ProOpDB: Prokaryotic Operon DataBase

The Prokaryotic Operon DataBase (ProOpDB, http://operons.ibt.unam.mx/OperonPredictor) constitutes one of the most precise and complete repositories of operon predictions now available. Using our novel and highly accurate operon identification algorithm, we have predicted the operon structures of more than 1200 prokaryotic genomes. ProOpDB offers diverse alternatives by which a set of operon predictions can be retrieved including: (i) organism name, (ii) metabolic pathways, as defined by the KEGG database, (iii) gene orthology, as defined by the COG database, (iv) conserved protein domains, as defined by the Pfam database, (v) reference gene and (vi) reference operon, among others. In order to limit the operon output to non-redundant organisms, ProOpDB offers an efficient method to select the most representative organisms based on a precompiled phylogenetic distances matrix. In addition, the ProOpDB operon predictions are used directly as the input data of our Gene Context Tool to visualize their genomic context and retrieve the sequence of their corresponding 5′ regulatory regions, as well as the nucleotide or amino acid sequences of their genes

Inferring modules of functionally interacting proteins using the Bond Energy Algorithm

<p>Abstract</p> <p>Background</p> <p>Non-homology based methods such as phylogenetic profiles are effective for predicting functional relationships between proteins with no considerable sequence or structure similarity. Those methods rely heavily on traditional similarity metrics defined on pairs of phylogenetic patterns. Proteins do not exclusively interact in pairs as the final biological function of a protein in the cellular context is often hold by a group of proteins. In order to accurately infer modules of functionally interacting proteins, the consideration of not only direct but also indirect relationships is required.</p> <p>In this paper, we used the Bond Energy Algorithm (<it>BEA</it>) to predict functionally related groups of proteins. With <it>BEA </it>we create clusters of phylogenetic profiles based on the associations of the surrounding elements of the analyzed data using a metric that considers linked relationships among elements in the data set.</p> <p>Results</p> <p>Using phylogenetic profiles obtained from the Cluster of Orthologous Groups of Proteins (<it>COG</it>) database, we conducted a series of clustering experiments using <it>BEA </it>to predict (upper level) relationships between profiles. We evaluated our results by comparing with <it>COG's </it>functional categories, And even more, with the experimentally determined functional relationships between proteins provided by the <it>DIP </it>and <it>ECOCYC </it>databases. Our results demonstrate that <it>BEA </it>is capable of predicting meaningful modules of functionally related proteins. <it>BEA </it>outperforms traditionally used clustering methods, such as <it>k</it>-means and hierarchical clustering by predicting functional relationships between proteins with higher accuracy.</p> <p>Conclusion</p> <p>This study shows that the linked relationships of phylogenetic profiles obtained by <it>BEA </it>is useful for detecting functional associations between profiles and extending functional modules not found by traditional methods. <it>BEA </it>is capable of detecting relationship among phylogenetic patterns by linking them through a common element shared in a group. Additionally, we discuss how the proposed method may become more powerful if other criteria to classify different levels of protein functional interactions, as gene neighborhood or protein fusion information, is provided.</p

A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum

Petri K, Walter F, Persicke M, Rückert C, Kalinowski J. A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum. BMC Genomics. 2013;14(1): 713.BACKGROUND: Arginine biosynthesis in Corynebacterium glutamicum consists of eight enzymatic steps, starting with acetylation of glutamate, catalysed by N-acetylglutamate synthase (NAGS). There are different kinds of known NAGSs, for example, "classical" ArgA, bifunctional ArgJ, ArgO, and S-NAGS. However, since C. glutamicum possesses a monofunctional ArgJ, which catalyses only the fifth step of the arginine biosynthesis pathway, glutamate must be acetylated by an as of yet unknown NAGS gene. RESULTS: Arginine biosynthesis was investigated by metabolome profiling using defined gene deletion mutants that were expected to accumulate corresponding intracellular metabolites. HPLC-ESI-qTOF analyses gave detailed insights into arginine metabolism by detecting six out of seven intermediates of arginine biosynthesis. Accumulation of N-acetylglutamate in all mutants was a further confirmation of the unknown NAGS activity. To elucidate the identity of this gene, a genomic library of C. glutamicum was created and used to complement an Escherichia coli DeltaargA mutant. The plasmid identified, which allowed functional complementation, contained part of gene cg3035, which contains an acetyltransferase domain in its amino acid sequence. Deletion of cg3035 in the C. glutamicum genome led to a partial auxotrophy for arginine. Heterologous overexpression of the entire cg3035 gene verified its ability to complement the E. coli DeltaargA mutant in vivo and homologous overexpression led to a significantly higher intracellular N-acetylglutamate pool. Enzyme assays confirmed the N-acetylglutamate synthase activity of Cg3035 in vitro. However, the amino acid sequence of Cg3035 revealed no similarities to members of known NAGS gene families. CONCLUSIONS: The N-acetylglutamate synthase Cg3035 is able to catalyse the first step of arginine biosynthesis in C. glutamicum. It represents a novel class of NAGS genes apparently present only in bacteria of the suborder Corynebacterineae, comprising amongst others the genera Corynebacterium, Mycobacterium, and Nocardia. Therefore, the name C-NAGS (Corynebacterineae-type NAGS) is proposed for this new family

