The stationary phase-specific expression of many genes in free living bacteria such as Escherichia coli is controlled at the level of transcription by the alternative sigma factor RpoS encoded by the rpoS gene. This central regulator of E coli's stationary phase regulon (and several stress response regulons) is known to be required for the induction of over 30 proteins in stationary phase cultures and proteins induced in response to environmental stresses such as carbon starvation or osmotic upshift. To date, several RpoS-dependent genes have been identified in the literature. However, since no single inducer exists to which all members of the regulon respond, identifying RpoS-dependent genes based on phenotypic screening (e.g. carbon starvation inducible genes) methods may not provide us with a complete enumeration of the regulon. The present study is a continuation of previous work done with a previously-generated bank of 5,000 promoter- lacZ operon fusion mutants which were screened for RpoS-dependence by introducing an rpoS null allele into these strains and scoring for reduced ß-galactosidase activity. The identities of several of these RpoS-dependent promoter-lacZ fusions were determined by DNA sequencing and subsequent sequence analysis using the BLAST algorithm. The RpoS- and growth-phase-dependence of several of the genes identified in this study was verified by Northern blot analysis. The genes identified here fall in into three groups: (i) genes previously shown to be RpoS-dependent; (ii) genes of known function that werenot previously known to be RpoS-dependent; and (iii) ORFs not previously known to be RpoS-dependent. Expression and activity of RpoS itself is subject to regulation that occurs at the levels of transcription, translation, and protein stability. How these different levels of control interact to affect the activation of RpoS and the RpoS regulon is only partially elucidated. This study identifies BarA as the first two-component transcriptional regulator required for the activity of rpoS and provides evidence that signal(s) may be present in exponentially growing cultures that lead to early exponential phase stimulation of rpoS and subsequently, the RpoS regulon. An E coli strain with a mutation in barA exhibits a hydrogen peroxide sensitive phenotype resulting from reduced levels of HPI and HPII catalase (which is under the control of RpoS). The reduction in HPII activity is a result of a reduction in the levels of katE message (encoding HPII) in a harA strain. Western blots probed with anti-RpoS antisera and Northern blots probed with an rpoS- specific probe demonstrate that this deficiency for the HPII catalase is caused by a decrease in the levels of the regulator, RpoS, present in the harA strain. Northern analysis and promoter-lacZ fusion expression data provide evidence for a model of early exponential phase expression of the RpoS regulon. Signal(s) responsible for this induction may be present in early exponential phase cultures and may ultimately lead to RpoS-dependent gene expression in stationary phase. / Thesis / Master of Science (MSc)

Escherichia coli has served as a model organism for studies in molecular genetics and physiology since the 1960s. Yet the function of 20% of the 4288 known and predicted proteins in E. coli is still not known. Almost two-thirds of these proteins have homologs in other microorganisms, but their function(s) is not known in any organism. One such protein is YciG. YciG was chosen as the focus of this study because, intriguingly, an ortholog of YciG is found in the genome of the fungus Neurospora crassa. The gene encoding YciG is predicted to be in an operon with two other genes, yciF and yciE. Genes in the same operon often encode proteins with related functions, so the study was extended to include YciF and YciE. To determine the function of these proteins, in-frame deletion alleles were constructed and strains lacking one or more of the three proteins were tested for mutant phenotypes. Expression of the yciGFE operon is induced by several stresses and is regulated by RpoS, which controls the general stress response in E. coli. Therefore, we tested the ability of the mutant strains to survive environmental stresses. Our results revealed that YciG was important for stationary-phase resistance to thermal stress, oxidative stress and, in particular, acid stress. Both RpoS-dependent and RpoS-independent acid resistance mechanisms are found in E. coli. YciG was shown to be required for RpoS-independent acid resistance, but further experiments are needed to determine whether YciG also is required for RpoS-dependent acid resistance. YciG was not required for normal exponential growth of E. coli, as mutants lacking YciG had the same growth rate as the wild-type parent. No mutant phenotypes have been found yet for mutants lacking YciF or YciE. yciE deletion mutants showed the same growth rate and the same level of acid resistance as wild-type cells. The acid resistance of yciF mutants has not yet been tested, and strains lacking YciE and/or YciF need to be assayed for their ability to survive stresses other than acid stress.

