EMBL Nucleotide Sequence Database Research Articles

Phylogenetic characterization and screening of halophilic bacteria from Algerian salt lake for the production of biosurfactant and enzymes

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).

Molecular determinants of pathogenicity and host specificity of highly pathogenic h5N1 BiH isolates

ABSTRACT Background: Towards preparation for a possible influenza pandemic, investigation of the molecular characteristics of the circulating avian H5N1 influenza virus strains is of crucial importance. These H5N1 viruses continue to spread, to infect animals and humans and to evolve and diversify providing so an ever-looming pandemic threat.Aim: To identify genetic structure and molecular biological characteristics of BiH's isolates of H5N1 HPAI as well as to assess the level of pathogenicity, phylogenetic origin and host- specificity of the isolates.Material and Methods: SPF embryonated chicken eggs were used for virus isolation. Viral RNA extracted using QIAamp viral RNA kit and manufacturer’s protocol (QIAGEN®) was used for PCR amplification. cDNA synthesis and PCR amplification of the coding region, using gene specific primer sets (primer sequences available on request), were carried out for all eight viral RNA segments separately. The Prism Big Dye Terminator v1.1 cycle sequencing kit (Applied Biosystems) was used and products were analyzed on an automatic ABI PRISM 3130 genetic analyzer (Applied Biosystems). Nucleotide sequences were analyzed using Bioedit software (v. 7.0.9.0) with an engine based on the ClustalW 1.4 algorithm. MEGA software (v. 4,0), using the neighbor joining tree inference analysis with the Tamura-Nei γ-model, was used to estimate phylogenies and calculate bootstrap values from the nucleotide sequences.Results: Full-length nucleotide sequences of the A/Cygnus olor/BIH/1/2006 (H5N1) strain were deposited in EMBL Nucleotide Sequence Database under accession nos. FN186008 to FN186014 and FM20943. The pathogenicity and host specificity of this strain, as polygenic traits, are determined in silico by the structure of its proteins, especially surface glycoproteins, HA and NA. Multibasic amino acid stretch PQGERRRKKR/GLF, marker of strains highly pathogenic to poultry, was present at the HA cleavage site of BiH strain. The RBS was typical for avian influenza viruses and contained Gln and Gly at positions 238 and 240 (H5 numbering) that is,226 and 228 according to H3 numbering with seven potential glycosylated sites but with increased binding to alpha2-6 sialoglycans thanks to substitutions, as follows, 110N, 171N, 171N, 172A, 205R and 251P. NA structure assigned this strain to the Z genotype, characterized also by the deletion of the five amino acid residues of the NS1 protein (positions 80-84). Amino acid residues, typical for the avian influenza viruses, were revealed in 40 out of 43 positions of M1, M2, NP, PA, PB2 and HA, determining the host range specificity. Phylogenetic analysis of the HA gene revealed that BiH isolates belonged to genetic clade 2.2., and presence of aspartic acid at the position of 403 of HA locate BiH isolates in 2.2.2. sub-clade.Conclusions: The BiH’s isolates were determined as HPAI virus with genes sequences closely related to A/Cygnus olor/Astrakhan/Ast05-2-10/2005 (H5N1). Three residues (M2 - 28V and 78K, NP - 33I), typical of human influenza viruses, were found, indicating a certain degree of intercurrent evolutionary adaptive changes in BiH isolates. Sequence comparison of HA and NA segments with relevant sequences in GenBank revealed that the BiH isolates and the ones from the southern Russia (Astrakhan region) group together phylogenetically, forming a monophyleticcluster in both genes indicating that these isolates have evolved from the same origin. Sequence derived phenotype markers of NA protein (E99, V129, D131, R136, H255 and Y256) as well as of M2 protein (26L, 27V, 30A, S31 and G34) showed that the isolates have an oseltamivir and amantadine sensitive genotype.

STUDIES ON ANTHOSTOMA DECIPIENS INVOLVED IN CARPINUS BETULUS DECLINE

Carpinus betulus L. (European or common hornbeam) is well known for its rustic nature and adaptability, which make it a very important tree in private and public green areas. During the last few years, many cases of decline and death of these trees were reported in different Italian regions, and because of its lethal outcome, this disease was termed “Carpinus betulus decline.” Laboratory analysis aimed at isolating the etiological agents of this disease ascertained the frequent presence of two different fungi in the same canker, and experimental infections confirmed that both fungi are hornbeam pathogens. The aim of this work is to describe the taxonomic determination of one of the fungi. On the bases of symptoms, morphocultural and micromorphological characteristics, this fungus, which produces red-spore masses on hornbeam bark, was identified as Anthostoma decipiens (DC.: Fr) Nitschke, anamorph Cytospora decipiens Sacc. To date, no Anthostoma- related DNA sequences have been published, and comparison of ITS1, ITS2, and partial beta-tubulin nucleotide sequences with those present in the international databases gave no help in identifying the pathogen. Therefore, the sequences obtained were deposited in the EMBL Nucleotide Sequence Database. A specific primer, to be used for PCR reactions in combination with the standard primer ITS4, and located 80 nucleotides next to the 3’-end of ITS1 primer, named REX, was selected, synthesized, and tested.