Short and long-term genome stability analysis of prokaryotic genomes.

BACKGROUND: Gene organization dynamics is actively studied because it provides useful evolutionary information, makes functional annotation easier and often enables to characterize pathogens. There is therefore a strong interest in understanding the variability of this trait and the possible correlations with life-style. Two kinds of events affect genome organization: on one hand translocations and recombinations change the relative position of genes shared by two genomes (i.e. the backbone gene order); on the other, insertions and deletions leave the backbone gene order unchanged but they alter the gene neighborhoods by breaking the syntenic regions. A complete picture about genome organization evolution therefore requires to account for both kinds of events. RESULTS: We developed an approach where we model chromosomes as graphs on which we compute different stability estimators; we consider genome rearrangements as well as the effect of gene insertions and deletions. In a first part of the paper, we fit a measure of backbone gene order conservation (hereinafter called backbone stability) against phylogenetic distance for over 3000 genome comparisons, improving existing models for the divergence in time of backbone stability. Intra- and inter-specific comparisons were treated separately to focus on different time-scales. The use of multiple genomes of a same species allowed to identify genomes with diverging gene order with respect to their conspecific. The inter-species analysis indicates that pathogens are more often unstable with respect to non-pathogens. In a second part of the text, we show that in pathogens, gene content dynamics (insertions and deletions) have a much more dramatic effect on genome organization stability than backbone rearrangements. CONCLUSION: In this work, we studied genome organization divergence taking into account the contribution of both genome order rearrangements and genome content dynamics. By studying species with multiple sequenced genomes available, we were able to explore genome organization stability at different time-scales and to find significant differences for pathogen and non-pathogen species. The output of our framework also allows to identify the conserved gene clusters and/or partial occurrences thereof, making possible to explore how gene clusters assembled during evolution.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803

Although the biogeochemistry of the two environmentally hazardous compounds arsenic and sulfide has been extensively investigated, the biological interference of these two toxic but potentially energy-rich compounds has only been hypothesized and indirectly proven. Here we provide direct evidence for the first time that in the photosynthetic model organism Synechocystis sp. strain PCC6803 the two metabolic pathways are linked by coregulated genes that are involved in arsenic transport, sulfide oxidation, and probably in sulfide-based alternative photosynthesis. Although Synechocystis sp. strain PCC6803 is an obligate photoautotrophic cyanobacterium that grows via oxygenic photosynthesis, we discovered that specific genes are activated in the presence of sulfide or arsenite to exploit the energy potentials of these chemicals. These genes form an operon that we termed suoRSCT, located on a transposable element of type IS4 on the plasmid pSYSM of the cyanobacterium. suoS (sll5036) encodes a light-dependent, type I sulfide:quinone oxidoreductase. The suoR (sll5035) gene downstream of suoS encodes a regulatory protein that belongs to the ArsR-type repressors that are normally involved in arsenic resistance. We found that this repressor has dual specificity, resulting in 200-fold induction of the operon upon either arsenite or sulfide exposure. The suoT gene encodes a transmembrane protein similar to chromate transporters but in fact functioning as an arsenite importer at permissive concentrations. We propose that the proteins encoded by the suoRSCT operon might have played an important role under anaerobic, reducing conditions on primordial Earth and that the operon was acquired by the cyanobacterium via horizontal gene transfer

