Characterization of water and wildlife strains as a subgroup of Campylobacter jejuni using DNA microarrays.

Campylobacter jejuni is the leading cause of human bacterial gastroenteritis worldwide, but source attribution of the organism is difficult. Previously, DNA microarrays were used to investigate isolate source, which suggested a non-livestock source of infection. In this study we analysed the genome content of 162 clinical, livestock and water and wildlife (WW) associated isolates combined with the previous study. Isolates were grouped by genotypes into nine clusters (C1 to C9). Multilocus sequence typing (MLST) data demonstrated that livestock associated clonal complexes dominated clusters C1-C6. The majority of WW isolates were present in the C9 cluster. Analysis of previously reported genomic variable regions demonstrated that these regions were linked to specific clusters. Two novel variable regions were identified. A six gene multiplex PCR (mPCR) assay, designed to effectively differentiated strains into clusters, was validated with 30 isolates. A further five WW isolates were tested by mPCR and were assigned to the C7-C9 group of clusters. The predictive mPCR test could be used to indicate if a clinical case has come from domesticated or WW sources. Our findings provide further evidence that WW C. jejuni subtypes show niche adaptation and may be important in causing human infection

Mycobacterium tuberculosis Transcriptional Adaptation, Growth Arrest and Dormancy Phenotype Development Is Triggered by Vitamin C

BACKGROUND: Tubercle bacilli are thought to persist in a dormant state during latent tuberculosis (TB) infection. Although little is known about the host factors that induce and maintain Mycobacterium tuberculosis (M. tb) within latent lesions, O(2) depletion, nutrient limitation and acidification are some of the stresses implicated in bacterial dormancy development/growth arrest. Adaptation to hypoxia and exposure to NO/CO is implemented through the DevRS/DosT two-component system which induces the dormancy regulon. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that vitamin C (ascorbic acid/AA) can serve as an additional signal to induce the DevR regulon. Physiological levels of AA scavenge O(2) and rapidly induce the DevR regulon at an estimated O(2) saturation of <30%. The kinetics and magnitude of the response suggests an initial involvement of DosT and a sustained DevS-mediated response during bacterial adaptation to increasing hypoxia. In addition to inducing DevR regulon mechanisms, vitamin C induces the expression of selected genes previously shown to be responsive to low pH and oxidative stress, triggers bacterial growth arrest and promotes dormancy phenotype development in M. tb grown in axenic culture and intracellularly in THP-1 cells. CONCLUSIONS/SIGNIFICANCE: Vitamin C mimics multiple intracellular stresses and has wide-ranging regulatory effects on gene expression and physiology of M. tb which leads to growth arrest and a 'dormant' drug-tolerant phenotype, but in a manner independent of the DevRS/DosT system. The 'AA-dormancy infection model' offers a potential alternative to other models of non-replicating persistence of M. tb and may be useful for investigating host-'dormant' M. tb interactions. Our findings offer a new perspective on the role of nutritional factors in TB and suggest a possible role for vitamin C in TB

RNase H enables efficient repair of R-loop induced DNA damage.

R-loops, three-stranded structures that form when transcripts hybridize to chromosomal DNA, are potent agents of genome instability. This instability has been explained by the ability of R-loops to induce DNA damage. Here, we show that persistent R-loops also compromise DNA repair. Depleting endogenous RNase H activity impairs R-loop removal in Saccharomyces cerevisiae, causing DNA damage that occurs preferentially in the repetitive ribosomal DNA locus (rDNA). We analyzed the repair kinetics of this damage and identified mutants that modulate repair. We present a model that the persistence of R-loops at sites of DNA damage induces repair by break-induced replication (BIR). This R-loop induced BIR is particularly susceptible to the formation of lethal repair intermediates at the rDNA because of a barrier imposed by RNA polymerase I

Nonconventional hydrolytic dehalogenation of 1-chlorobutane by dehydrated bacteria in a continuous solid-gas biofilter

