RESUMEN
Biofilms of the Gram-negative bacterium Pseudomonas aeruginosa are one of the major causes of complicated urinary tract infections with detrimental outcome. To develop novel therapeutic strategies the molecular adaption strategies of P. aeruginosa biofilms to the conditions of the urinary tract were investigated thoroughly at the systems level using transcriptome, proteome, metabolome and enzyme activity analyses. For this purpose biofilms were grown anaerobically in artificial urine medium (AUM). Obtained data were integrated bioinformatically into gene regulatory and metabolic networks. The dominating response at the transcriptome and proteome level was the adaptation to iron limitation via the broad Fur regulon including 19 sigma factors and up to 80 regulated target genes or operons. In agreement, reduction of the iron cofactor-dependent nitrate respiratory metabolism was detected. An adaptation of the central metabolism to lactate, citrate and amino acid as carbon sources with the induction of the glyoxylate bypass was observed, while other components of AUM like urea and creatinine were not used. Amino acid utilization pathways were found induced, while fatty acid biosynthesis was reduced. The high amounts of phosphate found in AUM explain the reduction of phosphate assimilation systems. Increased quorum sensing activity with the parallel reduction of chemotaxis and flagellum assembly underscored the importance of the biofilm life style. However, reduced formation of the extracellular polysaccharide alginate, typical for P. aeruginosa biofilms in lungs, indicated a different biofilm type for urinary tract infections. Furthermore, the obtained quorum sensing response results in an increased production of virulence factors like the extracellular lipase LipA and protease LasB and AprA explaining the harmful cause of these infections.
Asunto(s)
Adaptación Fisiológica , Biopelículas , Regulación Bacteriana de la Expresión Génica , Redes y Vías Metabólicas , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Infecciones Urinarias/microbiología , Alginatos/metabolismo , Aminoácidos Aromáticos/metabolismo , Metabolismo Energético , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/metabolismo , Hierro/metabolismo , Metaboloma , Proteoma , Percepción de Quorum , Estrés Fisiológico , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
UNLABELLED: In contrast to the rapidly increasing knowledge on genome content and diversity of bacterial viruses, insights in intracellular phage development and its impact on bacterial physiology are very limited. We present a multifaceted study combining quantitative PCR (qPCR), microarray, RNA-seq, and two-dimensional gel electrophoresis (2D-GE), to obtain a global overview of alterations in DNA, RNA, and protein content in Pseudomonas aeruginosa PAO1 cells upon infection with the strictly lytic phage LUZ19. Viral genome replication occurs in the second half of the phage infection cycle and coincides with degradation of the bacterial genome. At the RNA level, there is a sharp increase in viral mRNAs from 23 to 60% of all transcripts after 5 and 15 min of infection, respectively. Although microarray analysis revealed a complex pattern of bacterial up- and downregulated genes, the accumulation of viral mRNA clearly coincides with a general breakdown of abundant bacterial transcripts. Two-dimensional gel electrophoretic analyses shows no bacterial protein degradation during phage infection, and seven stress-related bacterial proteins appear. Moreover, the two most abundantly expressed early and late-early phage proteins, LUZ19 gene product 13 (Gp13) and Gp21, completely inhibit P. aeruginosa growth when expressed from a single-copy plasmid. Since Gp13 encodes a predicted GNAT acetyltransferase, this observation points at a crucial but yet unexplored level of posttranslational viral control during infection. IMPORTANCE: Massive genome sequencing has led to important insights into the enormous genetic diversity of bacterial viruses (bacteriophages). However, for nearly all known phages, information on the impact of the phage infection on host physiology and intracellular phage development is scarce. This aspect of phage research should be revitalized, as phages evolved genes which can shut down or redirect bacterial processes in a very efficient way, which can be exploited towards antibacterial design. In this context, we initiated a study of the human opportunistic pathogen Pseudomonas aeruginosa under attack by one its most common predators, the Phikmvlikevirus. By analyzing various stages of infection at different levels, this study uncovers new features of phage infection, representing a cornerstone for future studies on members of this phage genus.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Podoviridae/fisiología , Fagos Pseudomonas/fisiología , Pseudomonas aeruginosa/virología , Replicación Viral , Electroforesis en Gel Bidimensional , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The BRENDA (BRaunschweig ENzyme DAtabase) enzyme portal (http://www.brenda-enzymes.org) is the main information system of functional biochemical and molecular enzyme data and provides access to seven interconnected databases. BRENDA contains 2.7 million manually annotated data on enzyme occurrence, function, kinetics and molecular properties. Each entry is connected to a reference and the source organism. Enzyme ligands are stored with their structures and can be accessed via their names, synonyms or via a structure search. FRENDA (Full Reference ENzyme DAta) and AMENDA (Automatic Mining of ENzyme DAta) are based on text mining methods and represent a complete survey of PubMed abstracts with information on enzymes in different organisms, tissues or organelles. The supplemental database DRENDA provides more than 910 000 new EC number-disease relations in more than 510 000 references from automatic search and a classification of enzyme-disease-related information. KENDA (Kinetic ENzyme DAta), a new amendment extracts and displays kinetic values from PubMed abstracts. The integration of the EnzymeDetector offers an automatic comparison, evaluation and prediction of enzyme function annotations for prokaryotic genomes. The biochemical reaction database BKM-react contains non-redundant enzyme-catalysed and spontaneous reactions and was developed to facilitate and accelerate the construction of biochemical models.
