Para se adaptar a diversas condições ambientais Escherichia coli regula sua transcrição gênica. Um importante regulador de adaptação da bactéria é a subunidade sigma da RNA polimerase, responsável pela transcrição da maioria dos genes relacionados com a fase estacionária e situações de estresse. O gene rpoS possui elevado grau de polimorfismo, adquirindo mutações nulas ou de atenuação em cepas cultivadas em laboratório. Na natureza, entretanto, alelos rpoS não-funcionais são, aparentemente, menos comuns. Neste trabalho foi realizado uma análise temporal do perfil de RpoS em isolados ambientais de E. coli, a fim de correlacionar o status de RpoS com as alterações físico-químicas que ocorrem ao longo do ano em águas residuárias. Concluiu-se que RpoS é altamente persistente no ambiente, e os níveis da proteína variaram muito de uma cepa para outra. Não houve grande correlação entre os fenótipos e RpoS, mas os dados demostraram a diversidade fenotípica que pode existir no ambiente e que permitem sua sobrevivência por longos períodos fora do hospedeiro. / To adapt to various environmental conditions Escherichia coli regulates its gene transcription. An important regulator of bacterial adaptation is the sigma subunit of RNA polymerase, responsible for the transcription of most genes related to stationary phase and stress situations. The rpoS gene has a high degree of polymorphism, acquiring null or attenuation mutations in strains grown in the laboratory. In nature, however, nonfunctional rpoS alleles are apparently less common. In this work a temporal analysis of the profile of RpoS in environmental isolates of E. coli was carried out, in order to correlate the status of RpoS with the physico-chemical changes occurring throughout the year in wastewater. It was concluded that RpoS is highly persistent in the environment, and protein levels varied widely from one strain to another. There was no great correlation between the phenotypes and RpoS, but the data demonstrated the phenotypic diversity that may exist in the environment and that allow its survival for long periods outside the host.

Escherichia coli and several other bacteria express a stationary phase sigma factor, RpoS, for RNA polymerase that is responsible for inducing the expression of stress response genes. rpoS expression is induced during early exponential phase and the concentration of RpoS dramatically increases during the transition from log phase to stationary phase. The goal of this study was to test whether rpoS expression could be modulated using an inducible rpoS antisense and rpoS sense. In the first part of this study, a rpoS antisense under the control of an IPTG-inducible promoter was tested for its efficiency for modulating the expression of the RpoS regulon. RpoS-dependent and RpoS-independent lacZ fusions were utilized to quantify the effect of rpoS antisense expression on rpoS translation. Unlike an earlier study, the results of this study suggest that the rpoS antisense was not induced and/or that it was not inhibiting the expression of RpoS-dependent genes. In the second part of this study, an IPTG-inducible rpoS sense was used to test whether expression of certain members of the RpoS regulon were solely dependent on RpoS and not additional factors present in stationary phase. Thus, their expression could be induced in exponential phase. The rpoS sense was integrated into the E. coli chromosome at the ebg locus by homologous recombination utilizing a one-step PCR method. Some putative rpoS sense recombinants showed an increase in catalase expression, which is known to be RpoS-dependent. However, upon further verification, the 5kb PCR fragment encoding the inducible rpoS sense did not appear to have integrated to the intended site. / Thesis / Master of Science (MS)

<i>Escherichia coli</i> O157 is a food borne pathogen of increasing public health concern worldwide. Cattle have been implicated as the primary reservoir of <i>E. coli</i> O157. The fact that the livestock industry has rapidly expanded in Saskatchewan makes it imperative to have a clear scientific understanding of the prevalence of <i>E. coli</i> O157 in this province as well as its survival in soil under ambient conditions.<p>Longitudinal and point studies were employed to determine the prevalence of <i>E. coli</i> O157 among Saskatchewans cattle. During a 2-year period, 23 feedlot and cattle operations were examined and an overall prevalence of 15.6% was reported. The most important finding was that the prevalence rates were highly dependent on cattle density. All <i>E. coli</i> O157 isolates obtained from this study were characterized by using multiplex PCR, RAPD fingerprinting, a Vero cell cytotoxicity assay and antibiotic susceptibility tests. This characterization revealed a surprisingly highly virulent and heterogenous population of <i>E. coli</i> O157 isolates. <p>Subsequently, the survival characteristics of <i>E. coli</i> O157:H7 ATCC 43894 in sterile soil and manure-amended soil microcosms, as well as in situ under ambient environmental conditions were examined. Findings from this work indicated that desiccation had the most lethal effect on cell viability, whereas nutritionally-rich soils significantly increased survival times of the pathogen population. <p>A final study was designed to examine the survival strategy of hyper- and hypothermally adapted <i>E. coli</i> O157 cells exposed to high and low temperatures, with specific focus on the role of RpoS. Using wild type and its rpoS null allele <i>E. coli</i> O157 strains, in combination with 2D PAGE, It was found that both heat and cold post-acclimation stimulons consisted of two large sub-groups: (i) stress proteins, and (ii) housekeeping proteins. Comparative proteomic analyses revealed that the GroEL/S chaperonin complex and Pnp ribonuclease played a crucial role in growth resumption during high and low temperatures, respectively. Notably, RpoS had no control over key stress proteins in either stress stimulon. RpoS, however, showed a significantly more pronounced role during cold temperatures, where it was seen to regulate key proteins involved in homoeoviscous adaptation as well as various housekeeping proteins of both stress stimulons.