Petabyte-scale innovations at the European Nucleotide Archive

Dramatic increases in the throughput of nucleotide sequencing machines, and the promise of ever greater performance, have thrust bioinformatics into the era of petabyte-scale data sets. Sequence repositories, which provide the feed for these data sets into the worldwide computational infrastructure, are challenged by the impact of these data volumes. The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/embl), comprising the EMBL Nucleotide Sequence Database and the Ensembl Trace Archive, has identified challenges in the storage, movement, analysis, interpretation and visualization of petabyte-scale data sets. We present here our new repository for next generation sequence data, a brief summary of contents of the ENA and provide details of major developments to submission pipelines, high-throughput rule-based validation infrastructure and data integration approaches.

First Report of Solanum jasminoides Infected by Citrus exocortis viroid in Germany and the Netherlands and Tomato apical stunt viroid in Belgium and Germany.

Recent identifications of Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd) in Solanum jasminoides (3,4) prompted the testing of this plant species for infections with other pospiviroids. From autumn of 2006 to spring of 2007, samples from symptomless plants of S. jasminoides were collected in Belgium (3 samples ranging from 75 to 150 plants), Germany (3 samples ranging from 1 to 200 plants), and the Netherlands (3 samples ranging from 2 to 200 plants). Samples were tested for pospiviroids by reverse transcription (RT)-PCR assays using the Pospi1-FW/RE and Vid-FW/RE (2) and PSTV-Nb-FW (5'-ggatccccggggaaacctgga-3')/RE (5'-ggatccctgaagcgctcctcc-3') primer sets. Each set amplifies several but not all pospiviroids. The first and last primer sets amplified PCR products from six samples. The full-length genomes of all six isolates were amplified using primer pairs CEVd-FW1/RE1 (1) and CEVd-FW2 (5'-gtgctcacctgaccctgcagg-3')/RE2 (5'-accacaggaacctcaagaaag-3'), which are fully complementary to both Citrus exocortis viroid (CEVd) and Tomato apical stunt viroid (TASVd). Sequence analysis of the PCR products identified CEVd from two samples each from Germany and the Netherlands and TASVd from one sample each from Germany and Belgium (plants were imported from Israel). Although the sequences of the different CEVd isolates from S. jasminoides were not identical, all exhibited more than 95% identity with a CEVd isolate from Vicia faba (GenBank Accession No. EF494687). Both TASVd sequences were identical and showed 99.2% identity to a TASVd isolate from tomato (GenBank Accession No. AY 062121). Two nucleotide sequences of CEVd were submitted to the NCBI GenBank (Accession Nos. EU094207 and EU094208). The two other CEVd sequences and the TASVd sequence were submitted to the EMBL Nucleotide Sequence Database as Accession Nos. AM774356, AM774357, and AM777161. In addition to identification from S. jasminoides by sequence analysis, TASVd infection in the S. jasminoides sample from Germany and CEVd in one sample from the Netherlands was confirmed by mechanical inoculation to tomato followed by RT-PCR using the two CEVd-FW/RE primer pairs and analysis of the sequenced PCR product. Infection by CEVd and TASVd was also confirmed in the German samples by Northern hybridization and TASVd was confirmed in the Belgian sample by return-polyacrylamide gel electrophoresis. To our knowledge, these are the first reports of CEVd and TASVd in S. jasminoides. The viroids do not reduce the quality of S. jasminoides plants; however, the infected plants may act as infection sources for other crops.

B cells: B cell back catalogue (remastered)

B cells: B cell back catalogue (remastered)

Priorities for nucleotide trace, sequence and annotation data capture at the Ensembl Trace Archive and the EMBL Nucleotide Sequence Database.

The Ensembl Trace Archive (http://trace.ensembl.org/) and the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/), known together as the European Nucleotide Archive, continue to see growth in data volume and diversity. Selected major developments of 2007 are presented briefly, along with data submission and retrieval information. In the face of increasing requirements for nucleotide trace, sequence and annotation data archiving, data capture priority decisions have been taken at the European Nucleotide Archive. Priorities are discussed in terms of how reliably information can be captured, the long-term benefits of its capture and the ease with which it can be captured.