Rhodococcus erythropolis NCIMB 13064 and Xanthobacter autotrophicus GJ10 are able to catalyze the conversion of halogenated hydrocarbons to their corresponding alcohols. These strains are attractive biocatalysts for gas phase remediation of polluted gaseous effluents because of their complementary specificity for short or medium and for mono-, di-, or trisubstituted halogenated hydrocarbons (C2-C8 for Rhodococcus erythropolis and C1-C4 for Xanthobacter autotrophicus). After dehydration, these bacteria can catalyze the hydrolytic dehalogenation of 1-chlorobutane in a nonconventional gas phase system under a controlled water thermodynamic activity (aw). This process makes it possible to avoid the problems of solubility and bacterial development due to the presence of water in the traditional biofilters. In the aqueous phase, the dehalogenase activity of Rhodococcus erythropolis is less sensitive to thermal denaturation and the apparent Michaelis-Menten constants at 30°C were 0.4 mM and 2.40 μmol min−1 g−1 for Km and Vmax, respectively. For Xanthobacter autotrophicus they were 2.8 mM and 0.35 μmol min−1 g−1. In the gas phase, the behavior of dehydrated Xanthobacter autotrophicus cells is different from that observed with Rhododcoccus erythropolis cells. The stability of the dehalogenase activity is markedly lower. It is shown that the HCl produced during the reaction is responsible for this low stability. Contrary to Rhodococcus erythropolis cells, disruption of cell walls does not increase the stability of the dehalogenase activity. The activity and stability of lyophilized Xanthobacter autotrophicus GJ10 cells are dependant on various parameters. Optimal dehalogenase activity was determined for water thermodynamic activity (aw) of 0.85. A temperature of 30°C offers the best compromise between activity and stability. The pH control before dehydration plays a role in the ionization state of the dehalogenase in the cells. The apparent Michaelis-Menten constants Km and Vmax for the dehydrated Xanthobacter autotrophicus cells were 0.07 (1-chlorobutane thermodynamic activity) and 0.08 μmol min−1 g−1 of cells, respectively. A maximal transformation capacity of 1.4 g of 1-chlorobutane per day was finally obtained using 1g of lyophilized Xanthobacter autotrophicus GJ10 cell

The bacillary and macrophage response to hypoxia in tuberculosis and the consequences for T cell antigen recognition

M. tuberculosis is a facultative anaerobe and its characteristic pathological hallmark, the granuloma, exhibits hypoxia in humans and in most experimental models. Thus the host and bacillary adaptation to hypoxia is of central importance in understanding pathogenesis and thereby to derive new drug treatments and vaccines

Sublethal salinity stress contributes to habitat limitation in an endangered estuarine fish.

As global change alters multiple environmental conditions, predicting species' responses can be challenging without understanding how each environmental factor influences organismal performance. Approaches quantifying mechanistic relationships can greatly complement correlative field data, strengthening our abilities to forecast global change impacts. Substantial salinity increases are projected in the San Francisco Estuary, California, due to anthropogenic water diversion and climatic changes, where the critically endangered delta smelt (Hypomesus transpacificus) largely occurs in a low-salinity zone (LSZ), despite their ability to tolerate a much broader salinity range. In this study, we combined molecular and organismal measures to quantify the physiological mechanisms and sublethal responses involved in coping with salinity changes. Delta smelt utilize a suite of conserved molecular mechanisms to rapidly adjust their osmoregulatory physiology in response to salinity changes in estuarine environments. However, these responses can be energetically expensive, and delta smelt body condition was reduced at high salinities. Thus, acclimating to salinities outside the LSZ could impose energetic costs that constrain delta smelt's ability to exploit these habitats. By integrating data across biological levels, we provide key insight into the mechanistic relationships contributing to phenotypic plasticity and distribution limitations and advance the understanding of the molecular osmoregulatory responses in nonmodel estuarine fishes

\u3cem\u3eE. Coli\u3c/em\u3e Persister Cell Survival and Rhizobia Attachment to Soybean Roots

The theme of this thesis revolves around how bacteria respond and thrive during stress. Chapters 1-3 are about how bacteria deal with life-threatening antibiotics. Chapter 4 covers new research on how bacteria can move from a stressful individual lifestyle (free-living bacteria) to initiating a symbiotic relationship with a plant (a less stressful lifestyle). In Chapter 1, I briefly summarize the current state of knowledge in the field of antibiotic resistance and persistence. In Chapter 2, I add to this knowledge by providing new insights into several antibiotics\u27 potency and exploring the antibiotic Eagle effect. In Chapter 3, I use pyruvate to study the revival of persister cells. Finally, in Chapter 4, I switch gears and briefly discuss how I optimized the initial steps of soybean germination and rhizobia culturing techniques to monitor the root-bacterial attachment

Quantitative physiology of bacterial survival under carbon starvation and temperature stress