<i>Escherichia coli</i> O157 is a food borne pathogen of increasing public health concern worldwide. Cattle have been implicated as the primary reservoir of <i>E. coli</i> O157. The fact that the livestock industry has rapidly expanded in Saskatchewan makes it imperative to have a clear scientific understanding of the prevalence of <i>E. coli</i> O157 in this province as well as its survival in soil under ambient conditions.<p>Longitudinal and point studies were employed to determine the prevalence of <i>E. coli</i> O157 among Saskatchewans cattle. During a 2-year period, 23 feedlot and cattle operations were examined and an overall prevalence of 15.6% was reported. The most important finding was that the prevalence rates were highly dependent on cattle density. All <i>E. coli</i> O157 isolates obtained from this study were characterized by using multiplex PCR, RAPD fingerprinting, a Vero cell cytotoxicity assay and antibiotic susceptibility tests. This characterization revealed a surprisingly highly virulent and heterogenous population of <i>E. coli</i> O157 isolates. <p>Subsequently, the survival characteristics of <i>E. coli</i> O157:H7 ATCC 43894 in sterile soil and manure-amended soil microcosms, as well as in situ under ambient environmental conditions were examined. Findings from this work indicated that desiccation had the most lethal effect on cell viability, whereas nutritionally-rich soils significantly increased survival times of the pathogen population. <p>A final study was designed to examine the survival strategy of hyper- and hypothermally adapted <i>E. coli</i> O157 cells exposed to high and low temperatures, with specific focus on the role of RpoS. Using wild type and its rpoS null allele <i>E. coli</i> O157 strains, in combination with 2D PAGE, It was found that both heat and cold post-acclimation stimulons consisted of two large sub-groups: (i) stress proteins, and (ii) housekeeping proteins. Comparative proteomic analyses revealed that the GroEL/S chaperonin complex and Pnp ribonuclease played a crucial role in growth resumption during high and low temperatures, respectively. Notably, RpoS had no control over key stress proteins in either stress stimulon. RpoS, however, showed a significantly more pronounced role during cold temperatures, where it was seen to regulate key proteins involved in homoeoviscous adaptation as well as various housekeeping proteins of both stress stimulons.

