BBProF: An Asynchronous Application Server for Rapid Identification of Proteins Associated with Bacterial Bioleaching

Recovery of metal value, especially from low-grade ores and overburden minerals using acidophilic bacteria through the process of bioleaching is an environmentally benign and commercially scalable biotechnology. In recent years, while the “OMICS” landscape has been witnessing extensive application of computational tools to understand and interpret global biological sequence data, a dedicated bioinformatic server for analysis of bacterial information in the context of its bioleaching ability is not available. We have developed an on-line Bacterial Bioleaching Protein Finder (BBProF) System, which rapidly identifies novel proteins involved in a bacterial bioleaching process and also performs phylogenetic analysis of 16S rRNA genes. BBProF uses the features of Asynchronous Java Script and XML (AJAX) to provide an efficient and fast user experience with minimal requirement of network bandwidth. In the input module the server accepts any bacterial or archaeal complete genome sequence in RAW format and provides a list of proteins involved in the microbial leaching process. BBProF web server is integrated with the European Bioinformatics Institute (EBI) web services such as BLAST for homology search and InterProScan for functional characterization of output protein sequences. Studying evolutionary relationship of bacterial strains of interest using Muscle and ClustalW2 phylogeny web services from EBI is another key feature of our server, where 16S rRNA gene sequences are considered as input through a JQUERY interface along with the sequences present in the BBProF database library. Complete genome sequences of 24 bioleaching microorganism characterized by genomic and physiological study in the laboratory and their respective 16S rRNA gene sequences were stored in the database of the BBProF library. To our knowledge BBProF is the first integrated bioinformatic web server that demonstrates its utility in identifying potential bioleaching bacteria. We hope that the server facilitate ongoing comparative genomic studies on of bioleaching microorganisms and also assist in identification and design of novel microbial consortia that are optimally efficient bioleaching agents.     

Locating probable genes using Fourier transform approach

FTG is a web server for analyzing nucleotide sequences to predict the genes using Fourier transform techniques. This server implements the existing Fourier transform algorithms for gene prediction and allows the rapid visualization of analysis by output in GIF format.     

In silico simulation of fingerprinting techniques based on double endonuclease digestion of genomic DNA

We have developed an online generic tool for simulation of fingerprinting techniques based on the double endonuclease digestion of DNA. This tool allows modelling and modifications of already existing techniques, as well as new theoretical approaches not yet tried in the lab. It allows the use of any combination of recognition patterns and discrimination of end types yielded by restriction with non palindromic recognition sizes. Re-creation of experimental conditions in silico saves time and reduces laboratory costs. This tool allows simulation of Amplified Fragment Length Polymorphism (AFLP-PCR), Subtracted Restriction Fingerprinting (SRF), and additional novel fingerprinting techniques. Simulation may be performed against custom sequences uploaded to the server, or against all sequenced bacterial genomes. Different endonuclease types may be selected from a list, or a recognition sequence may be introduced in the form. After double digestion of DNA, four fragment types are yielded, and the program allows their customised selection. Selective nucleotides may be used in the experiment. Scripts for specific simulation of AFLP-PCR and SRF techniques are available, and both include a suggestion tool for the selection of endonucleases. This is the first program available for the simulation of SRF fingerprinting. Availability: This free online tool is available at http://www.in-silico.com/DDF/.     

OligoMatcher

OligoMatcher is a web-based tool for analysis and selection of unique oligonucleotide sequences for gene silencing by antisense oligonucleotides (ASOs) or small interfering RNA (siRNA). A specific BLAST server was built for analysing sequences of ASOs that target pre-mRNA in the cell nucleus. Tissue- and cell-specific expression data of potential cross-reactive genes are integrated in the OligoMatcher program, which allows biologists to select unique oligonucleotide sequences for their target genes in specific experimental systems. AVAILABILITY: The OligoMatcher web server is available at http://shelob.cs.iupui.edu:18081/oligomatch.php. The source code is freely available for non-profit use on request to the authors. CONTACT: Mathew Palakal (mpalakal@cs.iupui.edu) or Shuyu Li (li_shuyu_dan@lilly.com).     

