The dual transcriptional regulator CysR in Corynebacterium glutamicum ATCC 13032 controls a subset of genes of the McbR regulon in response to the availability of sulphide acceptor molecules

Background: Regulation of sulphur metabolism in Corynebacterium glutamicum ATCC 13032 has been studied intensively in the last few years, due to its industrial as well as scientific importance. Previously, the gene cg0156 was shown to belong to the regulon of McbR, a global transcriptional repressor of sulphur metabolism in C. glutamicum. This gene encodes a putative ROK-type regulator, a paralogue of the activator of sulphonate utilisation, SsuR. Therefore, it is an interesting candidate for study to further the understanding of the regulation of sulphur metabolism in C. glutamicum. Results: Deletion of cg0156, now designated cysR, results in the inability of the mutant to utilise sulphate and aliphatic sulphonates. DNA microarray hybridisations revealed 49 genes with significantly increased and 48 with decreased transcript levels in presence of the native CysR compared to a cysR deletion mutant. Among the genes positively controlled by CysR were the gene cluster involved in sulphate reduction, fpr2 cysIXHDNYZ, and ssuR. Gel retardation experiments demonstrated that binding of CysR to DNA depends in vitro on the presence of either O-acetyl-L-serine or O-acetyl-L-homoserine. Mapping of the transcription start points of five transcription units helped to identify a 10 bp inverted repeat as the possible CysR binding site. Subsequent in vivo tests proved this motif to be necessary for CysR-dependent transcriptional regulation. Conclusion: CysR acts as the functional analogue of the unrelated LysR-type regulator CysB from Escherichia coli, controlling sulphide production in response to acceptor availability. In both bacteria, gene duplication events seem to have taken place which resulted in the evolution of dedicated regulators for the control of sulphonate utilisation. The striking convergent evolution of network topology indicates the strong selective pressure to control the metabolism of the essential but often toxic sulphur-containing (bio-)molecules

RSAT: regulatory sequence analysis tools

The regulatory sequence analysis tools (RSAT, http://rsat.ulb.ac.be/rsat/) is a software suite that integrates a wide collection of modular tools for the detection of cis-regulatory elements in genome sequences. The suite includes programs for sequence retrieval, pattern discovery, phylogenetic footprint detection, pattern matching, genome scanning and feature map drawing. Random controls can be performed with random gene selections or by generating random sequences according to a variety of background models (Bernoulli, Markov). Beyond the original word-based pattern-discovery tools (oligo-analysis and dyad-analysis), we recently added a battery of tools for matrix-based detection of cis-acting elements, with some original features (adaptive background models, Markov-chain estimation of P-values) that do not exist in other matrix-based scanning tools. The web server offers an intuitive interface, where each program can be accessed either separately or connected to the other tools. In addition, the tools are now available as web services, enabling their integration in programmatic workflows. Genomes are regularly updated from various genome repositories (NCBI and EnsEMBL) and 682 organisms are currently supported. Since 1998, the tools have been used by several hundreds of researchers from all over the world. Several predictions made with RSAT were validated experimentally and published

Bioinformatic and Proteomic Investigation of Chloroplast Transit Peptide Motifs and Genesis

The eukaryotic mitochondrion was formed by the endosymbiotic association of an - proteobacterium and a primordial phagocytic eukaryote. A second, and later, endosymbiosis between the eukaryote and a cyanobacterium gave rise to the chloroplast of plants. Following each of these events most of the organellar DNA was exported to the nucleus. A system evolved wherein proteins produced on cytosolic ribosomes are targeted to organelle protein translocators by N-terminal targeting sequences. Protein sorting between the chloroplast and the mitochondrion in the plant cell by the general import pathways shows remarkable fidelity despite a lack of sequence conservation among transit peptides and pre-sequences and despite very little sequence difference between these two targeting peptides. There is evidence for a hydrophobic recognition motif in mitochondrial presequences, and a similar motif has been proposed for the chloroplast transit peptide. We have developed novel motif-finding methods and applied them to our own chloroplast proteome data and to literature mitochondrial data. We fail to find a hydrophobic motif that discriminates the chloroplast and the mitochondrion. Another little understood phenomenon of organelle protein trafficking is how the targeting sequence is acquired after transfer of organelle DNA to the nucleus. It has been hypothesized that the transit peptide is acquired by exon shuffling. We find no correlation of transit peptide lengths with exon boundaries. Furthermore, using highly expressed cyanobacterial proteins conserved in plants, we find that the transit peptide appears as likely to be attached within the primordial sequence as without, indicating a more stochastic process for the origin of the transit peptide