Der alternative Sigmafaktor RpoS ist der Masterregulator der generellen Stressantwort in Escherichia coli. Er kontrolliert ein Regulon von mehr als 500 Genen. In ungestressten Zellen ist die RpoS-Synthese gering, da RpoS nach Bindung an den spezifischen Erkennungsfaktor RssB von der ClpXP-Protease unter ATP-Verbrauch degradiert wird. RssB wird dabei nicht codegradiert und wirkt daher katalytisch. In gestressten Zellen wird RpoS stabilisiert. Die zentrale Fragestellung dieser Arbeit war, wann und wodurch RpoS in den beiden E. coli K12-Laborstämmen W3110 und MC4110 stabilisiert wird. Die Ergebnisse zeigen, dass die Verfügbarkeit von RssB und das Verhältnis von RpoS zu RssB die zentralen Schlüssel sind. In ungestressten Zellen sind die geringen Konzentrationen von RpoS und RssB durch homöostatisches Feedback aufeinander abgestimmt (RssB benötigt RpoS zu seiner Expression). In der postexponentiellen Wachstumsphase steigt der RpoS-Gehalt so stark an, dass die Zellen mit RpoS “überschwemmt“ werden und RssB austitriert wird. In der Stationärphase liegt RssB meist inaktiv vor. Zusammen mit dem Absinken des ATP-Gehalts führt das zur Stabilisierung von RpoS. Da parallel die Bindung von RpoD an die RNA-Polymerase (RNAP) durch Rsd inhibiert wird, (p)ppGpp die Bindung alternativer Sigmafaktoren an die RNAP fördert und Crl gezielt RpoS bei der Bindung an die RNAP unterstützt, kann RpoS nun erfolgreich mit den anderen Sigmafaktoren um die RNAP konkurrieren und die generelle Stressantwort initiieren. Im Stamm W3110 wird RpoS bereits in der späten postexponentiellen Phase stabilisiert und der hohe RpoS-Gehalt schafft in diesem Stamm die Basis für eine starke generelle Stressantwort. In MC4100 hingegen wird RpoS zweiphasig stabilisiert und wird am Ende der postexponentiellen Phase zunächst wieder destabilisiert. Das resultiert in einer geringeren Stressresistenz und Überlebensfähigkeit des MC4100 und belegt die stärkere Degenerierung des MC4100 im Vergleich zum W3110. / The alternative sigma factor RpoS is the master regulator of the general stress response in Escherichia coli, which controls a regulon of >500 genes. In unstressed cells RpoS levels are low, because upon binding to the specific RpoS recognition factor RssB, RpoS becomes degraded by the ATP-dependent ClpXP protease. RssB is not co-degraded and acts catalytically. In stressed cells RpoS becomes stabilized. The central question of this study was when and how RpoS is stabilized in the two E. coli K-12 laboratory strains W3110 and MC4110. The results show that the availability of RssB and the ratio of RpoS to RssB are the central key. In unstressed cells low levels of RpoS and RssB level are finely balanced due homeostatic feedback (the expression of RssB itself requires RpoS). During the post-exponential growth phase the RpoS level strongly increases, so that cells are ‘flooded‘ with RpoS which titrates RssB. During stationary phase RssB exists mainly in its inactive form. Along with a lower ATP level, this results in a stabilization of RpoS. Since in parallel binding of RpoD to RNA polymerase (RNAP) is inhibited by Rsd, (p)ppGpp promotes the binding of alternative sigma factors to the RNAP and Crl supports RpoS binding to RNAP, RpoS can successfully compete with other sigma factors for RNAP and initiate the general stress response. In strain W3110 RpoS is stabilized already during the late post-exponential phase and the high RpoS content in this strain creates the basis for a strong general stress response. In MC4100, however, RpoS is stabilized in two phases, and during the end of the post-exponential phase RpoS is even transiently destabilized again. This is reflected in reduced stress resistance and longterm survival of MC4100 and shows the stronger degeneration of strain MC4100 compared to W3110.