EMBL Nucleotide Sequence Database in 2006

The EMBL Nucleotide Sequence Database () at the EMBL European Bioinformatics Institute, UK, offers a large and freely accessible collection of nucleotide sequences and accompanying annotation. The database is maintained in collaboration with DDBJ and GenBank. Data are exchanged between the collaborating databases on a daily basis to achieve optimal synchrony. Webin is the preferred tool for individual submissions of nucleotide sequences, including Third Party Annotation, alignments and bulk data. Automated procedures are provided for submissions from large-scale sequencing projects and data from the European Patent Office. In 2006, the volume of data has continued to grow exponentially. Access to the data is provided via SRS, ftp and variety of other methods. Extensive external and internal cross-references enable users to search for related information across other databases and within the database. All available resources can be accessed via the EBI home page at . Changes over the past year include changes to the file format, further development of the EMBLCDS dataset and developments to the XML format.

A cytological and molecular study of the generaScorzoneraL. andPodospermum(L.) DC (Asteraceae)

The genus Scorzonera L. s.l. encompasses enormous morphological variation and has created problems for taxonomists. Podospermum has been treated as either a section of a broadly defined Scorzonera or as an independ- ent genus. In Italy, there are twelve Scorzonera species and three Podospermum species. The relationship between these species is not at all clear. To clarify these relationships, a survey, including thirteen of the fifteen taxa found in Italy, was carried out using: i) sequence analysis of ribosomal internal transcribed spacer (ITS) and external tran- scribed spacer (ETS) regions; ii) analysis of Amplified Fragment Length Polymorphisms (AFLPs); iii) chromosome counts. In addition, to broaden the scope of the work and allow taxonomically significant interpretation of the data to be made, sequences from a range of related species were obtained from the EMBL nucleotide sequence database. Our analyses clearly indicate that Scorzonera s.l. is a polyphyletic grouping. The three approaches used were consist- ent with each other although showing different levels of resolution. There was broad division, on the basis of chro- mosome number, either 2n = 12 or 14. This was supported by PCO analysis of AFLP fragments which indicate three groups. Finally, phylogenetic analysis of sequence data produced four to six highly supported clades that might ap- propriately be given independent taxonomic status.

Identification of the genesGPD1andGPD2ofPichia jadinii

Two genes coding for proteins with a high degree of sequence similarity to glycerol-3-phosphate dehydrogenases have been isolated from the yeast Pichiajadinii. Fragments of the genes were PCR-amplified with degenerated primers from genomic DNA of P. jadinii. Clones containing the full-length genes PjGPDI and PjGPD2 were isolated by screening genomic libraries. DNA sequencing revealed open reading frames (ORFs) of 1182 bp and 1185 bp for PjGpdlp and PjGpd2p, respectively. In a complementation study PjGPD1 rescued the growth defect of a Saccharomyces cerevisiae Agpdl mutant strain under osmotic stress, while complementation by PjGPD2 is temperature sensitive. The sequences of the PjGPD1 and PjGPD2 ORFs have been submitted to the EMBL Nucleotide Sequence Database under Accession No. AJ632339 and AJ632340, the sequences of the corresponding genomic DNA fragments under Accession No. AJ632341 and AJ635370, respectively.

EMBL Nucleotide Sequence Database: developments in 2005

The EMBL Nucleotide Sequence Database () at the EMBL European Bioinformatics Institute, UK, offers a comprehensive set of publicly available nucleotide sequence and annotation, freely accessible to all. Maintained in collaboration with partners DDBJ and GenBank, coverage includes whole genome sequencing project data, directly submitted sequence, sequence recorded in support of patent applications and much more. The database continues to offer submission tools, data retrieval facilities and user support. In 2005, the volume of data offered has continued to grow exponentially. In addition to the newly presented data, the database encompasses a range of new data types generated by novel technologies, offers enhanced presentation and searchability of the data and has greater integration with other data resources offered at the EBI and elsewhere. In stride with these developing data types, the database has continued to develop submission and retrieval tools to maximise the information content of submitted data and to offer the simplest possible submission routes for data producers. New developments, the submission process, data retrieval and access to support are presented in this paper, along with links to sources of further information.