A Survey of Codon and Amino Acid Frequency Bias in Microbial Genomes Focusing on Translational Efficiency

Unequal use of synonymous codons has been found in several prokaryotic and eukaryotic genomes. This bias has been associated with translational efficiency. The prevalence of this bias across lineages is currently unknown. Here, a new method (GCB) to measure codon usage bias is presented. It uses an iterative approach for the determination of codon scores and allows the computation of an index of codon bias suitable for interspecies comparison. A server to calculate GCB-values of individual genes as well as a list of compiled results are available at . The method was applied to complete bacterial genomes. The relation of codon usage bias with amino acid composition and the choice of stop codons were determined and discussed.     

FTMove: A Web Server for Detection and Analysis of Cryptic and Allosteric Binding Sites by Mapping Multiple Protein Structures

Protein mapping distributes many copies of different molecular probes on the surface of a target protein in order to determine binding hot spots, regions that are highly preferable for ligand binding. While mapping of X-ray structures by the FTMap server is inherently static, this limitation can be overcome by the simultaneous analysis of multiple structures of the protein. FTMove is an automated web server that implements this approach. From the input of a target protein, by PDB code, the server identifies all structures of the protein available in the PDB, runs mapping on them, and combines the results to form binding hot spots and binding sites. The user may also upload their own protein structures, bypassing the PDB search for similar structures. Output of the server consists of the consensus binding sites and the individual mapping results for each structure - including the number of probes located in each binding site, for each structure. This level of detail allows the users to investigate how the strength of a binding site relates to the protein conformation, other binding sites, and the presence of ligands or mutations. In addition, the structures are clustered on the basis of their binding properties. The use of FTMove is demonstrated by application to 22 proteins with known allosteric binding sites; the orthosteric and allosteric binding sites were identified in all but one case, and the sites were typically ranked among the top five. The FTMove server is publicly available at https://ftmove.bu.edu.     

List mania: interpreting microarray results with the L2L server

A new server for interpreting microarray results, list to list(L2L), is described. This tool offers a unique approach to understandthe meaning of microarray results, based on comparing them topreviously identified lists of differentially expressed genes.The usefulness of the server is demonstrated by studying differentialexpression in primary tumours versus metastases in colon cancer.     

STRUCLA: a WWW meta-server for protein structure comparison and evolutionary classification

MOTIVATION: Evolutionary relationships of proteins have long been derived from the alignment of protein sequences. But from the view of function, most restraints of evolutionary divergence operate at the level of tertiary structure. It has been demonstrated that quantitative measures of dissimilarity in families of structurally similar proteins can be applied to the construction of trees from a comparison of their three-dimensional structures. However, no convenient tool is publicly available to carry out such analyses. RESULTS: We developed STRUCLA (STRUcture CLAssification), a WWW tool for generation of trees based on evolutionary distances inferred from protein structures according to various methods. The server takes as an input a list of PDB files or the initial alignment of protein coordinates provided by the user (for instance exported from SWISS PDB VIEWER). The user specifies the distance cutoff and selects the distance measures. The server returns series of unrooted trees in the NEXUS format and corresponding distance matrices, as well as a consensus tree. The results can be used as an alternative and a complement to a fixed hierarchy of current protein structure databases. It can complement sequence-based phylogenetic analysis in the 'twilight zone of homology', where amino acid sequences are too diverged to provide reliable relationships.     

ASAP: analysis of peptide composition

SUMMARY: ASAP is a web tool designed to search for specific dipeptides, tripeptides and tetrapeptides in a protein sequence database. The server allows for: (a) identification of frequent and infrequent peptides and the creation of peptide probability tables for a given database of sequences (GenerNet program), (b) determination of the compatibility of an amino-acid sequence to the given peptide probability tables (ClonErrNet program); and (c) comparison of different protein databases based on peptide composition (CompNet program). ASAP server can be useful in protein engineering and/or protein classification studies.     