Asunto(s)
Bases de Datos de Proteínas , Enzimas/química , Enzimas/metabolismo , Enfermedad , Enzimas/clasificación , Enzimas/genética , Internet , Cinética , LigandosRESUMEN
The bacterial phytochrome of Pseudomonas aeruginosa (PaBphP) is an in vitro-active red/far-red light sensor histidine kinase of a two-component regulatory system. Despite solid biochemical data, its function in this heterotrophic, opportunistic pathogen is still unknown. Previous studies established that the genes encoding the two necessary phytochrome components BphO, a chromophore-producing haem oxygenase, and BphP, the apo-phytochrome, are co-transcribed in a bicistronic operon. Transcription has been shown to be induced in the stationary phase and to be dependent on the alternative sigma factor RpoS. Here we show an additional regulation of bphP expression through the quorum-sensing (QS) regulator LasR. This regulation is also reflected in a combination of expression profile experiments and proteome analyses of wild-type and phytochrome-deficient strains. While PaBphP has a pleiotropic effect on global gene expression, 66â% of the downregulated genes in the phytochrome mutant display a link to the Las QS system. Most of these genes seem to be indirectly regulated by LasR through BphP and the unknown response regulator BphR. A model of phytochrome function within the Las QS network is presented.
Asunto(s)
Proteínas Bacterianas/metabolismo , Fitocromo/metabolismo , Pseudomonas aeruginosa/crecimiento & desarrollo , Percepción de Quorum , Factor sigma/metabolismo , Transactivadores/metabolismo , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Fitocromo/genética , Proteoma , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor sigma/genética , Transactivadores/genéticaRESUMEN
The BRENDA (BRaunschweig ENzyme Database, http://www.brenda-enzymes.org) enzyme information system is the main collection of enzyme functional and property data for the scientific community. The majority of the data are manually extracted from the primary literature. The content covers information on function, structure, occurrence, preparation and application of enzymes as well as properties of mutants and engineered variants. The number of manually annotated references increased by 30% to more than 100,000, the number of ligand structures by 45% to almost 100,000. New query, analysis and data management tools were implemented to improve data processing, data presentation, data input and data access. BRENDA now provides new viewing options such as the display of the statistics of functional parameters and the 3D view of protein sequence and structure features. Furthermore a ligand summary shows comprehensive information on the BRENDA ligands. The enzymes are linked to their respective pathways and can be viewed in pathway maps. The disease text mining part is strongly enhanced. It is possible to submit new, not yet classified enzymes to BRENDA, which then are reviewed and classified by the International Union of Biochemistry and Molecular Biology. A new SBML output format of BRENDA kinetic data allows the construction of organism-specific metabolic models.
Asunto(s)
Bases de Datos de Proteínas , Enzimas/química , Enzimas/genética , Enzimas/metabolismo , Ligandos , Redes y Vías Metabólicas , Conformación Proteica , Programas InformáticosRESUMEN
BTO, the BRENDA Tissue Ontology (http://www.BTO.brenda-enzymes.org) represents a comprehensive structured encyclopedia of tissue terms. The project started in 2003 to create a connection between the enzyme data collection of the BRENDA enzyme database and a structured network of source tissues and cell types. Currently, BTO contains more than 4600 different anatomical structures, tissues, cell types and cell lines, classified under generic categories corresponding to the rules and formats of the Gene Ontology Consortium and organized as a directed acyclic graph (DAG). Most of the terms are endowed with comments on their derivation or definitions. The content of the ontology is constantly curated with â¼1000 new terms each year. Four different types of relationships between the terms are implemented. A versatile web interface with several search and navigation functionalities allows convenient online access to the BTO and to the enzymes isolated from the tissues. Important areas of applications of the BTO terms are the detection of enzymes in tissues and the provision of a solid basis for text-mining approaches in this field. It is widely used by lab scientists, curators of genomic and biochemical databases and bioinformaticians. The BTO is freely available at http://www.obofoundry.org.