SPdb – a signal peptide database

BackgroundThe signal peptide plays an important role in protein targeting and protein translocation in both prokaryotic and eukaryotic cells. This transient, short peptide sequence functions like a postal address on an envelope by targeting proteins for secretion or for transfer to specific organelles for further processing. Understanding how signal peptides function is crucial in predicting where proteins are translocated. To support this understanding, we present SPdb signal peptide database , a repository of experimentally determined and computationally predicted signal peptides.ResultsSPdb integrates information from two sources (a) Swiss-Prot protein sequence database which is now part of UniProt and (b) EMBL nucleotide sequence database. The database update is semi-automated with human checking and verification of the data to ensure the correctness of the data stored. The latest release SPdb release 3.2 contains 18,146 entries of which 2,584 entries are experimentally verified signal sequences; the remaining 15,562 entries are either signal sequences that fail to meet our filtering criteria or entries that contain unverified signal sequences.ConclusionSPdb is a manually curated database constructed to support the understanding and analysis of signal peptides. SPdb tracks the major updates of the two underlying primary databases thereby ensuring that its information remains up-to-date.

The EMBL Nucleotide Sequence Database

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl), maintained at the European Bioinformatics Institute (EBI) near Cambridge, UK, is a comprehensive collection of nucleotide sequences and annotation from available public sources. The database is part of an international collaboration with DDBJ (Japan) and GenBank (USA). Data are exchanged daily between the collaborating institutes to achieve swift synchrony. Webin is the preferred tool for individual submissions of nucleotide sequences, including Third Party Annotation (TPA) and alignments. Automated procedures are provided for submissions from large-scale sequencing projects and data from the European Patent Office. New and updated data records are distributed daily and the whole EMBL Nucleotide Sequence Database is released four times a year. Access to the sequence data is provided via ftp and several WWW interfaces. With the web-based Sequence Retrieval System (SRS) it is also possible to link nucleotide data to other specialist molecular biology databases maintained at the EBI. Other tools are available for sequence similarity searching (e.g. FASTA and BLAST). Changes over the past year include the removal of the sequence length limit, the launch of the EMBLCDSs dataset, extension of the Sequence Version Archive functionality and the revision of quality rules for TPA data.

VBASE2, an integrative V gene database

The database VBASE2 provides germ-line sequences of human and mouse immunoglobulin variable (V) genes. It acts as an interconnecting platform between several existing self-contained data systems: VBASE2 integrates genome sequence data and links to the V genes in the Ensembl Genome Browser. For a single V gene sequence, all references to the EMBL nucleotide sequence database are provided, including references for V(D)J rearrangements. Furthermore, cross-references to the VBASE database, the IMGT database and the Kabat database are available. A DAS server allows the display of VBASE2 V genes within the Ensembl Genome Browser. VBASE2 can be accessed either by a web-based text query or by a sequence similarity search with the DNAPLOT software. VBASE2 is available at http://www.vbase2.org, and the DAS server is located at http://www.dnaplot.com/das.

Integr8 and Genome Reviews: integrated views of complete genomes and proteomes

Integr8 is a new web portal for exploring the biology of organisms with completely deciphered genomes. For over 190 species, Integr8 provides access to general information, recent publications, and a detailed statistical overview of the genome and proteome of the organism. The preparation of this analysis is supported through Genome Reviews, a new database of bacterial and archaeal DNA sequences in which annotation has been upgraded (compared to the original submission) through the integration of data from many sources, including the EMBL Nucleotide Sequence Database, the UniProt Knowledgebase, InterPro, CluSTr, GOA and HOGENOM. Integr8 also allows the users to customize their own interactive analysis, and to download both customized and prepared datasets for their own use. Integr8 is available at http://www.ebi.ac.uk/integr8.

The EMBL Nucleotide Sequence Database.

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/), maintained at the European Bioinformatics Institute (EBI), incorporates, organizes and distributes nucleotide sequences from public sources. The database is a part of an international collaboration with DDBJ (Japan) and GenBank (USA). Data are exchanged between the collaborating databases on a daily basis to achieve optimal synchrony. The web-based tool, Webin, is the preferred system for individual submission of nucleotide sequences, including Third Party Annotation (TPA) and alignment data. Automatic submission procedures are used for submission of data from large-scale genome sequencing centres and from the European Patent Office. Database releases are produced quarterly. The latest data collection can be accessed via FTP, email and WWW interfaces. The EBI's Sequence Retrieval System (SRS) integrates and links the main nucleotide and protein databases as well as many other specialist molecular biology databases. For sequence similarity searching, a variety of tools (e.g. FASTA and BLAST) are available that allow external users to compare their own sequences against the data in the EMBL Nucleotide Sequence Database, the complete genomic component subsection of the database, the WGS data sets and other databases. All available resources can be accessed via the EBI home page at http://www.ebi.ac.uk.

