Gene regulation in bacteria is highly complex and requires the activity of sigma factors that function as transcriptional regulators. In Pseudomonas syringae pv. syringae B728a, 14 sigma factors have been identified. One of the more interesting is rpoN, encoding Sigma 54, which was initially described for its role in nitrogen utilization and later shown to be involved in regulating adhesion, motility, toxin production, and pathogenicity. The only commonality identified amongst these genes is that gene regulation by Sigma 54 is not essential for normal growth and development because mutational inactivation of rpoN is not lethal. Unlike Sigma 70, which recognizes promoter sites located at positions -10/-35 upstream of the transcription initiation site, Sigma 54 recognizes sites located at positions -12/-24. P.s. pv. syringae B728a encodes an RpoN that shares 80-98% identity with other Pseudomonas species. Promoter scans were conducted on the B728a genome to look for probable binding sites of RpoN. Analysis revealed that RpoN may be involved in regulating genes encoding ABC transporters, drug efflux pumps, flagella proteins, nitrate transporters, and several regulatory proteins. An insertional mutation in the rpoN gene was constructed in the B728a genome and a phenotypic analysis was initiated. Decreased swarming and adhesion ability of the rpoN mutant was observed as compared to B728a. The ability to utilize sole nitrogen sources was also affected. The rpoN mutant showed little or no growth on sole nitrogen sources such as alanine, histidine, lysine, and serine. Pathogenicity was shown to require a functional RpoN, as both HR and disease development was effected by an rpoN mutation. Pseudomonas syringae pv. syringae is most known for the production of two phytotoxins. Unlike RpoN in other species, in P.s. pv. syringae B728a it appears to indirectly down regulate toxin production of syringomycin and syringopeptin. The goal of this study was to characterize some of the important roles RpoN is known to possess and to understand its role in the plant pathogenic and epiphytic lifestyle of P. s. pv. syringae B728a.

Doctor of Philosophy / Department of Biology / Lynn Hancock / Enterococci are the third leading cause of nosocomial infections including urinary tract infections (UTI), surgical site infections (SSI) and blood stream infections. Enterococci are also found in the gastrointestinal tracts of humans, and other mammals. We elucidated the influence of the transcriptional regulator RpoN on enterococcal biofilm formation, virulence potential and cell wall architecture and proposed a potential involvement for carbohydrate metabolism in these processes. Biofilms are held together by matrix (BM) components such as extracellular DNA (eDNA) released by cell death from a sub-population of cells. The rpoN mutant (ΔrpoN) was resistant to autolysis as well as fratricide-mediated cell death and eDNA was not detected in planktonic as well as biofilm cultures. Unlike the parental strain V583, the ΔrpoN mutant formed proteinase K sensitive biofilms, suggesting that protein as well as eDNA serves as an important matrix component. The rabbit model of endocarditis was used to assess the effect of rpoN deletion on enterococcal virulence. Rabbits infected with ΔrpoN had reduced bacterial burden in heart, blood, liver, kidney and vegetation in comparison to the parental strain. The growth defect of ΔrpoN in physiologically relevant glucose levels (5 mM) partially explains the reduced bacterial burdens observed in the virulence study. Microarray analysis of ΔrpoN showed that 10% of the genome is differentially regulated by RpoN. Deletion of rpoN also protects Enterococcus faecalis from lysis in the absence of known modulators of cellular lytic events such as O-acetylation and D-alanylation. Of the four identified enhancer binding proteins in E. faecalis, MptR regulates the RpoN-dependent mannose/glucose uptake system (MptABCD) and the ΔmptR mutant phenocopied the ΔrpoN mutant in the eDNA release and growth assays. Because MptC and MptD have been shown to be the cellular receptors for class IIa and IIc bacteriocins, we are presently testing the hypothesis that these receptors may serve as a global receptor for bacteriocins. In conclusion, our data demonstrates that alterations in the metabolic state of the bacterium, as observed in the ΔrpoN mutant could be responsible for the switch in biofilm matrix composition, and this switch in turn likely influences the virulence potential of the bacterium.