Escherichia coli has served as a model organism for studies in molecular genetics and physiology since the 1960s. Yet the function of 20% of the 4288 known and predicted proteins in E. coli is still not known. Almost two-thirds of these proteins have homologs in other microorganisms, but their function(s) is not known in any organism. One such protein is YciG. YciG was chosen as the focus of this study because, intriguingly, an ortholog of YciG is found in the genome of the fungus Neurospora crassa. The gene encoding YciG is predicted to be in an operon with two other genes, yciF and yciE. Genes in the same operon often encode proteins with related functions, so the study was extended to include YciF and YciE. To determine the function of these proteins, in-frame deletion alleles were constructed and strains lacking one or more of the three proteins were tested for mutant phenotypes. Expression of the yciGFE operon is induced by several stresses and is regulated by RpoS, which controls the general stress response in E. coli. Therefore, we tested the ability of the mutant strains to survive environmental stresses. Our results revealed that YciG was important for stationary-phase resistance to thermal stress, oxidative stress and, in particular, acid stress. Both RpoS-dependent and RpoS-independent acid resistance mechanisms are found in E. coli. YciG was shown to be required for RpoS-independent acid resistance, but further experiments are needed to determine whether YciG also is required for RpoS-dependent acid resistance. YciG was not required for normal exponential growth of E. coli, as mutants lacking YciG had the same growth rate as the wild-type parent. No mutant phenotypes have been found yet for mutants lacking YciF or YciE. yciE deletion mutants showed the same growth rate and the same level of acid resistance as wild-type cells. The acid resistance of yciF mutants has not yet been tested, and strains lacking YciE and/or YciF need to be assayed for their ability to survive stresses other than acid stress.

During entry into stationary phase, Escherichia coli expresses many genes which impart cellular resistance to numerous environmental stresses such as oxidative or acid stress. Many ofthese genes are regulated by the alternative sigma factor, RpoS To identify additional genes regulated by RpoS, a phenotype independent genetic screen was previously employed (L. Wei Masters thesis). The identities ofthe ten most highly RpoSdependent fusions were determined by DNA sequencing and subsequent sequence analysis using the BLAST algorithm Three fusions map to genes previously known to be RpoSdependent while the remaining seven represent new members ofthe regulon The expression of many ofthe RpoS-dependent fusions remained growth phase dependent even in the rpoS background This suggests that other growth phase regulatory factors in addition to RpoS may coordinate stationary phase gene expression Upon sequencing the remaining rsd fusions, three mutants mapped to narY which is part ofthe narZYWV operon encoding the secondary nitrate reductase Z (NRZ) This operon was selected for further investigation since NRZ has been previously reported to be constitutively expressed Expression studies using promoter lacZ fusions and nitrate reductase assays reveal that NRZ is induced ten-fold at the onset ofstationary phase and twenty-fold in the presence of nitrate Like other rsd fusions, growth phase dependent expression was observed in an rpoS background indicating that other regulatory factors may be involvedn the regulation of NRZ Northern analyses using probes specific to NRZ confirmed that transcription of NRZ is indeed dependent on RpoS. These results suggest that RpoS mediated regulation of NRZ may be an important physiological adaptation to reduced oxygen levels during transition to stationary phase / Thesis / Master of Science (MSc)

This thesis is missing page 129. Other copies of this thesis do not have the page either. -Digitization Centre / E. coli expresses an alternative sigma factor, RpoS, in response to starvation and environmental stresses. RpoS is a global regulator and it controls numerous genes, which aids in counteracting these stresses. The RpoS regulon is large but is not completely characterized. We have previously identified over one hundred RpoS-dependent fusions in a genetic screen based on the differential expression of an operon-lacZ fusion bank in rpoS mutant and wild type backgrounds. Forty-eight independent gene fusions were identified including several in well-characterized RpoS-regulated genes such as osmY, katE and otsA. Many of the fusions mapped to genes of unknown function or to genes that were not previously known to be under RpoS control. Based on the homology to other known bacterial genes, some of the RpoS regulated genes with unknown functions may be important for nutrient scavenging. To gain a better insight into the functions of these poorly characterized genes, we tested the ability of the fusion mutants to utilize various carbon sources and to utilize individual amino acids as carbon and nitrogen sources. The results indicate that most of the strains in rpos-backgrounds exhibited better growth in succinate and fumarate and in several amino acids than did the corresponding strains in wild-type backgrounds. / Thesis / Master of Science (MS)