Untersuchung des Argininmetabolismus und dessen Regulation in Corynebacterium glutamicum ATCC 13032

Haasner K. Untersuchung des Argininmetabolismus und dessen Regulation in Corynebacterium glutamicum ATCC 13032. Bielefeld: Universitätsbibliothek Bielefeld; 2013.Arginin ist eine semi-essentielle Aminosäure und aufgrund der vielfältigen Anwendungsmöglichkeiten von großem wirtschaftlichem Interesse. In Corynebacterium glutamicum, einem industriell bedeutsamen Produzenten von Aminosäuren, gibt es ein Argininoperon, welches aus den Genen argCJBDFRGH besteht und dessen Transkription ausgehend von einem Promotor stromaufwärts von argC durch ein leaderless Transkript initiiert wird. Die Argininbiosynthese besteht aus insgesamt acht aufeinanderfolgenden enzymatischen Schritten, und beginnt mit der Acetylierung von Glutamat. Reguliert wird die Biosynthese zum einen durch den transkriptionellen Regulator ArgR, der als Repressor wirkt und u.a. im Promotorbereich von argC bindet, und zum anderen durch eine feedback Inhibition von ArgB, dem zweiten Enzym der Argininbiosynthese. Durch Northern Blots sowie durch die RNAseq-Daten konnte ein zweiter Promotor verifiziert werden, der den Transkriptionsstart eines argGH-Suboperons kennzeichnet. Überdies zeigten die Northern Blots, dass es viele Prozessierungsstellen innerhalb des arg-Operons gibt, die wahrscheinlich durch Endoribonukleasen oder andere RNA-modifizierende Enzyme gespalten werden. Die RNAseq-Daten offenbarten die Existenz einer bislang unbekannten antisense RNA (asRNA) gegenüber dem 5’ Ende von argC, die AsaC genannt wurde. Transkriptionelle Analysen mit einer ΔasaC Mutante zeigten, dass die Transkription von argC negativ durch die asRNA beeinflusst wird und dass der Wirkmechanismus von AsaC transkriptionelle Interferenz ist. Aus diesen Ergebnissen konnte die biologische Bedeutung von AsaC abgeleitet werden: AsaC fungiert als zusätzlicher transkriptioneller Regulationsmechanismus der Argininbiosynthese und inhibiert die zufälligen Transkriptionsinitiationen am argC-Promotor, zu denen es kommt, wenn die RNA-Polymerase trotz der Bindung von ArgR sporadischen Zugang zur Promotorregion erhält. Bei inaktivem ArgR wird die transkriptionelle Interferenz jedoch umgekehrt. Somit ist AsaC nicht das dominierende regulatorische Element, jedoch bewirkt es durch seine Funktion als transkriptioneller Rauschunterdrücker eine Energieeinsparung für die Zelle. In einem weiteren Teil dieser Arbeit wurde die Argininbiosynthese von C. glutamicum mittels Metabolom-profiling von definierten Deletionmutanten, die die zu erwartenden korrespondierenden intrazellulären Metabolite anstauen sollten, analysiert. Die Ergebnisse der HPLC-ESI-qTOF-Messungen gaben einen detaillierten Einblick in den Argininmetabolismus, indem sechs von sieben Intermediaten der Argininbiosynthese detektiert werden konnten. Die Akkumulation von N-Acetylglutamat in allen Mutanten war dabei eine Bestätigung der bislang unbekannten NAGS-Aktivität, die für den ersten Schritt der Argininbiosynthese benötigt wird. Überdies konnte durch diese Ergebnisse herausgefunden werden, dass das von einer anderen Gruppe vorgeschlagene NAGS-Gen cg1722 nicht fähig ist, diese Reaktion auszuführen. Aus anderen Organismen sind verschiedene Arten von NAGS-Genen bekannt, z.B. gibt es das “klassische” ArgA, das bifunktionelle ArgJ, ArgO und S-NAGS. Um das entsprechende Gen in C. glutamicum zu identifizieren, wurde eine genomische Bibliothek erzeugt und für die Komplementation einer E. coli ΔargA Mutante verwendet. Das komplementierende Plasmid enthielt einen Teil des Gens cg3035, dessen Protein eine Acetyltransferase-Domäne aufweist. Eingehende Untersuchungen zeigten, dass eine Deletion von cg3035 im C. glutamicum Genom zu einer partiellen Auxotrophie für Arginin führt. Weiterhin verifizierte eine heterologe Überexpression des kompletten Cg3035 in vivo dessen Fähigkeit eine E. coli ΔargA Mutante zu komplementieren und eine homologe Überexpression führte zu einer signifikanten Erhöhung des intrazellulären N-Acetylglutamatpools. Zusätzlich bestätigten Enzymassays dessen N-Acetylglutamat Synthase-Funktion in vitro. Die Aminosäure-Sequenz von Cg3035 besitzt jedoch keine Ähnlichkeit zu Mitgliedern bekannter NAGS-Genfamilien, so dass Cg3035 eine neue Klasse von NAGS-Genen etabliert. Da Vertreter dieser neuen Klasse nur in Bakterien der Unterordnung Corynebacterineae gefunden wurden, wird der Name C-NAGS (Corynebacterineae NAGS) für diese Familie empfohlen