In enterohemorrhagic E. coli (EHEC) sigma factor N (σN) regulates glutamate-dependent acid resistance (GDAR) and the locus of enterocyte effacement (LEE), discrete genetic systems required for transmission and virulence of this intestinal pathogen. Regulation of these systems requires nitrogen regulatory protein C, NtrC, and is a consequence of NtrC/σN-dependent reduction in the activity of sigma factor S (σS). This study elucidates pathway components and stimuli for σN-directed regulation of GDAR and the LEE in EHEC. Deletion of fliZ, the product of which reduces σS activity, phenocopies rpoN (σN) and ntrC null strains for GDAR and LEE control, acid resistance and adherence. Upregulation of fliZ by NtrC/σN is indirect, requiring an intact flagellar regulator flhDC. Activation of flhDC by NtrC/σN and FlhDC-dependent regulation of GDAR and the LEE is dependent on σN-promoter flhDP2, and a newly described NtrC upstream activator sequence. While the addition of ammonium significantly alters GDAR and LEE expression, acid resistance and adherence, it does so independently of rpoN, ntrC and the NtrC sensor kinase ntrB. Altering the availability of NtrC phosphodonor acetyl phosphate by growth without glucose, with acetate addition, or by deletion of acetate kinase, ackA, abrogates NtrC/σN-dependent control of flhDC, fliZ, GDAR and LEE genes.

Chlamydia is a clinically significant organism that exhibits a unique stage-specific developmental cycle, involving the interconversion between two metabolically distinct forms. The completion of eight chlamydial genome sequences identified three different RNA polymerase sigma factor (ó) genes. Temporal gene expression analysis has predicted that each ó may play an integral role in controlling the development cycle. This thesis examines the role of the chlamydial alternate sigma factor, ó54 (rpoN) and the potential mechanism for the control the developmental cycle and disease pathogenesis. To achieve this, we searched the genome for putative ó54 promoters, validated the findings by DNA-binding assays, and examined the roles of the genes predicted to be regulated by ó54. This study applied a bioinformatics approach to search for additional ó54 regulated genes in C. trachomatis L2. A reduced consensus sequence (TGGCACnnnnnTTGC) identified two previously published ó54 promoter sequences upstream of CT652.1 and CT683. A modified consensus sequence (TGG-N9-TGC) was applied to the C. trachomatis D genome in Findpatterns yielded 512 potential targets of which 20 by virtue of sequence orientation and distance upstream of the predicted ORF start codon Primer extension analysis of total RNA isolated at 24 hours post-infection mapped the 5' RNA end upstream for acpS (CT100), yhf0_1 (CT258), SAM (CT404), lpxA (CT531), hypothetical proteins CT652.1 and CT683, and htrA (CT823) to the predicted ó54 promoters. Three candidates (CT291, CT404, and CT847) were mapped to putative ó70-like ó66 promoters. No transcript start sites were detected for the remaining ó54 promoter candidates. Two transcripts were detected from predicted ó66 and ó54 tandem promoters upstream of CT404. Primer extension analysis of the CT404 transcripts from RNA isolated at 4, 8, 12, 24 and 32 hours post-infection showed a decrease between 12 hours and 24 hours post-infection in transcripts thought to be generated from the predicted ó66 promoter. Transcripts from the predicted ó54 promoter were identified throughout development. Temporal gene expression profiles of the candidate genes with predicted ó54 promoters (CT652.1, CT683, CT100, CT258, CT531 and CT823) were resolved throughout the C. trachomatis L2 developmental cycle using real-time PCR. Transcripts for CT608 and CT609 were detected early in the cycle, while strong transcript levels were detected for CT258, CT531 and CT823 after the appearance of CT609 (rpoN). Low levels of CT652.1 and CT683 were measured, in the mid to late phase of the cycle, and transcripts for CT100 appeared at lower levels during the middle phase of the cycle. The functional assay of the predicted ó54 promoters required the generation of recombinant C. trachomatis L2 ó54 (rRpoN). The C. trachomatis rpoN was cloned into a bacterial expression system (pQE70) and the recombinant proteins purified for subsequent DNA mobility shift assays. Expression of rRpoN was hampered by low copy numbers, and unusual physical characteristics. DNA binding and mobility shift assays using rRpoN extracts against the chlamydial CT652.1 ó54 promoter, plus two characterised E. coli ó54 promoters (hypA and hycA), were successful if E. coli core RNA polymerase was added to the assay. All 20 candidates with predicted ó54 promoters were analysed with EMSA using rRpoN extract. The promoters upstream of CT100, CT223, CT258, CT322, CT652.1 and CT683 showed affinity towards the recombinant rRpoN-E. coli core RNA polymerase holoenzyme complex. Searches for potential chlamydial ó54 transcription initiation activators were made using the Multiple Em for Motif Elucidation (MEME) software, looking to identify the DNA binding motifs. The upstream promoter regions of CT100, CT223, CT258, CT322, CT531, CT652.1, CT683 and CT823 in C. trachomatis L2 and orthologs found in other species of Chlamydia were analysed. The software identified a near palindromic sequence upstream of CT100 orthologs in C. trachomatis D and C. trachomatis MoPn (CAACCCAAC and CACCACAAC) where as a CT531- and CT823-specific motif was also discovered (CCGTTGTAGAATCTC). It is beginning to emerge that ó54 may regulate the expression of proteins required for the formation of the cell wall. Since the expression of the ó54 transcript, rpoN, coincides with the morphological change from the non-infectious RB to the infectious EB, predictions could be made concerning which genes are potentially regulated by ó54.