A large number of the bacteria on Earth live for long periods in states of very low metabolic activity and little or no growth due to starvation and other environmental stresses. Within millions of years, bacteria have developed several strategies to adapt to many different environments, where they survive and evolve to optimize their fitness and to undergo rapid division cycles when conditions become favourable. However, many of these survival strategies are still a puzzle and relatively little is known about the mechanisms that underpin the dominant modes of bacterial existence. This is particularly alarming, as the growth-arrest phase has become crucial to understand the contribution of microorganisms to human physiology and predisposition to disease as well as microbial tolerance and resistance to antibiotics. The dearth of information is mainly due to the difficulties in defining, reproducing and measuring bacterial behaviours in growth-arrest states, which may often seem erratic and unpredictable, while cell physiology is similarly diverse and often specific to the particular environmental conditions. Thus, determining how molecular contributions affect survival is challenging. This explains why, in the last century, bacteria have been mainly studied during the exponential growth phase, which is, on the contrary, a well-defined and reproducible steady state of constant growth, gene expression and molecular compositions. As a result, an increasing combined use of experiments and predictive models focused on this phase has provided a deep understanding of bacterial physiology and gene regulation during growth. A similar quantitative approach that focuses on the growth-arrest phase is largely missing. In this thesis, we contribute to fill this gap by developing new quantitative approaches to investigate bacterial physiology in hostile environments where stresses, such as lack of nutrients and additional environmental perturbations, like temperature increase, force the cells to activate strategies of survival. To do so, we choose to work with the bacterium Escherichia coli (E. coli) that, among the estimated 10^12 microbial species living in our planet, is one of the most studied thanks to its hardiness, versatility and ease of handling. In Chapter 1, we provide an overview of the physiology of E. coli life cycle and of the main quantitative methods so far used to study it, especially focusing on its behaviour during the growth-arrest phase. In Chapter 2, we establish the missing quantitative approach to study E. coli physiology in the death phase. We show that in carbon starvation, an exponential decay of viability emerges as a collective phenomenon, with viable cells recycling nutrients from dead cells to maintain viability. The observed collective death rate is determined by the maintenance rate of viable cells and the amount of nutrients recovered from dead cells, the yield. Using this relation, we study the cost of a wasteful enzyme during starvation and the benefit of the stress response sigma factor RpoS. While the enzyme activity increases maintenance and thereby the death rate, RpoS improves biomass recycling, decreasing the death rate. Our approach thus enables quantitative analyses of how cellular components affect the survival of non-growing cells. In Chapter 3, we use the quantitative approach developed in the previous chapter to study how survival of E. coli in carbon starvation depends on the previous culture conditions. We show that environments that support only slow growth lead to longer survival in starvation because of a decrease of maintenance rate, meaning that slower growing cells need less energy to survive. Our results suggest a physiological trade-off between the ability to proliferate fast and the ability to survive long that could shed light on the long-standing question of why bacteria outside of laboratory environments are not optimized for fast growth. In Chapter 4, we study E. coli physiology under the combined stresses of carbon starvation and high temperatures, characterizing a thermal fuse that leads to a dormant and antibiotic persistent sub-population. This fuse is implemented by a thermally unstable enzyme, MetA, in the methionine synthesis pathway. The combination of a positive feed-back in the methionine system and a dual-use of methionine for protein synthesis and as a methyl-donor results in the bacterial population splitting into two distinct states at elevated temperatures, growing and dormant. We then reveal that these dormant bacteria not only survive antibiotic treatment, but also heat shocks, suggesting that the thermal fuse has originally evolved as a ''bet-hedging'' strategy to ensure survival in heat shocks. Our findings, summarized in Chapter 5, pave the way for the development of a new theoretical framework and experimental approach to understand bacterial physiology in the growth-arrest phase, by linking phenomenological modeling to molecular mechanisms.Eine große Anzahl der Bakterien auf der Erde lebt über große Zeiträume in einem Zustand mit sehr geringer Stoffwechselaktivität und nur geringem oder keinem Wachstum. Ein Grund dafür sind widrige Umwelteinflüsse und die damit einhergehenden Belastungen wie beispielsweise Ressourcenmangel. Innerhalb von Millionen von Jahren haben Bakterien diverse Strategien zur Anpassung an verschiedene Umgebungen, in denen sie überleben und sich weiterentwickeln, entwickelt, um ihre Fitness zu optimieren und bei günstigen Bedingungen schnelle