Role of a novel iron transport system in Brucella pathogenicity and intracellular survival

The mbfA (membrane bound ferritin A) gene is carried by all Brucella species and is widely conserved in α-Proteobacteria. mbfA encodes a novel iron-export protein comprising an N-terminal peroxide reductase (erythrin) domain and a C-terminal, membrane-embedded, iron-export domain (vacuolar iron transport, VIT). In this thesis, for the first time, the role of mbfA in Brucella suis 1330 and Brucella melitensis 16M was investigated, and the contributions of the two domains towards a novel mechanism of oxidative-stress resistance was considered. In addition, any part played by MbfA in enhancing Brucella survival within the macrophage ‘Brucella-containing vacuole’ (BCV) was investigated. The isolated erythrin domain was shown to bind two iron atoms (as expected) or zinc which stabilized the protein, but no substantive peroxidase activity was exhibited, presumably due to lack of a reduction partner. mbfA complemented an E. coli mutant that was devoid of catalases/alkyl-hydroperoxidases, enhanced growth of a fur mutant with deregulated iron uptake, and impaired growth of a mutant lacking iron-uptake capacity. These findings support a role for MbfA in peroxide resistance and iron export. MbfA was shown to export 55Fe when expressed in E. coli, and this activity was found to be O2 and H2O2 dependent. Similar results were obtained using a Burkholderia multivorans mbfA mutant. In addition, MbfA mediated the decomposition of exogenous H2O2, which was mainly achieved by degradation of exogenous peroxide by exported iron. MbfA provided peroxide resistance in wildtype E. coli but only when endogenous catalases/peroxidases were inhibited by NO. Thus, MbfA is not markedly NO inhibited. MbfA iron export was found to be partly dependent on the presence of iron stores, in the form of bacterioferritin. A naturally-occurring single-nucleotide substitution in the proximal coding region of the mbfA gene renders this gene cryptic in B. melitensis (although mbfA appears functional in all other Brucella spp.). Thus, a B. melitensis mbfA mutant exhibited no observable phenotype whereas a B. suis mbfA mutant displayed enhanced sensitivity to both Fe2+- and H2O2-mediated oxidative stress. It is hypothesized that MbfA exports ferric iron into the luminal space of the endosomal Brucella-containing vacuole (eBCV) to counteract both the respiratory burst generated by NADPH oxidase as well as the export of iron into the macrophage cytoplasm by NRAMP1. Exported iron causes disproportionation of peroxide externally, thus reducing the potential for harmful oxygen- and nitrogen-radical formation within the bacterial cell. Macrophage survival assays show that the absence of functional mbfA decreases intracellular survival during the early colonization phase in the eBCV, suggesting a reduced capacity to resist the redox stress of the endosomal and transient lysosomal compartments in the macrophage, which precede transition to the replicative BCV. Additionally, in vivo, mbfA contributes significantly to the maintenance of chronic infection within the murine liver and spleen. Further, MbfA provided a greater advantage in NRAMP1+ macrophages, than in NRAMP1- macrophages. This finding supports a role for MbfA in countering the iron export role of NRAMP1 within the phagolysosome, such that the purpose of this NRAMP1 actiy would be to preserve peroxide against iron-catalysed degradation. In summary, the results are therefore consistent with a role for mbfA in intracellular survival and redox stress resistance in Brucella