In silico identification of NF-kappaB-regulated genes in pancreatic beta-cells

BACKGROUND: Pancreatic beta-cells are the target of an autoimmune attack in type 1 diabetes mellitus (T1DM). This is mediated in part by cytokines, such as interleukin (IL)-1β and interferon (IFN)-γ. These cytokines modify the expression of hundreds of genes, leading to beta-cell dysfunction and death by apoptosis. Several of these cytokine-induced genes are potentially regulated by the IL-1β-activated transcription factor (TF) nuclear factor (NF)-κB, and previous studies by our group have shown that cytokine-induced NF-κB activation is pro-apoptotic in beta-cells. To identify NF-κB-regulated gene networks in beta-cells we presently used a discriminant analysis-based approach to predict NF-κB responding genes on the basis of putative regulatory elements. RESULTS: The performance of linear and quadratic discriminant analysis (LDA, QDA) in identifying NF-κB-responding genes was examined on a dataset of 240 positive and negative examples of NF-κB regulation, using stratified cross-validation with an internal leave-one-out cross-validation (LOOCV) loop for automated feature selection and noise reduction. LDA performed slightly better than QDA, achieving 61% sensitivity, 91% specificity and 87% positive predictive value, and allowing the identification of 231, 251 and 580 NF-κB putative target genes in insulin-producing INS-1E cells, primary rat beta-cells and human pancreatic islets, respectively. Predicted NF-κB targets had a significant enrichment in genes regulated by cytokines (IL-1β or IL-1β + IFN-γ) and double stranded RNA (dsRNA), as compared to genes not regulated by these NF-κB-dependent stimuli. We increased the confidence of the predictions by selecting only evolutionary stable genes, i.e. genes with homologs predicted as NF-κB targets in rat, mouse, human and chimpanzee. CONCLUSION: The present in silico analysis allowed us to identify novel regulatory targets of NF-κB using a supervised classification method based on putative binding motifs. This provides new insights into the gene networks regulating cytokine-induced beta-cell dysfunction and death

Engineering the pregnane X receptor and estrogen receptor alpha to bind novel small molecules using negative chemical complementation

Nuclear receptors are ligand-activated transcription factors that play significant roles in various biological processes within the body, such as cell development, hormone metabolism, reproduction, and cardiac function. As transcription factors, nuclear receptors are involved in many diseases, such as diabetes, cancer, and arthritis, resulting in approximately 10-15% of the pharmaceutical drugs presently on the market being targeted toward nuclear receptors. Structurally, nuclear receptors consist of a DNA-binding domain (DBD), responsible for binding specific sequences of DNA called response elements, fused to a ligand-binding domain (LBD) through a hinge region. The LBD binds a small molecule ligand. Upon ligand binding, the LBD changes to an active conformation leading to the recruitment of coactivator (CoAC) proteins and initiation of transcription. As a result of their involvement in disease, there is an emphasis on engineering nuclear receptors for applications in gene therapy, drug discovery and metabolic engineering.Ph.D.Committee Chair: Bahareh Azizi; Committee Chair: Donald Doyle; Committee Co-Chair: Andreas Bommarius; Committee Co-Chair: Loren Williams; Committee Member: Adegboyega Oyelere; Committee Member: Nick Hud; Committee Member: Sheldon Ma

Functional Roles of Nucleases in DNA Metabolism and Genome Stability

The work outlined in this dissertation focuses on two distinct areas that are important for genome stability. Both areas focus on DNA repair pathways that require the action of nucleases, specifically Exonuclease 5 and Ribonuclease H2. First, I describe the biochemical and molecular characterization of the novel Exonuclease 5 family of enzymes from S. cerevisiae, S. pombe, and humans. The Exo5 family consists of bi-directional single-strand DNA specific exonucleases that all contain an iron-sulfur cluster as a structural motif and all have various roles in DNA metabolism. In the Saccharomycetales order that includes the budding yeast, S. cerevisiae, Exo5 is a mitochondrial protein that is essential for mitochondrial genome maintenance. In an unrelated yeast species, Schizosaccharomyes pombe, Exo5 is important for both nuclear and mitochondrial DNA metabolism. The human ortholog is important for nuclear genome stability, and for DNA repair. The work outlined in Chapter II of this Dissertation establishes Exo5 as a protein that is important for DNA metabolism. The second area of study outlined in Chapters III and IV is related to the phenomenon of ribonucleotide incorporation into the genome by replicative polymerases, and these chapters focus on the enzymes that remove these noncanonical nucleotides. Ribonucleotides are incorporated into DNA by the replicative DNA polymerases at frequencies of about 2 per kb, which makes them by far the most abundant form of potential DNA damage in the cell. Their removal is essential for restoring a stable intact chromosome. In Chapter III, I present a complete biochemical reconstitution of the ribonucleotide excision repair (RER) pathway with enzymes purified from Saccharomyces cerevisiae. I highlight the requirement for RNase H2 in the process of RER and investigate the redundancies at different steps of repair. Also outlined in this dissertation is the dissection of the different functions of RNase H2 in RER and in the removal of RNA-loops in DNA, and implications for genome instability in human diseases that are affected for these activities. Chapter IV of this dissertation discusses work on an alternative pathway for ribonucleotide removal from the genome by Topoisomerase I. In S. cerevisiae, deletion of rnh201, the catalytic subunit of RNase H2, results in the persistence of ribonucleotides remain in the genome, which leads to ~100-fold increase in the frequency of 2-5 bp deletions at di-nucleotide repeat sequences. These deletions are dependent on topoisomerase I (Top1) activity. Here we present an in vitro reconstitution of the mechanism of Top1-dependent deletions at di-nucleotide repeat sequences and a mechanism for Top1-initiated removal of ribonucleotides outside of the context of these repeat sequences in S. cerevisiae. Top1 attack at a ribonucleotide leads to the formation of a 2\u27, 3\u27 cyclic phosphate terminated ssDNA nick, followed by subsequent formation of a Top1-cleavage complex (Top1-cc) upstream of the 2\u27, 3\u27 cyclic phosphate. If the ribonucleotide is in the context of a di-nucleotide repeat, there can be realignment of the DNA allowing for religation and release of Top1, leading to a 2-nucleotide deletion. If the ribonucleotide resides outside a repeat sequence, the realignment is not possible and a different pathway must repair the Top1-cc. Tdp1-dependent repair of Top1-cc requires prior proteolytic processing of the Top1-cc before it can be removed leaving a 3\u27-phosphate that can be removed by Tpp1, Apn1, or Apn2 forming a substrate suitable for repair by DNA polymerase δ, FEN1 and DNA ligase