Teilungszyklen zu durchlaufen. Viele dieser Überlebensstrategien sind jedoch immer noch ein Rätsel und es ist nur relativ wenig über die Mechanismen bekannt, die den dominanten Formen der bakteriellen Existenz zu Grunde liegen. Dies ist von besonderer Bedeutung, da die Phase unterdrückten Wachstums entscheidend ist, um den Beitrag von Mikroorganismen zur menschlichen Physiologie und Anfälligkeit für Krankheiten, sowie zur mikrobiellen Verträglichkeit und Antibiotikaresistenz zu verstehen. Der Mangel an Informationen ist hauptsächlich auf die Schwierigkeiten bei der Definition, Reproduktion und Messung des Verhaltens von Bakterien in Zuständen des Wachstumsstillstands zurückzuführen, die oft unberechenbar und unvorhersehbar erscheinen, während die Zellphysiologie ähnlich vielfältig und oft spezifisch für die jeweiligen Umgebungsbedingungen ist. Daher ist es schwierig zu bestimmen, wie sich molekulare Mechanismen auf das Überleben auswirken. Dies erklärt, warum im letzten Jahrhundert Bakterien hauptsächlich während der exponentiellen Wachstumsphase untersucht wurden, die im Gegenteil ein genau definierter und reproduzierbarer Gleichgewichtszustand des konstanten Wachstums, der Genexpression und der molekularen Zusammensetzung ist. Infolgedessen hat eine zunehmende Kombination von Experimenten und Vorhersagemodellen, die sich auf diese Phase konzentrieren, ein tiefes Verständnis der bakteriellen Physiologie und Genregulation während des Wachstums geliefert. Ein ähnlicher quantitativer Ansatz, der sich auf die Phase der Stagnation konzentriert, fehlt weitgehend. In dieser Doktorarbeit tragen wir dazu bei, diese Lücke durch die Entwicklung neuer quantitativer Ansätze zur Untersuchung der bakteriellen Physiologie in ungünstigen Umgebungen zu füllen, in denen Stressfaktoren, wie beispielsweise Nährstoffmangel, auftreten und zusätzliche umweltbedingte Störungen, wie eine Temperaturerhöhung, die Zellen zwingen, Strategien zum Überleben zu aktivieren. Dazu arbeiten wir mit dem Bakterium Escherichia coli (E. coli), das unter den circa 10^12 mikrobiellen Spezies, die auf unserem Planeten leben, wegen seiner Widerstandsfähigkeit, Vielseitigkeit und einfachen Handhabung eines der am besten untersuchten Bakterien darstellt. In Kapitel 1, geben wir einen Überblick über die Physiologie des Lebenszyklus von E. coli und über die wichtigsten bisher verwendeten quantitativen Methoden, wobei wir uns auf das Verhalten während der Wachstumsphase konzentrieren. In Kapitel 2, stellen wir den fehlenden quantitativen Ansatz zur Untersuchung der Physiologie von E. coli während der Sterbephase fest. Wir zeigen, dass bei Kohlenstoffmangel ein exponentieller Zerfall der Lebensfähigkeit als kollektives Phänomen auftritt, wobei lebensfähige Zellen Nährstoffe aus toten Zellen recyceln, um die Lebensfähigkeit aufrechtzuerhalten. Die beobachtete kollektive Sterberate wird durch die Erhaltungsrate lebensfähiger Zellen und die Menge an Nährstoffen, die aus toten Zellen als Ertrag gewonnen werden, bestimmt. Unter Verwendung dieser Beziehung untersuchen wir die Kosten einer verschwenderischen Enzymaktivität während des Hungerns und den Nutzen des Sigma Faktors RpoS für die Stressreaktion. Während diese Aktivität die Instandhaltung und damit die Sterblichkeitsrate erhöht, verbessert RpoS das Recycling der Biomasse und senkt die Sterblichkeitsrate. Unser Ansatz ermöglicht daher quantitative Analysen darüber, wie sich zelluläre Komponenten auf das Überleben nicht wachsender Zellen auswirken. In Kapitel 3, verwenden wir den im vorherigen Kapitel entwickelten quantitativen Ansatz, um zu untersuchen, wie das Überleben von E. coli bei Kohlenstoffmangel von den vorherigen Kulturbedingungen abhängt. Wir zeigen, dass Umgebungen, die nur langsames Wachstum unterstützen, aufgrund einer verringerten Erhaltungsrate zu einem längeren Überleben führen, was bedeutet, dass langsamer wachsende Zellen weniger Energie zum Überleben benötigen. Unsere Ergebnisse legen einen physiologischen Kompromiss zwischen der Fähigkeit, sich schnell zu vermehren, und der Fähigkeit, lange zu überleben, nahe, der Auschluss darüber geben könnte, warum Bakterien außerhalb von Laborumgebungen nicht für schnelles Wachstum optimiert sind. In Kapitel 4, untersuchen wir die Physiologie von E. coli unter dem kombinierten Stress von Kohlenstoffmangel und hohen Temperaturen und charakterisieren eine thermische Sicherung, die zu einer ruhenden und antibiotisch persistierenden Subpopulation führt. Diese Sicherung wird durch ein thermisch instabiles Enzym, MetA, im Methioninsyntheseweg implementiert. Die Kombination aus einer positiven Rückkopplung im Methioninsystem und einer doppelten Verwendung von Methionin für die Proteinsynthese und als Methyldonor führt dazu, dass sich die Bakterienpopulation bei erhöhten Temperaturen in zwei verschiedene Zustände aufspaltet, wobei jeweils eine Subpopulation wächst und die Andere schläft. Wir zeigen dann, dass diese ruhenden Bakterien nicht nur eine Antibiotikabehandlung, sondern auch Hitzeschocks überstehen, was darauf hindeutet, dass sich die thermische Sicherung ursprünglich als eine ''bet-hedging'' Strategie entwickelt hat, um das Überleben bei Hitzeschocks sicherzustellen. Unsere Ergebnisse, die in Kapitel 5 zusammengefasst sind, ebnen den Weg für die Entwicklung eines neuen theoretischen Rahmens und experimentellen Ansatzes zum Verständnis der Bakterienphysiologie in der Phase des Wachstumsstopps, indem phänomenologische Modelle mit molekularen Mechanismen verknüpft werden