In der vorliegenden Arbeit wurde zunächst die Konservierung bekannter RpoS-assoziierter Funktionen für das V. cholerae Homolog untersucht. Dabei ergab die phänotypische Analyse der rpoS-Deletionsmutante, dass analog zu der Bedeutung als Regulator des Stationärphasen-Wachstums in E. coli, definierte Zelldichte-abhängige Eigenschaften in V. cholerae gleichermaßen der Kontrolle von RpoS unterliegen. In weiterführenden Experimenten konnte daraufhin die Konservierung der entsprechenden Promotorstrukturen über die funktionelle Komplementierung rpoS-abhängiger Gene durch das jeweils speziesfremde Protein aufgedeckt werden. Dahingegen konnte die Bedeutung von RpoS bei der Ausprägung der generellen Stress-Resistenz u. a. in E. coli für das V. cholerae Homolog über den gewählten experimentellen Ansatz nicht belegt werden. So wurden in Survival-Assays für keine der getesteten Stress-Bedingungen signifikante Unterschiede zwischen rpoS-Mutante und Wildtyp ermittelt. Die in E. coli gezeigte intrazelluläre Anreicherung des Sigmafaktors unter diversen Stress-Situationen konnte ebenfalls nicht nachgewiesen werden. Hinsichtlich der potentiellen Stellung von RpoS als globaler Regulator für Virulenz-assoziierte Gene, unterstützen und ergänzen die Ergebnisse der vorliegenden Arbeit die gegenwärtige Theorie, wonach RpoS das Ablösen der V. cholerae Zellen vom Darm-Epithel fördert. Die postulierte Bedeutung des alternativen Sigmafaktors in der letzten Phase der Pathogenese wurde über die RpoS-abhängige Sekretion der Mukin-degradierenden Protease HapA und die hier unabhängig nachgewiesene Transkriptionskontrolle von Chemotaxis-Genen bestätigt. In E. coli gilt als entscheidender Parameter für die dargelegten RpoS-Funktionen die intrazelluläre Konzentration des Masterregulators. Deshalb war ein weiteres zentrales Thema dieser Arbeit die Regulation des RpoS-Levels in V. cholerae. Neben der Identifizierung von Bedingungen, welche die RpoS-Expression beeinflussen, wurde vorrangig der Mechanismus der Proteolyse analysiert. Dabei wurden als RpoS-degradierende Komponenten in V. cholerae die Homologe des Proteolyse-Targetingfaktors RssB und des Protease-Komplexes ClpXP identifiziert. Die weitere Untersuchung der RpoS-Proteolyse ergab außerdem, dass bestimmte Stress-Signale den Abbau stark verzögern. Interessanterweise resultierten die gleichen Signale jedoch nicht in der Akkumulation von RpoS. Als weiterer Unterschied zu der bekannten Proteolysekaskade in E. coli zeigte sich, dass das V. cholerae Homolog der RssB-aktivierenden Kinase ArcB (FexB) an der RpoS-Proteolyse nicht beteiligt ist. Indessen deuten die Ergebnisse weiterführender Experimente auf den Einfluss der Kinasen CheA-1 und CheA-3 des V. cholerae Chemotaxis-Systems auf die RpoS-Degradation. Aus diesem Grund wurde in der vorliegenden Arbeit ein zu E. coli abweichendes Modell der RpoS-Proteolyse postuliert, in welchem die aktiven CheA-Kinasen den Targetingfaktor RssB phosphorylieren und somit den Abbau einleiten. Die Beteiligung von MCP-Rezeptoren an der Kontrolle der intrazellulären RpoS-Konzentration und damit an der Transkription der Chemotaxisgene selbst, beschreibt erstmalig ein Regulationssystem, wonach innerhalb der Chemotaxis-Kaskade die Rezeptoraktivität wahrscheinlich über einen positiven „Feedback-Loop“ mit der eigenen Gen-Expression gekoppelt ist. Darüber hinaus deutete sich die Beteiligung der ATP-abhängigen Protease Lon an der RpoS-Proteolyse-Kaskade in V. cholerae an. Die Inaktivierung der in E. coli unter Hitzeschock-Bedingungen induzierten Protease resultierte in einem extrem beschleunigten RpoS-Abbau. Ein letztes Teilprojekt dieser Arbeit adressierte die Regulationsmechanismen der V. cholerae Osmostress-Adaptation. Während in E. coli der alternative Sigmafaktor dabei eine zentrale Rolle spielt, konnte die Beteiligung des V. cholerae RpoS an der Osmostress-Regulation jedoch nicht aufgedeckt werden. Dafür ergab die Funktionsanalyse eines neu definierten Osmostress-Sensors (OsmRK) die Kontrolle von ompU durch dieses Zwei-Komponentensystems unter hypertonen Bedingungen. Dieses Ergebnis überraschte, da bislang nur der Virulenzfaktor ToxR als Regulator für das Außenmembranporin beschrieben wurde. Die nachgewiesene ompU-Transkriptionskontrolle durch zwei Regulatoren führte zu der Hypothese eines unbekannten regulativen Netzwerkes, welchem mindestens 52 weitere Gene zugeordnet