The bacterial RNA binding protein Hfq is a key component for bacterial sRNA mediated riboregulation of mRNA expression. A kinetic and thermodynamic analysis of Hfq binding to its sRNA targets DsrA, RprA, and OxyS, and to its mRNA target rpoS was carried out. The ability of Hfq to significantly enhance the stability of the DsrA-rpoS and RprA-rpoS complex was demonstrated, and the entire untranslated leader region of rpoS was shown to be important for Hfq binding and in Hfq facilitated sRNA-mRNA duplex formation. Hfq was not shown to enhance OxyS binding to rpoS. DsrA and OxyS were shown to bind mostly to the proximal surface region of Hfq, while RprA bound to both proximal and distal surface regions. The rpoS leader region was shown to possess at least two distinct Hfq binding sites, with one site binding the proximal region and the other to the distal region of Hfq. These sites were shown to be important for Hfq to stimulate DsrA-rpoS binding. The outer-circumference region and the C-terminal tail of Hfq does not play a major role in binding DsrA, RprA, OxyS and rpoS, and in stimulating DsrA-rpoS binding. Evidence was obtained implicating Hfq to bind DsrA, RprA, OxyS, and oligo rA18 in a 1:1 protein to RNA stoichiometry. Binding properties of Hfq to E. coli genomic DNA were examined. Double stranded DNA was shown to bind mostly on the distal surface region and the C-terminal tail of Hfq with an affinity 10 fold less than Hfq targeted RNA. Single stranded DNA binds Hfq more tightly than double stranded DNA and binding seems to be sequence specific. Evidence indicates Hfq binds certain sequences of the E. coli genome.

Serratia sp. ATCC 39006 (S39006) is a Gram-negative bacterium that is virulent in plant (potato) and animal (Caenorhabditis elegans) models. It produces two secondary metabolite antibiotics, prodigiosin and a carbapenem, and the plant cell wall degrading exoenzymes, pectate lyase and cellulase. A complex regulatory network controls production of prodigiosin, including a quorum sensing (QS) system, and the role of post-transcriptional regulation was investigated. It was hypothesized that Hfq-dependent small regulatory RNAs (sRNAs) might also play a role. Hfq is an RNA chaperone involved in post-transcriptional regulation that plays a key role in stress response and virulence in other bacterial species. An S39006 ∆hfq mutant was constructed and in the mutants production of prodigiosin and carbapenem was abolished, while production of the QS molecule, butanoyl homoserine lactone (BHL), was unaffected. Using transcriptional fusions, it was found that Hfq regulated the QS response regulators, SmaR and CarR. Additionally, exoenzyme production and swimming motility were decreased in the ∆hfq mutant, and virulence was attenuated in potato and C. elegans. It was also shown that the phenotype of an hfq mutant is independent of its role in regulating the stationary phase sigma factor, rpoS. In order to define the complete regulon of Hfq and identify relevant potential sRNAs, deep sequencing of strand-specific cDNAs (RNA-seq) was used to analyse the whole transcriptome of S39006 WT and the ∆hfq mutant. The regulon of another post-transcriptional regulator, RsmA, also involved in regulating prodigiosin production, was investigated by performing RNA-seq on an rsmA mutant. Moreover, global changes in the proteome of the hfq mutant was analysed using an LC-MS/MS approach with isobaric tags for relative and absolute quantification (iTRAQ). This study confirms a role for Hfq in pathogenesis and the regulation of antibiotic production in S39006, and begins to provide a systems-level understanding of Hfq and RsmA regulation using a combination of transcriptomics and proteomics.