Genomic BLAST: custom-defined virtual databases for complete and unfinished genomes

BLAST (Basic Local Alignment Search Tool) searches against DNA and protein sequence databases have become an indispensable tool for biomedical research. The proliferation of the genome sequencing projects is steadily increasing the fraction of genome-derived sequences in the public databases and their importance as a public resource. We report here the availability of Genomic BLAST, a novel graphical tool for simplifying BLAST searches against complete and unfinished genome sequences. This tool allows the user to compare the query sequence against a virtual database of DNA and/or protein sequences from a selected group of organisms with finished or unfinished genomes. The organisms for such a database can be selected using either a graphic taxonomy-based tree or an alphabetical list of organism-specific sequences. The first option is designed to help explore the evolutionary relationships among organisms within a certain taxonomy group when performing BLAST searches. The use of an alphabetical list allows the user to perform a more elaborate set of selections, assembling any given number of organism-specific databases from unfinished or complete genomes. This tool, available at the NCBI web site http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi, currently provides access to over 170 bacterial and archaeal genomes and over 40 eukaryotic genomes.     

The alpha/beta fold family of proteins database and the cholinesterase gene server ESTHER.

ESTHER (for esterases, alpha/betahydrolase enzyme and relatives) is a database of sequences phylogenetically related to cholinesterases. These sequences define a homogeneous group of enzymes (carboxylesterases, lipases and hormone-sensitive lipases) sharing a similar structure of a central beta-sheet surrounded by alpha-helices. Among these proteins a wide range of functions can be found (hydrolases, adhesion molecules, hormone precursors). The purpose of ESTHER is to help comparison of structures and functions of members of the family. Since the last release, new features have been added to the server. A BLAST comparison tool allows sequence homology searches within the database sequences. New sections are available: kinetics and inhibitors of cholinesterases, fasciculin-acetylcholinesterase interaction and a gene structure review. The mutation analysis compilation has been improved with three-dimensional images. A mailing list has been created.     

PISCES: a protein sequence culling server

PISCES is a public server for culling sets of protein sequences from the Protein Data Bank (PDB) by sequence identity and structural quality criteria. PISCES can provide lists culled from the entire PDB or from lists of PDB entries or chains provided by the user. The sequence identities are obtained from PSI-BLAST alignments with position-specific substitution matrices derived from the non-redundant protein sequence database. PISCES therefore provides better lists than servers that use BLAST, which is unable to identify many relationships below 40% sequence identity and often overestimates sequence identity by aligning only well-conserved fragments. PDB sequences are updated weekly. PISCES can also cull non-PDB sequences provided by the user as a list of GenBank identifiers, a FASTA format file, or BLAST/PSI-BLAST output.     

Identification and mechanism of evolution of new alleles coding for the AIDA-I autotransporter of porcine pathogenic Escherichia coli

Autotransporters are a large family of virulence factors of Gram-negative bacterial pathogens. The autotransporter adhesin involved in diffuse adherence (AIDA-I) is an outer membrane protein of Escherichia coli, which allows binding to epithelial cells as well as the autoaggregation of bacteria. AIDA-I is glycosylated by a specific heptosyltransferase encoded by the aah gene that forms an operon with the aidA gene. aidA is highly prevalent in strains that cause disease in pigs. Nevertheless, there are only two published whole-length sequences for this gene. In this study, we sequenced the aah and aidA genes of 24 aidA-positive porcine strains harboring distinct virulence factor profiles. We compared the obtained sequences and performed phylogenetic and pulsed-field electrophoresis analyses. Our results suggest that there are at least 3 different alleles for aidA, which are associated with distinct virulence factor profiles. The genes are found on high-molecular-weight plasmids and seem to evolve via shuffling mechanisms, with one of the sequences showing evidence of genetic recombination. Our work suggests that genetic plasticity allows the evolution of aah-aidA alleles that are selected during pathogenesis.     

Identifying coding exons by similarity search: alu-derived and other potentially misleading protein sequences.