werden konnten. Insgesamt ist festzuhalten, dass die in dieser Arbeit durchgeführte molekulare Charakterisierung der RpoS-Proteolyse in V. cholerae Beweise für eine mögliche Verbindung zwischen der Transkriptionskontrolle für Motilitäts- und Chemotaxisgene mit der Chemotaxis-Reizwahrnehmung erbrachte. Eine derartige intermolekulare Verknüpfung wurde bislang für keinen anderen Organismus beschrieben und stellt somit eine neue Variante der Signaltransduktion innerhalb der Virulenz-assoziierten Genregulation dar. / In the present work conserved function of RpoS in E. coli was approached for its homolog in V. cholerae. Comprehensive phenotypical analysis of rpoS-mutant and wildtype revealed the involvement of RpoS in growth-phase-dependent processes, according to RpoS-function as stationary phase regulator in E. coli. In further experiments the conservation of RpoS-promoters in both species could be shown. To the contrary, the well-known function of E. coli RpoS as general stress-regulator could not be demonstrate for V. cholerae: By testing several stress conditions in survival assays, no significant differences were determined between rpoS mutant and wildtype. Additionally, the intracellular mode of RpoS accumulation in E. coli due to different stress conditions was also not observed in V. cholerae. Regarding the putative role of RpoS as a regulator for virulence-associated genes, the inhere described data support and complement the current theory of RpoS being involved in mucosal detachment of V. cholerae cells. In E. coli the intracellular concentration of RpoS is a decisive parameter for its described function. So far the homologs of the proteolysis targeting factor RssB and the ATP-depending terminal protease complex ClpXP were identified to be involved in V. cholerae RpoS-proteolysis. Further characterization also unravelled, that various stress signals slow down that degradation. But such conditions did not yield in the RpoS accumulation. Based on these differences to the E. coli dynamics of RpoS-degradation additional investigations were performed to gain more insights into the regulatory path of RpoS degradation in V. cholerae. In E. coli the ArcB kinase ist the sensor kinase for regulating the activity of RssB. In this study fexB was identified as arcB homolog in V. cholerae. But by monitoring the RpoS stability in the corresponding knock-out mutant no effect could be observed. Therefore the ArcB-system is not influencing RpoS stability in V. cholerae. Knowing, that RpoS is a major regulator for motility and chemotaxis in V. cholerae, it was investigated next whether other signal-kinases are involved in RpoS proteolysis. Thereby, the known chemotaxis kinases were tested. Knockout mutants of cheAs and subsequent analysis of RpoS half-life revealed, that cheA-1 and cheA-3 did alter RpoS proteolysis to slow down the degradation, whereas cheA-2 mutant did not. Therefore, it can be postulated, that a different mode of RpoS-proteolysis is operating in V. cholerae in which active CheA-1 and CheA-3 may be responsible for RssB phosphorylation, hence leading to RpoS degradation. That kind of interaction may also include the output signalling of the MCP-receptors regulating CheA kinase activity. Since the cheA genes are also under transcriptional control by RpoS a new regulation system can be postulated, where MCP signal output links transcriptional regulation of motility and chemotaxis via RpoS stability in a “positive feedback loop”. Additionally, data are presented, where the ATP depending protease Lon is also involved in RpoS proteolysis in an inverted manner. Lon, which in E. coli is a heat shock induced protease, seems to recognize and degrade substrates in V. cholerae operating in RpoS degradation in the RssB-depending branch. That phenotype was observed as an accelerated RpoS degradation in a lon background. Finally, the complex regulatory pathway of osmo-regulation was characterized. In E. coli RpoS plays a central role. However, in V. cholerae RpoS could not be identified to participate in osmo-regulation, instead a new defined osmostress-sensor (OsmRK) was characterized. In first analysis, it was found that osmRK knockout mutants showed a deregulated ompU expression under hyperosmotic conditions. Considering, that so far only the well known virulence regulator ToxR was identified to act on the ompU promoter, a novel regulatory network was suggested, which regulates at least further 52 genes. In summary, the components of RpoS proteolysis in V. cholerae were unravelled and characterized. Additionally, evidence could be gathered, which indicates a linkage between transcriptional control of motility and chemotaxis genes and the chemotaxis-signalling pathway. So far, such an regulatory pathway has not been described before and would represent a novel branch of signal transduction in bacteria.