L'étude qu'on a entreprise a pour but d'analyser les facteurs de virulence des espèces "Classiques" et "nouvelles" de Brucella. Dans cette perspective, on a analysé les génomes des espèces récemment découvertes : Brucella inopinata BO1 et Brucella inopinata-like BO2, isolés pour la première fois de patients humains sans réservoir animal connu. On a découvert que ces deux espèces possèdent des profils de restriction uniques. De plus, BO2 possède deux chromosomes de taille identique, un profil jamais décrit pour une autre espèce de Brucella. L'analyse de la réplication intracellulaire de ces deux espèces révèle que BO2 ne se réplique pas dans les macrophages humains et murins alors que BO1 se réplique d'une façon similaire à Brucella suis 1330, ce qui confirme la potentielle implication de BO1 dans la pathogenèse chez l'homme. Sur un autre niveau d'analyse, on a été à la recherche de facteurs de virulence potentiels dans d'autres espèces de Brucella notamment Brucella microti et Brucella suis sur les niveaux génomique et post-transcriptionnel. Sur le niveau génomique, on a découvert que le système GAD (glutamate decarboxylase) confère une résistance à l'acidité à Brucella microti lors de son passage dans l'estomac. Sur le niveau post-transcriptionnel, on a isolé, séquencé et identifié les petits ARNs noncodant associés à la protéine chaperone Hfq, qui joue un rôle important dans la virulence de Brucella. / We have undertaken in this study a multidimensional analysis of the virulence factors of "Classical" and new "Brucella species". In this objective, we have analysed the genomes of newly described species Brucella inopinata BO1 and Brucella inopinata-like BO2 isolated for the first time from human patients with no known animal reservoir. We found that these two species have unique restriction profiles. In addition, BO2 has a unique chromosomal distribution with two chromosomes of the same size, never seen before in Brucella. Analysis of the intracellular replication of these strains reveals that BO2 is unable to replicate in neither human nor mouse macrophages while BO1 successfully entered and replicated as efficiently as Brucella suis 1330 confirming the potential virulence of this species for humans. On an other level of analysis, we looked for potential virulence factors in other Brucella species including Brucella microti and Brucella suis at the genomic and post-transcriptional level. At the genomic level we discovered that the glutamate decarboxylase system confers resistance to acidity to Brucella miroti during its transit in the stomach. On the post-transcriptional level, we isolated, sequenced and identified small noncoding RNAs associated to the chaperone protein Hfq, known to play a role in the virulence of Brucella.