HCVDB

To date, more than 30 000 hepatitis C virus (HCV) sequences have been deposited in the generalist databases DNA Data Bank of Japan (DDBJ), EMBL Nucleotide Sequence Database (EMBL) and GenBank. The main difficulties with HCV sequences in these databases are their retrieval, annotation and analyses. To help HCV researchers face the increasing needs of HCV sequence analyses, we developed a specialised database of computer-annotated HCV sequences, called HCVDB. HCVDB is re-built every month from an up-to-date EMBL database by an automated process. HCVDB provides key data about the HCV sequences (e.g. genotype, genomic region, protein names and functions, known 3-dimensional structures) and ensures consistency of the annotations, which enables reliable keyword queries. The database is highly integrated with sequence and structure analysis tools and the SRS (LION bioscience) keywords query system. Thus, any user can extract subsets of sequences matching particular criteria or enter their own sequences and analyse them with various bioinformatics programs available on the same server. HCVDB is available from http://hepatitis.ibcp.fr.

Biosphere

The growing use of DNA microarrays in biomedical research has led to the proliferation of analysis tools. These software programs address different aspects of analysis (e.g. normalisation and clustering within and across individual arrays) as well as extended analysis methods (e.g. clustering, annotation and mining of multiple datasets). Therefore, microarray data analysis typically requires the interoperability of multiple software programs involving different analysis types and methods. Such interoperation is often hampered by the heterogeneity inherent in the software tools (which may function by implementing different interfaces and using different programming languages). To address this problem, we employed the simple object access protocol (SOAP)-based web service approach that provides a uniform programmatic interface to these heterogeneous software components. To demonstrate this approach in the microarray context, we created a web server application, Biosphere, which interoperates a number of web services that are geographically widely distributed. These web services include a clustering web service, which is a suite of different clustering algorithms for analysing microarray data; XEMBL, developed at the European Bioinformatics Institute (EBI) for retrieving EMBL Nucleotide Sequence Database sequence data; and three gene annotation web services: GetGO, GetHAPI and GetUMLS. GetGO allows retrieval of Gene Ontology (GO) annotation, and the other two web services retrieve annotation from the biomedical literature that is indexed based on the Medical Subject Headings (MeSH) terms. With these web services, Biosphere allows the users to do the following: (i) cluster gene expression data using seven different algorithms; (ii) visualise the clustering results that are grouped statistically in colour; and (iii) retrieve sequence, annotation and citation data for the genes of interest. Biosphere and its web services described in Web Service Description Language (WSDL) can be accessed at http://rook.cecid.hku.hk:8280/BiosphereServer.

The EMBL sequence version archive.

The EMBL Nucleotide Sequence Database, maintained at the European Bioinformatics institute, is Europe's primary nucleotide sequences database. Its entries are subject to changes, but only the most recent versions are preserved in the database. The EMBL Sequence Version Archive is a new publicly available database retaining also the earlier versions of these entries. http://www.ebi.ac.uk/embl/sva/

Identification of a triacylglycerol lipase gene family in Candida deformans: molecular cloning and functional expression.

The yeast Candida deformans CBS 2071 produces an extracellular lipase which was shown to catalyse the production of various esters by the esterification of free fatty acids, even in the presence of a large molar excess of water. To clone the gene encoding this extracellular lipase, Saccharomyces cerevisiae was transformed with C. deformans genomic libraries and screened for lipolytic activity on a medium containing rapeseed oil emulsion and rhodamine B. Three members of a lipase gene family (CdLIP1, CdLIP2 and CdLIP3) were cloned and characterized. Each deduced lipase sequence has a Gly-His-Ser-Leu-Gly-(Gly/Ala)-Ala conserved motif, eight cysteine residues and encodes an N-terminal signal sequence. MALDI-TOF mass spectrometry analysis of a proteolytic digest of the lipase produced was used to obtain experimental evidence that the CdLIP1 gene encoded the extracellular lipase. Recombinant expression studies confirmed that the cloned genes encoded functional lipases. The three lipases are very similar to lipases from the related species Yarrowia lipolytica. Significant homologies were also found with several yeast and fungal lipases. As C. deformans CBS 2071 was previously considered to be synonymous with Y. lipolytica, the strains were compared for the extent of nucleotide divergence in the variable regions (D1/D2) at the 5'-end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region has diverged sufficiently to suggest that C. deformans is a separate species. The nucleotide sequences of the CdLIP1, CdLIP2 and CdLIP3 genes will appear in the EMBL nucleotide sequence database under Accession Nos AJ428393, AJ428394 and AJ428395, respectively.