In cold temperatures, the survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) requires the action of cold shock protein A (CspA) paralogues. These are thought to melt misfolded ribonucleic acids, facilitating their translation at low temperatures. However, through phenotypic analysis of our SL1344 csp null mutant (lacking all CspA paralogues), it has been shown that CspA paralogues function during other environmental stresses, outwith temperature reduction, and play an essential role in colony formation of an SL1344 rpoS mutant at 37°C. The general stress σ subunit, RpoS, plays an important role in adapting cells to a number of stresses including oxidative stress, temperature changes, low pH and stationary phase. Under such conditions, RpoS acts as an ‘emergency co-ordinator’, subsequently inducing the transcription of necessary stress response genes. In Escherichia coli, RpoS is regulated posttranscriptionally by at least three small RNAs (sRNAs): OxyS, DsrA and RprA; that require interactions with the Sm-like RNA chaperone, Hfq. In S. Typhimurium, the stability of the RpoS protein itself is regulated by ClpXP, an ATP-dependent protease responsible for RpoS degradation, and a specific recognition factor that targets RpoS to this protease, MviA. The present study has shown that the CspA paralogues of S. Typhimurium are involved in the expression of RpoS and aims to elucidate the role of these proteins in RpoS production. Comparative phenotypic tests were carried out in strains carrying mutations in rpoS, hfq and the csp genes to gain insight into the interactions of Hfq and CspA paralogues, with respect to RpoS expression. Both significant phenotypic overlaps, such as peroxide sensitivity, and phenotypes unique to certain mutant strains, such as cold acclimation in the csp null strain, were observed. CspA paralogues and Hfq are functionally distinct, not only in their involvement in RpoS expression, but also in RpoS-independent processes, such as cold acclimation, motility and to some extent, growth at 37°C. The roles of Hfq and the CspA paralogues, in RpoS expression, were also assessed at the molecular level. A combination of qRT-PCR analysis, transcriptional fusions and immunoblotting (with anti-σ antibodies) has shown that DsrA and RprA are not essential for RpoS expression in S. Typhimurium, during stationary phase or exponential cold shock, and do not require Hfq under these conditions. Contrary to reports in E. coli, DsrA is not induced upon cold shock in SL1344. Northern blots have shown that neither Hfq nor the CspA paralogues are involved in regulating rpoS transcription during either stationary phase at 37°C or cold shock in exponential phase. Immunoblotting and translational fusions have identified different pathways for the regulation of RpoS during stationary phase at 37°C and cold shock in exponential phase. Hfq is involved during the former condition only, whilst CspA paralogues are involved in both. Protein stability experiments have shown that the CspA paralogues do not play a major role in stabilising RpoS protein against degradation. Together, these results have pointed to a role for both the CspA paralogues and Hfq in facilitating the efficient translation of rpoS mRNA. An SL1344 csp null rpoS mutant is unable to form colonies on LB agar at 37°C, a phenomenon found when introducing combinations of mutations to SL1344 for phenotypic assessment. A conditional rpoS mutant revealed that the SL1344 csp null rpoS strain is viable but non-culturable. From the csp gene family, only cspA and cspB were able to restore colony forming ability to the rpoS mutant. Further complementation experiments pointed to faulty cell division, due to abnormal RNase E activity, as the cause.