Das phylogenetisch weit verbreitete RNA-bindende Protein Hfq ist an einer Vielzahl von Prozessen innerhalb des bakteriellen RNA-Metabolismus, insbesondere im Rahmen der post-transkriptionellen Genregulation durch kleine RNAs (sRNAs) beteiligt. Hfq-Proteine zählen zu der Familie der Sm- und Lsm-Proteine und zeichnen sich strukturell durch die funktionelle Ausbildung ringförmiger Homohexamere aus. Cyanobakterielle Orthologe zeigen gegenüber den gut untersuchten Hfq-Proteinen aus E. coli und anderen Proteobakterien eine schwache Sequenzkonservierung und bieten auch daher einen interessanten Ansatzpunkt für die Untersuchung riboregulatorischer Prozesse in diesen Organismen. In der vorliegenden Arbeit werden einleitende Untersuchungen zu Funktion und Struktur des orthologen Hfq-Proteins aus dem einzelligen Modell-Cyanobakterium Synechocystis sp. PCC 6803 vorgestellt. Die Inaktivierung des hfq-Gens (ssr3341) führte in diesem Organismus zum Verlust der phototaktischen Motilität. Mithilfe elektronenmikroskopischer Analysen konnte dieser Phänotyp auf das Fehlen von Typ IV Pili zurückgeführt werden. Microarray-Analysen wiesen in der deltahfq-Mutante für 31 Gene eine veränderte, in den meisten Fällen reduzierte Transkriptakkumulation nach. Am stärksten betroffenen waren Gene bzw. Operone, welche dem Regulon des cAMP-Rezeptorproteins Sycrp1 zugeordnet werden und zum Teil nachweislich an der Motilität von Synechocystis-Zellen beteiligt sind. Weitere vergleichende Expressionsanalysen identifizierten mithilfe eines speziellen Tiling-arrays ferner zwei „intergenisch“ kodierte potenzielle sRNAs, Hpr1 und Hpr3, deren Transkriptmengen signifikant von der hfq-Inaktivierung beeinflusst werden. Kristallstrukturdaten deuten zusammen mit den Ergebnissen aus in vitro-Bindungsstudien und genetischen Komplementierungsexperimenten - trotz starker Konservierung zentraler struktureller Charakteristika - neuartige biochemische und funktionelle Eigenschaften des Hfq-Proteins aus Synechocystis sp. PCC 6803 an. Funktionelle Implikationen werden im strukturellen und phylogenetischen Kontext diskutiert. / The phylogenetically conserved RNA binding protein Hfq is a key player in bacterial RNA metabolism, particularly with regard to sRNA-mediated post-transcriptional gene regulation. Hfq proteins belong to the well-conserved family of Sm- and Lsm proteins and are characterized by the formation of homo-hexameric ring-shaped structures. In comparison with well-studied Hfq proteins from E.coli and other proteobacteria the cyanobacterial orthologues show rather poor sequence conservation. Therefore, they provide a quite interesting background for analyzing riboregulatory processes in these organisms. In this work, the orthologous Hfq protein from the unicellular model cyanobacterium Synechocystis sp. PCC 6803 has been initially characterized on the functional and structural level. Insertional inactivation of the hfq gene (ssr3341) led to a non-phototactic phenotype that was due to the loss of type IV pili on the cell surface, as demonstrated by electron microscopy. Microarray analyses revealed a set of 31 genes with altered transcript levels in the knock-out mutant. Among the most strongly affected genes, there were members of two operons that had previously been shown to be involved in motility, controlled by the cAMP receptor protein Sycrp1. Further comparative transcriptional analyses using custom tiling arrays revealed two putative sRNAs (Hpr1 and Hpr3) from intergenic regions, whose transcript levels appeared to be significantly affected by hfq-inactivation. Structural analyses, genetic complementation as well as RNA-binding studies in vitro indicate that the Hfq orthologue from Synechocystis sp. PCC 6803 exhibits novel biochemical and functional properties, though retaining general structural features of its proteobacterial counterparts. Functional implications are discussed with regard to structural und phylogenetic considerations.

Metabolically versatile bacteria have evolved diverse strategies to adapt to different environmental niches and respond to fluctuating physico-chemical parameters. In order to survive in soil and water habitats, they employ specific and global regulatory circuits to integrate external and internal signals to counteract stress and optimise their energy status. One strategic endurance mechanism is the ability to choose the most energetically favourable carbon source amongst a number on offer. Pseudomonas putida strains possess large genomes that underlie much of their ability to use diverse carbon sources as growth substrates. Their metabolic potential is frequently expanded by possession of catabolic plasmids to include the ability to grow at the expense of seemingly obnoxious carbon sources such as phenols. However, this ability comes with a metabolic price tag. Carbon source repression is one of the main regulatory networks employed to subvert use of these expensive pathways in favour of alternative sources that provide a higher metabolic gain. This thesis identifies some of the key regulatory elements and factors used by P. putida to supress expression of plasmid-encoded enzymes for degradation of phenols until they are beneficial. I first present evidence for a newly identified DNA and RNA motif within the regulatory region of the gene encoding the master regulator of phenol catabolism – DmpR. The former of these motifs functions to decrease the number of transcripts originating from the dmpR promoter, while the latter mediates a regulatory checkpoint for translational repression by Crc – the carbon repression control protein of P. putida. The ability of Crc to form repressive riboprotein complexes with RNA is shown to be dependent on the RNA chaperone protein Hfq – a co-partnership demonstrated to be required for many previously identified Crc-targets implicated in hierarchical assimilation of different carbon sources in P. putida. Finally, I present evidence for a model in which Crc and Hfq co-target multiple RNA motifs to bring about a two-tiered regulation to subvert catabolism of phenols in the face of preferred substrates – one at the level of the regulator DmpR and another at the level of translation of the catabolic enzymes.

No description available.