Escherichia coli possède une grande plasticité génomique comme en témoigne la diversité des souches à l’intérieur de cette espèce bactérienne. Bien que la majorité des souches soient inoffensives ou à tout le moins opportunistes, plusieurs ont acquis des facteurs de virulence spécifiques leur procurant un pouvoir pathogénique. Les souches pathogènes comme E. coli O157 :H7 sont responsables de cas de morbidité, mortalité et pertes économiques importantes dans l’industrie agro-alimentaire dans le monde entier. L’évolution bactérienne est un mécanisme continuel qui se fait via l’échange d’éléments génétiques mobiles, de mutations ponctuelles et autres réarrangements génétiques. Ces changements génétiques peuvent procurer des avantages sélectifs permettant une adaptation bactérienne rapide face aux stress et changements environnementaux et favorisant le développement de pathogènes émergents. Dans la première partie de ce projet, nous avons étudié la région intergénique mutS-rpoS, qui est une des plus grandes sources de polymorphisme chromosomique chez les entérobactéries. Notre analyse génomique comparative a permis de confirmer le polymorphisme à l’intérieur même d’un ensemble de souches Escherichia spp., Salmonella spp. et Shigella spp. De plus, nous avons pu confirmer que certains types de polymorphismes dans la région mutS-rpoS étaient fortement associés à certains types de pathogènes chez E. coli. Dans notre analyse, nous avons ressorti un groupe de gènes à l’intérieur de la région mutS-rpoS qui pourraient sevir comme marqueur chromosomique intéressant pour les E. coli extra-intestinaux (ExPEC), un groupe comprennant des souches hautement pathogènes et difficiles à définir par les tests actuelllement disponibles. Dans notre analyse bio-informatique, nous avons isolé ce groupe de gènes associé aux ExPEC et nous l’avons caractérisé in sillico. Nous avons également inclus dans l’analyse deux souches hypermutables du genre Escherichia spp. de notre collection, isolées d’animaux de ferme. L’hypermutabilité ou la capacité d’acquérir des mutations plus rapidement que la normale accélère le processus d’évolution et la capacité d’adaptation de ces souches. La région mutS-rpoS est reliée au système de réparation de l’ADN bactérien (MMRS) et pourrait être impliquée dans l’apparition du phénotype d’hypermutabilité. Durant les dernières années, de plus en plus d’espèces du genre Escherichia ont été isolées de cas cliniques d’animaux et d’humains. Ces souches atypiques ont un potentiel de virulence très élevé, des combinaisons de gènes de virulence et des variants génétiques différents des souches typiques, et certaines souches ont même évolué en tant que pathogènes. Les souches de l’espèce E. albertii ont été isolées récemment et ont un grand potentiel de virulence autant chez les humains que chez les oiseaux. Ces souches sont souvent confondues avec d’autres organismes pathogènes comme E. coli dans les tests biochimiques, et le manque de connaissances sur E. albertii rend son identification difficile. Dans la deuxième partie de ce projet, nous avons identifié des gènes spécifiques aux souches d’E. albertii ainsi que des gènes de virulence présents chez E. albertii par comparaisons génomiques, ce qui a permis de développer et optimiser un test PCR (réaction en chaîne par polymérase) visant l’identification génomique rapide et fiable d’E. albertii.

E. coli K-12 peut survivre lors d'une incubation prolongée en carence en Pi, en condition aérobie, grâce à l'induction par RpoS de poxB et gadB. Les mutants rpoS survivent en utilisant une nouvelle stratégie. Le métabolisme des mutants rpoS nécessite l'activité du répresseur Fur pour diminuer la synthèse du petit ARN RyhB, qui inhibe la synthèse de protéines riches en fer (Sdh et FumA). L'activité Fur et l’absence de RpoS permettent ensemble qu’une activité substantielle du cycle TCA se poursuive en phase stationnaire, ce qui diminue la production d'acide acétique et, finalement, permet la croissance sur l'acide acétique et le Pi excrété dans le milieu. Lors d'une évolution expérimentale dans un milieu minimum limitant en Pi, les bactéries de type sauvage donnent rapidement naissance à des mutants qui détoxiquent l'acide acétique du milieu et survivent lors d'une incubation prolongée. Une souche évoluée ajoutée en minorité dans une culture mixte peut croître entre les jours 1 et 3 d’incubation grâce à l’acquisition de deux mutations : une délétion de 1 pb qui inactive RpoS et une délétion de 8 bp qui active PhnE, un transporteur de produits phosphorylés qui est normalement cryptique. Les mutants phnE+ rpoS- peuvent croître dans des cultures contenant une majorité de bactéries de type sauvage (phnE- rpoS+) grâce à 1°) l'activité PhnE, qui pourrait récupérer des produits phosphorylés excrétés par les bactéries de type sauvage carencées en Pi, et 2°) le manque d'activité RpoS qui, d’une part favorise l'induction du régulon PHO et donc de phnE, et d’autre part maintient l’entrée du flux métabolique dans le cycle TCA. / E. coli K-12 can survive prolonged incubation under aerobic, Pi starvation conditions as a result of the induction by RpoS of poxB and gadB. However, rpoS mutants survived Pi starvation by using a new strategy. Metabolism in rpoS mutants required the activity of Fur in order to decrease the synthesis of the small RNA RyhB that might otherwise inhibit the synthesis of iron-rich proteins such as SDH and FumA. Fur activity and the lack of RpoS activity allow a substantial activity of the TCA cycle to continue in stationary phase in rpoS mutants, which decreases the production of acetic acid and, eventually, allows growth on acetic acid and Pi excreted into the medium. During experimental evolution experiments conducted in Pi-limiting medium containing no glutamate, wt cells rapidly evolved mutants that detoxified acetic acid from the medium and survived during prolonged incubation. One evolved strain, added as a minority in mixed culture, could grow between days 1 and 3 of incubation provided that the PHO regulon was induced. One mutation that inactivated RpoS activity and one that activated the PhnE phosphorylated products-transport activity (normally cryptic) were necessary and sufficient to provide such phenotype. phnE+ rpoS- double mutants can grow in cultures containing a majority of wt cells (phnE- rpoS+) probably because PhnE activity scavenges low levels of phosphorylated products excreted by wt cells, when the lack of RpoS activity helps to maintain induction of the PHO regulon and a substantial activity of the TCA cycle in slow growing cells.