Asunto(s)
Bases de Datos Factuales , Enzimas , Vocabulario Controlado , Línea Celular , Células/clasificación , Células/enzimología , Diccionarios como Asunto , Integración de Sistemas , Interfaz Usuario-ComputadorRESUMEN
The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and biofilm formation during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network in response to oxygen tension and NO. Little is known about all members of the Anr and Dnr regulons and the mediated immediate response to oxygen depletion. Comprehensive transcriptome and bioinformatics analyses in combination with a limited proteome analyses were used for the investigation of the P. aeruginosa response to an immediate oxygen depletion and for definition of the corresponding Anr and Dnr regulons. We observed at first the activation of fermentative pathways for immediate energy generation followed by induction of alternative respiratory chains. A solid position weight matrix model was deduced from the experimentally identified Anr boxes and used for identification of 170 putative Anr boxes in potential P. aeruginosa promoter regions. The combination with the experimental data unambiguously identified 130 new members for the Anr and Dnr regulons. The basis for the understanding of two regulons of P. aeruginosa central to biofilm formation and infection is now defined.
Asunto(s)
Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , Pseudomonas aeruginosa/fisiología , Regulón , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Anaerobiosis , Proteínas Bacterianas/genética , Fermentación , Análisis por Micromatrices , Datos de Secuencia Molecular , Nitrato-Reductasa/metabolismo , Óxido Nítrico/metabolismo , Oxígeno/metabolismo , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
The BRENDA (BRaunschweig ENzyme DAtabase) (http://www.brenda-enzymes.org) represents the largest freely available information system containing a huge amount of biochemical and molecular information on all classified enzymes as well as software tools for querying the database and calculating molecular properties. The database covers information on classification and nomenclature, reaction and specificity, functional parameters, occurrence, enzyme structure and stability, mutants and enzyme engineering, preparation and isolation, the application of enzymes, and ligand-related data. The data in BRENDA are manually curated from more than 79,000 primary literature references. Each entry is clearly linked to a literature reference, the origin organism and, where available, to the protein sequence of the enzyme protein. A new search option provides the access to protein-specific data. FRENDA (Full Reference ENzyme DAta) and AMENDA (Automatic Mining of ENzyme DAta) are additional databases created by continuously improved text-mining procedures. These databases ought to provide a complete survey on enzyme data of the literature collection of PubMed. The web service via a SOAP (Simple Object Access Protocol) interface for access to the BRENDA data has been further enhanced.
Asunto(s)
Bases de Datos de Proteínas , Enzimas/química , Enzimas/fisiología , Enzimas/clasificación , Internet , PubMed , Programas InformáticosRESUMEN
To provide an integrated bioinformatics platform for a systems biology approach to the biology of pseudomonads in infection and biotechnology the database SYSTOMONAS (SYSTems biology of pseudOMONAS) was established. Besides our own experimental metabolome, proteome and transcriptome data, various additional predictions of cellular processes, such as gene-regulatory networks were stored. Reconstruction of metabolic networks in SYSTOMONAS was achieved via comparative genomics. Broad data integration is realized using SOAP interfaces for the well established databases BRENDA, KEGG and PRODORIC. Several tools for the analysis of stored data and for the visualization of the corresponding results are provided, enabling a quick understanding of metabolic pathways, genomic arrangements or promoter structures of interest. The focus of SYSTOMONAS is on pseudomonads and in particular Pseudomonas aeruginosa, an opportunistic human pathogen. With this database we would like to encourage the Pseudomonas community to elucidate cellular processes of interest using an integrated systems biology strategy. The database is accessible at http://www.systomonas.de.