Altered modulation of lamin A/C-HDAC2 interaction and p21 expression during oxidative stress response in HGPS

Defects in stress response are main determinants of cellular senescence and organism aging. In fibroblasts from patients affected by Hutchinson-Gilford progeria, a severe LMNA-linked syndrome associated with bone resorption, cardiovascular disorders, and premature aging, we found altered modulation of CDKN1A, encoding p21, upon oxidative stress induction, and accumulation of senescence markers during stress recovery. In this context, we unraveled a dynamic interaction of lamin A/C with HDAC2, an histone deacetylase that regulates CDKN1A expression. In control skin fibroblasts, lamin A/C is part of a protein complex including HDAC2 and its histone substrates; protein interaction is reduced at the onset of DNA damage response and recovered after completion of DNA repair. This interplay parallels modulation of p21 expression and global histone acetylation, and it is disrupted by LMNAmutations leading to progeroid phenotypes. In fact, HGPS cells show impaired lamin A/C-HDAC2 interplay and accumulation of p21 upon stress recovery. Collectively, these results link altered physical interaction between lamin A/C and HDAC2 to cellular and organism aging. The lamin A/C-HDAC2 complex may be a novel therapeutic target to slow down progression of progeria symptoms

Understanding the Molecular Basis of HPV-16 Neoplastic Progression Using an Organotypic Raft Model

High-risk Human Papillomavirus (HPV) types such as HPV-16 cause a spectrum of pre-cancerous neoplastic changes in epithelial sites including that of the cervix. HPV-induced cervical lesions that display low to moderate neoplastic changes are graded as low-grade squamous intraepithelial neoplasia (LSIL). HPV-induced lesions that display high levels of neoplasia are graded as high-grade squamous intraepithelial neoplasia (HSIL). The previous analyses of cervical lesions of LSIL and HSIL grades have shown that the timing of early and late viral gene expression changes, as the lesion becomes more neoplastic. In HSIL lesions, the HPV-16 E7 surrogate marker MCM-7 persists throughout the epithelium, which coincides with a delay in late gene expression including L1 capsid expression. HSIL lesions often do not support a complete virus life cycle. The work in this thesis has shown that the Normal Immortalized Human Keratinocyte (NIKS) organotypic raft culture system, which is commonly used to study the HPV-16 life cycle, surprisingly recapitulates the spectrum of gene expression patterns identified in both LSIL and HSIL lesions. In addition, this model also demonstrates pathological changes, which correlate with both LSIL and HSIL phenotypes. This has rendered this system a potential model of episomal HPV-16 induced neoplasia. This auspicious finding led us to make use of this system to explore viral characteristics that potentially underlie HSIL-like gene expression patterns and neoplastic changes. Monolayer LSIL-like and HSIL-like HPV-16 cell lines were subsequently used to evaluate viral copy number, early oncogene expression and growth potential. In these experiments we have found that episomal HSIL-like HPV-16 cell lines bypass density-dependent growth arrest, which coincides with a rise in E6 and E7 oncogene levels. Finally, we have found that high E6 and E7 expression levels and growth potential at cell-cell contact correlate with MCM-7 persistence in the HPV-16 raft epithelium