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.

Levels of macromolecules fluctuate both spatially and temporally in individual cells. Such heterogeneity could be exploited for bet hedging in uncertain environments, or be suppressed by negative feedback if perturbations are deleterious. For the master stress-response regulator in Escherichia coli, RpoS, both of these scenarios have been suggested. RpoS levels are also exceedingly low and controlled by the ClpXP protease, which reportedly displays extreme spatial heterogeneity. However, little is known quantitatively about RpoS dynamics. This is partly because no functional protein fusions exist, but also because the quantitative tools for studying fluctuations and localizations are limited, particularly ones that can be independently validated. Here I develop such methods and begin applying them to RpoS. Protein localization measurements increasingly rely on fluorescent protein fusions and are difficult to verify independently. I designed a non-intrusive method for validating localization patterns in live bacterial cells by exploiting post-division heterogeneity in downstream processes. Applying this assay to the ClpXP protease, widely reported to form biologically relevant foci, revealed in fact that the protease molecules are not specifically localized inside cells, as confirmed by four independent methods. I further evaluated 20+ commonly used fluorescent reporters and found that many cause severe mislocalization when fused to homo-oligomers, likely due to avidity effects. Further reinvestigating other foci-forming proteins strongly suggests that the previously reported foci were all caused by the fluorescent proteins used. For mRNAs – which are often present in low numbers per cell and major sources of non-genetic heterogeneity – existing single-cell assays have unknown accuracy: the experimental counting errors could completely over-shadow the natural variation. I therefore optimized and cross-evaluated two single-molecule mRNA detection methods. Several problems were identified and solutions discussed. I succeeded in building a functional RpoS protein fusion, and used bulk methods to show that the RpoS feedback loop is effectively not operating during exponential- phase growth. Mathematical analyses and initial experiments in a microfluidic device further suggest that the RpoS system has several unusual properties contributing towards extremely fast stress response. A stochastic analysis further suggests that the RpoS feedback loop cannot suppress spontaneous fluctuations, and preliminary experiments indicate that large deviations might indeed play important roles.

abstract: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative enteric pathogen that causes self-limiting gastroenteritis in healthy individuals and can cause systemic infections in those who are immunocompromised. During its natural lifecycle, S. Typhimurium encounters a wide variety of stresses it must sense and respond to in a dynamic and coordinated fashion to induce resistance and ensure survival. Salmonella is subjected to a series of stresses that include temperature shifts, pH variability, detergent-like bile salts, oxidative environments and changes in fluid shear levels. Previously, our lab showed that cultures of S. Typhimurium grown under physiological low fluid shear (LFS) conditions similar to those encountered in the intestinal tract during infection uniquely regulates the virulence, gene expression and pathogenesis-related stress responses of this pathogen during log phase. Interestingly, the log phase Salmonella mechanosensitive responses to LFS were independent of the master stress response sigma factor, RpoS, departing from our conventional understanding of RpoS regulation. Since RpoS is a growth phase dependent regulator with increased stability in stationary phase, the current study investigated the role of RpoS in mediating pathogenesis-related stress responses in stationary phase S. Typhimurium grown under LFS and control conditions. Specifically, stationary phase responses to acid, thermal, bile and oxidative stress were assayed. To our knowledge the results from the current study demonstrate the first report that the mechanical force of LFS globally alters the S. Typhimurium χ3339 stationary phase stress response independently of RpoS to acid and bile stressors but dependently on RpoS to oxidative and thermal stress. This indicates that fluid shear-dependent differences in acid and bile stress responses are regulated by alternative pathway(s) in S. Typhimurium, were the oxidative and thermal stress responses are regulated through RpoS in LFS conditions. Results from this study further highlight how bacterial mechanosensation may be important in promoting niche recognition and adaptation in the mammalian host during infection, and may lead to characterization of previously unidentified pathogenesis strategies. / Dissertation/Thesis / Masters Thesis Biology 2016