The search for significant local similarities with known protein sequences is a powerful method for interpreting anonymous cDNA sequences or locating coding exons within genomic DNA sequences at a stage where the average contig size is still very small. The BLASTx program, implemented on the National Center for Biotechnology Information server, allows a sensitive search of all putative translations of a nucleotide query sequence against all known proteins in a matter of seconds. From an analysis of the current databases, I report a set of protein sequences exhibiting high local similarity to Alu repeat or vector sequences. These entries can lead to misleading interpretations of similarity searches. During the course of this study, the protease of a human spumaretrovirus was found to have integrated the 3' end half of the U2 snRNA.     

AliasServer: a web server to handle multiple aliases used to refer to proteins

SUMMARY: AliasServer provides services that facilitate the assembly of data or datasets that make use of different identifiers for refering to the same protein. This resource relies on a database which contains, for a given organism, a non-redundant list of protein sequences associated with a set of aliases. AVAILABILITY: AliasServer is available as an interactive Web server at http://cbi.labri.fr/outils/alias/ and as a web service using a SOAP interface. The complete tool, including sources and data, is available for local installations upon request. SUPPLEMENTARY INFORMATION: Technical documentation is available at http://cbi.labri.fr/outils/alias/asdoc.pdf     

SLAM web server for comparative gene finding and alignment

SLAM is a program that simultaneously aligns and annotates pairs of homologous sequences. The SLAM web server integrates SLAM with repeat masking tools and the AVID alignment program to allow for rapid alignment and gene prediction in user submitted sequences. Along with annotations and alignments for the submitted sequences, users obtain a list of predicted conserved non-coding sequences (and their associated alignments). The web site also links to whole genome annotations of the human, mouse and rat genomes produced with the SLAM program. The server can be accessed at http://bio.math.berkeley.edu/slam.     

Plant codon optimized cry genes of Bacillus thuringiensis can be expressed as soluble proteins in Escherichia coli BL21 Codon Plus strain as NusA-Cry protein fusions

For optimal expression of delta-endotoxins from Bacillus thuringiensis in plants, preferential changes in the codon sequences, and reduction in overall AT content in the nucleotide sequence of the genes is important. Reports suggest that sequences with such modifications cannot be overexpressed in bacteria. We report here that the modified genes can be overexpressed in a strain of Escherichia coli carrying extra tRNA genes for some of the codons occurring at high frequency in plant genes and less preferred in E. coli. We also demonstrate that proteins when expressed as fusion products with NusA protein, are obtained as soluble fraction rather than in inclusion bodies. This allows easy and accurate LC50 analysis on insect pests.     

Identifying coding exons by similarity search: Alu-derived and other potentially misleading protein sequences

The search for significant local similarities with known protein sequences is a powerful method for interpreting anonymous cDNA sequences or locating coding exons within genomic DNA sequences at a stage where the average contig size is still very small. The BLASTx program, implemented on the National Center for Biotechnology Information server, allows a sensitive search of all putative translations of a nucleotide query sequence against all known proteins in a matter of seconds. From an analysis of the current databases, I report a set of protein sequences exhibiting high local similarity to Alu repeat or vector sequences. These entries can lead to misleading interpretations of similarity searches. During the course of this study, the protease of a human spumaretrovirus was found to have integrated the 3′ end half of the U2 snRNA.     

PRISE (PRImer SElector): software for designing sequence-selective PCR primers

This report presents PRImer Selector (PRISE), a new software package that implements several features that improve and streamline the design of sequence-selective PCR primers. The PRISE design process involves two main steps. In the first step, target and non-target DNA sequences are identified. In the second step, primers are designed to amplify target (but not non-target) sequences. One important feature of PRISE is that it automates the task of placing primer-template mismatches at the 3' end of the primers - a property that is crucial for sequence selectivity. Once a list of candidate primers has been produced, sorting tools in PRISE speed up the selection process by allowing a user to sort the primers by properties such as amplicon length, GC content and sequence selectivity. PRISE can be used to design primers with a range of specificities, targeting individual sequences as well as diverse assemblages of genes. PRISE also allows user-defined primers to be analyzed, enabling their properties to be examined in relation to target and non-target sequences. The utility of PRISE was demonstrated by using it to design sequence-selective PCR primers for an rRNA gene from the fungus Pochonia chlamydosporia.