Asunto(s)
Bases de Datos Genéticas , Pseudomonas/genética , Biología de Sistemas , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Sistemas de Administración de Bases de Datos , Redes Reguladoras de Genes , Genoma Bacteriano , Genómica , Internet , Redes y Vías Metabólicas , Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Integración de Sistemas , Interfaz Usuario-ComputadorRESUMEN
A novel program suite was implemented for the functional interpretation of high-throughput gene expression data based on the identification of Gene Ontology (GO) nodes. The focus of the analysis lies on the interpretation of microarray data from prokaryotes. The three well established statistical methods of the threshold value-based Fisher's exact test, as well as the threshold value-independent Kolmogorov-Smirnov and Student's t-test were employed in order to identify the groups of genes with a significantly altered expression profile. Furthermore, we provide the application of the rank-based unpaired Wilcoxon's test for a GO-based microarray data interpretation. Further features of the program include recognition of the alternative gene names and the correction for multiple testing. Obtained results are visualized interactively both as a table and as a GO subgraph including all significant nodes. Currently, JProGO enables the analysis of microarray data from more than 20 different prokaryotic species, including all important model organisms, and thus constitutes a useful web service for the microbial research community. JProGO is freely accessible via the web at the following address: http://www.jprogo.de.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Genes Arqueales , Genes Bacterianos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Programas Informáticos , Archaea/genética , Bacterias/genética , Gráficos por Computador , Interpretación Estadística de Datos , Internet , Interfaz Usuario-Computador , Vocabulario ControladoRESUMEN
SUMMARY: A new online framework for the accurate and integrative prediction of transcription factor binding sites (TFBSs) in prokaryotes was developed. The system consists of three interconnected modules: (1) The PRODORIC database as a comprehensive data source and extensive collection of TFBSs with corresponding position weight matrices. (2) The pattern matching tool Virtual Footprint for the prediction of genome based regulons and for the analysis of individual promoter regions. (3) The interactive genome browser GBPro for the visualization of TFBS search results in their genomic context and links to gene and regulator-specific information in PRODORIC. The aim of this service is to provide researchers a free and easy to use collection of interconnected tools in the field of molecular microbiology, infection and systems biology. AVAILABILITY: http://www.prodoric.de/vfp.
Asunto(s)
Biología Computacional/métodos , Bases de Datos de Proteínas , Algoritmos , Proteínas Bacterianas , Secuencia de Bases , Sitios de Unión , Bases de Datos Genéticas , Genes Bacterianos , Genoma , Genoma Humano , Genómica , Humanos , Almacenamiento y Recuperación de la Información , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Elementos de Respuesta , Sensibilidad y Especificidad , Programas Informáticos , Factores de Transcripción/genética , Interfaz Usuario-ComputadorRESUMEN
A novel method for the adaptation of target gene codon usage to most sequenced prokaryotes and selected eukaryotic gene expression hosts was developed to improve heterologous protein production. In contrast to existing tools, JCat (Java Codon Adaptation Tool) does not require the manual definition of highly expressed genes and is, therefore, a very rapid and easy method. Further options of JCat for codon adaptation include the avoidance of unwanted cleavage sites for restriction enzymes and Rho-independent transcription terminators. The output of JCat is both graphically and as Codon Adaptation Index (CAI) values given for the pasted sequence and the newly adapted sequence. Additionally, a list of genes in FASTA-format can be uploaded to calculate CAI values. In one example, all genes of the genome of Caenorhabditis elegans were adapted to Escherichia coli codon usage and further optimized to avoid commonly used restriction sites. In a second example, the Pseudomonas aeruginosa exbD gene codon usage was adapted to E.coli codon usage with parallel avoidance of the same restriction sites. For both, the degree of introduced changes was documented and evaluated. JCat is integrated into the PRODORIC database that hosts all required information on the various organisms to fulfill the requested calculations. JCat is freely accessible at http://www.prodoric.de/JCat.
Asunto(s)
Codón , Proteínas Recombinantes/genética , Programas Informáticos , Algoritmos , Animales , Caenorhabditis elegans/genética , Escherichia coli/genética , Expresión Génica , Internet , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/biosíntesis , Especificidad de la Especie , Interfaz Usuario-ComputadorRESUMEN
We have developed PrediSi (Prediction of Signal peptides), a new tool for predicting signal peptide sequences and their cleavage positions in bacterial and eukaryotic amino acid sequences. In contrast to previous prediction tools, our new software is especially useful for the analysis of large datasets in real time with high accuracy. PrediSi allows the evaluation of whole proteome datasets, which are currently accumulating as a result of numerous genome projects and proteomics experiments. The method employed is based on a position weight matrix approach improved by a frequency correction which takes in to consideration the amino acid bias present in proteins. The software was trained using sequences extracted from the most recent version of the SwissProt database. PrediSi is accessible via a web interface. An extra Java package was designed for the integration of PrediSi into other software projects. The tool is freely available on the World Wide Web at http://www.predisi.de.