Pseudomonas aeruginosa est une bactérie pathogène opportuniste extracellulaire. La souche PAO1 de P. aeruginosa possède trois loci codant des Systèmes de Sécrétion de Type VI (T6SS) indépendants, nommés H1 à H3-T6SS. Le premier d'entre eux, H1-T6SS, a été largement décrit dans la littérature scientifique et injecte des toxines à activité antiprocaryote dans d'autres bactéries permettant une défense active contre l'attaque d'un T6SS d'une autre bactérie. Les deux autres T6SS de P. aeruginosa sont probablement des facteurs de virulence de cette bactérie, mais aucun effecteur sécrété, ni cible intracellulaire ou rôle n'ont été proposés jusqu'ici. Dans ce travail de thèse, nous avons mis en évidence que la machinerie H2-T6SS permet l'invasion de cellules non-phagocytaires et participe à la virulence de P. aeruginosa. Cette entrée requière la dynamique du réseau de microtubules, via VgrG2b injectée par la machinerie H2-T6SS dans les cellules épithéliales, et capable de cibler les complexes de gamma-tubulines, centre nucléateur des microtubules. Il s'agit d'un mécanisme d'internalisation tout à fait original et jamais encore décrit. Nous avons également exploré la régulation croisée entre H2 et H3-T6SS. Enfin par une analyse de « RNAseq », nous avons observé entre les souches PAO1 et PA14 de P. aeruginosa une expression différentielle de nombreux gènes, certains codant des facteurs de virulence. Pour près de 30% d'entre eux, l'origine de cette variation reposerait sur une quasi-absence d'expression dans la souche PA14 de qslA, codant un anti-activateur du Quorum-Sensing. Ceci serait une nouvelle explication du phénotype hyper-virulent de la souche PA14 de P. aeruginosa. / Pseudomonas aeruginosa is an opportunistic extracellular pathogen. The PAO1 strain of P. aeruginosa harbors three loci encoding independent Type VI Secretion Systems (T6SS), named H1 to H3-T6SS. H1-T6SS has been widely described in the literature and injects toxins with antiprokaryotic activity in target bacteria, allowing an active defense against the attack of a T6SS from another bacterium. The other two T6SS of P. aeruginosa are likely virulence factors, but no secreted effector or intracellular target or role have been proposed so far.In this work, we demonstrated that the H2-T6SS machinery triggered the invasion of non-phagocytic epithelial cells and is involved in the virulence of P. aeruginosa. This uptake is mediated by the microtubule network dynamics, and via VgrG2b, injected by the H2-T6SS machinery in epithelial cells, to target the gamma-tubulin complex, the microtubule nucleating center. This is an original mechanism of internalization never described before. We also studied the cross-regulation between H2 and H3-T6SS. Finally through a « RNAseq » analysis, we observed differential expression of many genes, including some encoding virulence factors, between PAO1 and PA14 strains of P. aeruginosa. For nearly 30% of them, the origin of this variation is an almost lack of expression in the PA14 strain of qslA, encoding an anti-Quorum Sensing activator. This can be a new explanation of hyper-virulent phenotype of the PA14 strain of P. aeruginosa.

Extreme acid resistance contributes to the successful transmission of enterohemorrhagic E. coli (EHEC) through acidic food matrices and the stomach, allowing it to gain access to the intestine and elicit disease in humans. Alternative sigma factor N (σN, encoded by rpoN) was previously identified as a novel regulator of extreme acid resistance in EHEC. This study investigated the role for σN and co-expressed products of the rpoN operon in the acid resistance phenotype of EHEC. The results revealed that σN primarily controls acid resistance through repression of the glutamate-dependent acid resistance (GDAR) system through control of the σS-directed GadXW pathway. σN was also determined to repress additional acid resistance systems, including arginine-dependent acid resistance, and an anaerobic acid resistance mechanism. Two gene products of the rpoN operon, hpf and ptsN, were also determined to negatively affect GDAR, as well as expression of the σN dependent genes glnA, astA, and pspA. Mutation of hpf and ptsN did not however alter the transcription of rpoN. Transcript levels of rpoN operon genes were observed to be differential, and inconsistent with the hypothesis of expression as a single transcriptional unit. Together this data signifies the importance of rpoN operon genes in the negative regulation of extreme acid resistance systems, and suggests that the products of hpf and ptsN control the activity of σN at its promoters.

