for NaCl salt tolerance found previously in this wild soybean genotype. The DNA markers closely associated with the QTLs might be useful for marker-assisted selection to pyramid tolerance genes in soybean for both alkaline and saline stresses.     

Two extracellular endoxylanases from alkaliphilic Bacillus firmus differ in their synthesis

To investigate the synthesis of two extracellular endoxylanases, xylan-binding and unbound xylanases from an alkaliphilic Bacillus firmus, washed cells were incubated in alkaline mineral salt media containing various carbon sources. The 23 kDa xylan-binding endoxylanase (XBE), which hydrolyses insoluble xylan, was produced before the 45 kDa, unbound endoxylanase. All the carbon sources tested at 5 mg ml^–1, including glucose, induced production of XBE but the unbound xylanase was totally repressed by glucose. The production of XBE increased when glucose concentration increased but was not synthesized until the glucose in the medium was less than 1 mg ml^–1.     

Molecular Cloning,Nucleotide Sequence,and Expression of the Structural Gene for Alkaline Serine Protease from Alkaliphilic Bacillus sp. 221

The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus suhtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6—61.70/0). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.     

Alkaliphilic Soil Actinomycetes

The actinomycete complex of alkaline soils was found to be dominated by alkaliphilic streptomycetes, which showed maximal radial rates of colony growth at pH 8. At pH values of 7 and 10, the growth of these streptomycetes was poor. Alkaliphilic streptomycetes can be morphologically differentiated from other actinomycetes based on their high radial rates of colony growth and increased spore formation in alkaline media as compared to neutral media.     

Comparative analysis of enzymatic stability and amino acid sequences of thermostable alkaline proteases from two haloalkaliphilic bacteria isolated from Coastal region of Gujarat, India

Thermostable alkaline proteases from two haloalkaliphilic bacteria, Oceanobacillus iheyensis O.M.A₁8 (EU680961) and Haloalkaliphilic bacterium O.M.E₁2 (EU680960) were studied for enzymatic properties and amino acid sequences in comparative manner. The bacteria were isolated from salt enriched soil located in Okha, Coastal Gujarat, India. The unique aspect of the study was that alkaline protease from Haloalkaliphilic bacterium O.M.A₁8 optimally catalyzed the reaction over a wide range of temperature, 50-90 °C, with a half-life of 36 h at 90 °C. The molecular weights of O.M.A₁8 and O.M.E₁2 were 35 kDa and 25 kDa, respectively. The enzyme secretion was over the broader range of pH 8-11, with an optimum at 11. The alkaline proteases from the two haloalkaliphilic strains isolated from the same site reflected quite different characteristics features. To the best of our knowledge, we have not come across with any such report on the thermal stability of alkaline proteases from haloalkaliphiles. Amino acid sequences for both enzymes were deduced from the nucleotide sequences of their corresponding genes followed by the analysis of physico-chemical properties of the enzymes.     

Alkaliphilic soil actinomycetes

The actinomycete complex of alkaline soils was found to be dominated by alkaliphilic streptomycetes, which showed maximal radial rates of colony growth at pH 8. At pH values of 7 and 10, the growth of these streptomycetes was poor. Alkaliphilic streptomycetes can be morphologically differentiated from other actinomycetes based on their high radial rates of colony growth and increased spore formation in alkaline media as compared to neutral media.     

The alteration of mRNA expression of SOD and GPX genes,and proteins in tomato (Lycopersicon esculentum Mill) under stress of NaCl and/or ZnO nanoparticles

Five cultivars of tomato having different levels of salt stress tolerance were exposed to different treatments of NaCl (0, 3 and 6 g L⁻¹) and ZnO-NPs (0, 15 and 30 mg L⁻¹). Treatments with NaCl at both 3 and 6 g L⁻¹ suppressed the mRNA levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) genes in all cultivars while plants treated with ZnO-NPs in the presence of NaCl, showed increments in the mRNA expression levels. This indicated that ZnO-NPs had a positive response on plant metabolism under salt stress. Superior expression levels of mRNA were observed in the salt tolerant cultivars, Sandpoint and Edkawy while the lowest level was detected in the salt sensitive cultivar, Anna Aasa. SDS–PAGE showed clear differences in patterns of protein expression among the cultivars. A negative protein marker for salt sensitivity and ZnO-NPs was detected in cv. Anna Aasa at a molecular weight of 19.162 kDa, while the tolerant cultivar Edkawy had two positive markers at molecular weights of 74.991 and 79.735 kDa.     

汉滩病毒截短核蛋白在大肠杆菌中的表达及其初步应用

利用逆转录 聚合酶链反应 (RT PCR)从急性期HFRS病人外周血清总RNA中扩增获得汉坦病毒S片段部分编码基因 (SA) ,使用NCBIBlast软件对排分析证实为汉滩型 ,申请GenBank号 :AY42 5 61 2。将该片段克隆入原核表达载体 ,并在大肠杆菌中高效表达。所表达的蛋白分子量约为 490 0 0kDa,重组蛋白在菌体中主要以包涵体形式存在。Western blot分析表明该核蛋白具有较好的抗原活性。该蛋白在肾综合征出血热 (HFRS)的血清学诊断中具有一定的价值。     

Cloning,over expression and functional attributes of serine proteases from Oceanobacillus iheyensis O.M.A18 and Haloalkaliphilic bacterium O.M.E12

Cloning, over-expression, characterization and structural and functional analysis of two alkaline proteases from the newly isolated haloalkaliphilic bacteria: Oceanobacillus iheyensis O.M.A₁8 and Haloalkaliphilic bacterium O.M.E₁2 were carried out. The cloned protease genes were over-expressed in Escherichia coli within 6 h of the IPTG induction. The protease genes were sequenced and the sequence submitted to the GenBank with the accession numbers, HM219179 and HM219182. The recombinant proteases were active in the range of pH 8–11 and temperature 30–50 °C. The amino acid sequences of the alkaline proteases displayed hydrophobic character and stable configurations. The amino acids Asp 141, His 171 and Ser 324 formed the catalytic triad, while Ile, Leu and Ser were other amino acid moieties present in the active site. The characteristics of the recombinant proteases were compared and found to be similar to their native counterparts. On the basis of the in-silico analysis and inhibitor studies, the enzymes were confirmed as serine proteases. The study hold significance as only limited enzymes from the haloalkaliphilic bacteria have been cloned, sequenced and analyzed for the structure and function analysis.     

Enzymatic properties of the highly thermophilic and alkaline pectate lyase Pel-4B from alkaliphilic Bacillus sp. strain P-4-N and the entire nucleotide and amino acid sequences

We cloned two genes for alkaline pectate lyase, pel-4A and pel-4B, from alkaline pectinase-producing alkaliphilic Bacillus sp. strain P-4-N. The pel-4B gene product Pel-4B was purified to homogeneity and characterized. The purified enzyme had an isoelectric point of pH 9.6 and a molecular mass of 35 kDa, values close to those of the pel-4A gene product Pel-4A. The pH and temperature optima for activity were as high as 11.5 and 70 degrees C, respectively, which are the highest among the pectate lyases reported to date. The mature Pel-4B (304 amino acids; 33,868 Da) was structurally related to the enzymes in the polysaccharide lyase family 1 and showed 35.6% identity with Pel-4A on the amino acid level. It showed significant homology to other pectate lyases in the same family, such as the enzymes from alkaliphilic Bacillus sp. strains KSM-P7 and KSM-P103 and the fungi Aspergillus nidulans and Colletotrichum gloeosporioides f. sp. malvae.