No description available.

<p> Sigma S (RpoS), encoded by rpoS, is a subunit of RNA polymerase holoenzyme that controls the expression of many genes in stationary phase of various gram negative bacteria. Escherichia coli expresses these genes to withstand environmental stress and nutrient starvation. Several naturally occurring mutant alleles of the gene have been reported and indicate key differences from laboratory strains. We sought to explore the role of natural alleles of the rpoS gene (from non- K12 strains) and thus the sigma subunit relative to the K12 allele. To study the effect of the rpoS polymorphism on gene expression ofRpoS regulon members, rpoS alleles from ECOR- 21, ECOR-28, ECOR-37 and ECOR-40 as well as MG1655 were cloned into the same background, MG1655 ΔrpoS:cat osmY-lacZ. Sequence analysis showed rpoS alleles from all the natural strains tested were different from MG1655 and each other. The strain with rpoS allele from ECOR-28 had increased expression of osmY and katE similar to MG1655. In contrast, rpoS allele from ECOR- 37 showed low expression of osmYbut not as low as ECOR-21 and ECOR-40 which had expression similar to the rpoS mutant. Not surprisingly, recombinant strains with rpoS alleles from ECOR-21, ECOR-37 and ECOR-40 showed no expression of katE (HPII). These suggest that RpoS in ECOR-28 has high activity similar to wildtype K12 strain while RpoS in ECOR-21, ECOR-37 and ECOR-40 has very low or no activity. We conclude that natural E. coli strains have polymorphism in their rpoS ORF which cause variation in the regulatory activities of RpoS on its regulon. </p> / Thesis / Master of Science (MSc)

This thesis is missing page 53, no other copies have this page. -Digitization Centre / Bacterial adaptation to changing conditions and to the host environment requires coordinated changes in gene expression that permit more efficient utilization of metabolites and increased survival. An important form of gene control is through the use of alternative sigma factors that direct RNA polymerase to recognize a distinct group of genes. One such sigma factor is RpoS, which is widely present in Proteobacteria including many serious human pathogens. As a key stress response regulator, RpoS plays an important role in adaptation, but its effect on virulence varies in different species. RpoS contributes to virulence through either enhancing survival against host defense systems or directly regulating expression of virulence factors in some pathogens, while RpoS is dispensable, or even inhibitory, to virulence in others. The primary objective of this study is to understand the mechanism of RpoS control in gene expression and pathogenesis of Escherichia coli. This thesis first describes the characterization of RpoS regulon in laboratory and pathogenic E. coli strains by transcriptome profiling analysis. Comparison of RpoS regulons identifies a core set of RpoS-controlled genes as well as strain-specific groups of genes, including many implicated in virulence. The contribution of RpoS to enteropathogenesis in vivo was tested using a Citrobacter rodentium (CR)mouse infection model that is commonly used to simulate E. coli infection in human intestine. Mutations in rpoS result in reduced colonization and delay in mortality, indicating RpoS is important for full virulence. Clinical and natural E. coli isolates exhibit variable abilities in stress resistance and virulence, which is partly attributable to attenuating polymorphisms of rpoS commonly found in E. coli populations. A possible mechanism responsible for the occurrence of rpoS polymorphisms in pathogenic E. coli is addressed. Using a group of representative enterohemorrhagic E. coli strains, we report that growth-enhanced mutants can be selected during growth on succinate and other poor carbon sources under both aerobic and anaerobic conditions. The majority of these mutants carry nonsense or missense mutations in rpoS. Phenotypic microarray analysis reveals that rpoS mutations result in increased utilization of 92 nitrogen and 8 carbon sources. Therefore, the occurrence of rpoS polymorphisms may increase the fitness of the population as a whole for better nutrient scavenging. In conclusion, RpoS may be viewed as a transient regulator that orchestrates the temporal expression of a large regulon for better adaptation under specific conditions including natural and host environments. Under conditions not requiring RpoS, its functions can be turned off through decreasing expression, rapid proteolysis, inhibition of RpoS activity, or selection of attenuating mutations. The final part of this thesis reviews the distinct and niche-dependent involvement of RpoS in virulence of many rpoS-bearing pathogens. - / Thesis / Doctor of Philosophy (PhD)