Im Fokus dieser Arbeit stand die Analyse von Faktoren, die den Zusammenbau des Flagellenapparates von Legionella pneumophila (Lp) regulieren. Mit einem kombinierten Replikations-/ Überlebensversuchs mit Lp Corby oder Lp Paris und ihren zugehörigen Regulationsmutanten wurde eine verminderte Fitness für dfliA und erstmals für drpoN, dfleQ defiziente Stämme nachgewiesen. Zur Validierung von Microarray-Daten aus Lp Paris wurden wachstumsphasenabhängige Transkriptions- und Translationsanalysen mit Lp Corby Wildtyp und drpoN, dfleQ, dfliA und dflaA defizienten Stämmen durchgeführt. Es wurde gezeigt, dass die basale Expression von fliA in den späteren Phasen unabhängig von RpoN und FleQ stattfindet. In dieser Arbeit konnte erstmals der Transkriptionsstartpunkt des Hauptregulators FliA bestimmt werden. Es zeigte sich eine putative RpoD (S70) Bindungsstelle. Ein Modell zur Regulation der fliA Expression wurde weiterentwickelt. Demnach kommt es in der exponentiellen Phase durch das Zusammenwirken von RpoD und DksA, aber unabhängig von FleQ, zur basalen fliA Promotoraktivität. Durch den Übergang in die transmissive Phase und direkte oder indirekte Interaktion mit FleQ sowie dem Alarmon ppGpp scheint es zu einem Austausch des Sigmafaktors S70 gegen SS und zu einer Aktivierung der fliA Expression zu kommen. Elektronenmikroskopische Studien zeigten, dass drpoN und dfleQ defiziente Mutanten wahrscheinlich aufgrund des fehlenden Basalkörpers nicht flagelliert sind. Mutanten für dfliA, dflaA und dfliD hatten ebenfalls keine Flagelle, zeigten aber eine ungewöhnliche, gerade Hook Struktur, die den Zusammenbau des Basalkörpers demonstriert. Weiterhin wurden durch in silico Studien 15 Legionella Spezies in Bezug auf das Flagellensystem und ein putatives Chemotaxissystem untersucht. So konnte L. oakridgensis als erste Art ohne beide Systeme sequenziert werden. Andererseits konnten mit LLAP12, L. bozemanii, L. gormanii und L. lytica Stämme beschrieben werden, die beide Systeme tragen. / This work focused on the analysis of factors contributing to the regulation of the flagellum self-assembly of Legionella pneumophila (Lp). With a combined replication/survival assay with Lp Corby or Lp Paris and their corresponding regulatory mutants a reduced fitness could be verified for dfliA and for the first time for drpoN, dfleQ deficient strains. For validation of microarray-data for Lp Paris with strain Lp Corby a growth phase dependent analysis of transcription and translation rates was done with wild-type and the drpoN, dfleQ, dfliA and dflaA deficient strains. A regulation of basal fliA expression independently from RpoN and FleQ was shown in the later growth phases. Furthermore the transcriptional start site of fliA could be shown for the first time. A RpoD (S70) binding site could be identified. According to a further developed model for the regulation of the fliA expression RpoD and DksA lead to a basal fliA promotor activity, independently from FleQ. Most likely, during transition to stationary phase, direct or indirect interaction with FleQ and the alarmone ppGpp results in the exchange of the sigma factor S70 and the binding of RpoS. This leads to the activation of fliA expression. Electron microscopic studies revealed that drpoN and dfleQ deficient mutants are not flagellated caused by the missing basal body. Mutants of dfliA, dflaA and dfliD were also aflagellated, but there was a uncommon straight hook structure visible which demonstrates a filament-independent assembly of the basal body. Furthermore, in silico analysis was done with 15 Legionella species with regard to the flagellum regulation system and a putative chemotaxis system. Analysis revealed that the strain L. oakridgensis is the first strain lacking both systems. On the other hand the strains LLAP12, L. bozemanii, L. gormanii and L. lytica could be characterized as strains carrying both systems.