Streptococcus gordonii is a primary coloniser of the tooth surface where it efficiently ferments carbohydrates at pH levels above 6.0. By not being able to maintain the pH of dental plaque to a level required for enamel dissolution, the dominance of S. gordonii in dental plaque is considered a sign of a healthy oral cavity. However, upon entering the bloodstream and encountering a rise in pH, S. gordonii may become pathogenic, being one of the major causative organisms associated with infective endocarditis. Proteome analyses of S. gordonii grown at steady state in a chemostat allowed the phenotypic changes associated with alterations in pH levels characteristic of these two environments to be determined. As an initial starting point to this study, a two-dimensional electrophoresis (2- DE) reference map of S. gordonii grown at pH 7.0 was produced. Although only 50% of the S gordonii genome was available in an annotated form during the course of this study, the closely related Streptococcus pneumoniae genome (with which S. gordonii shares 97.24% DNA sequence homology) had been completed in 2001. The use of both of these databases allowed many of the S. gordonii proteins to be identified by mass spectrometry. Four hundred and seventy six protein spots, corresponding to 250 different proteins, or 12.5% of the S. gordonii proteome, were identified, giving rise to the first comprehensive proteome reference map of this oral bacterium. Of the 250 different proteins, 196 were of cellular origin while 68 were identified from the extracellular milieu. Only 14 proteins were common to both compartments. Of particular interest among the 54 uniquely identified extracellular proteins was a homologue of a peptidoglycan hydrolase that has been associated with virulence in S. pneumoniae. Among the other proteins identified were ones involved in transport and binding, energy metabolism, translation, transformation, stress response and virulence. Twelve cell envelope proteins were identified as well as 25 others that were predicted to have a membrane association based on the presence of at least one transmembrane domain. The study also confirmed the existence of 38 proteins previously designated as �hypothetical� or with no known function. Mass spectral data for over 1000 protein spots were accumulated and archived for future analysis when sequencing of the S. gordonii genome is finally completed. Following the mapping of the proteome of S. gordonii, alterations in protein spots associated with growth of the bacterium at pH intervals of 0.5 units in the pH range 5.5 - 7.5 were determined. Only 16 protein spots were shown to be significantly altered in their level of expression despite the range of pH studied. Among the differentially expressed proteins was a manganese-dependent inorganic pyrophosphatase (PpaC), which regulates expression of adhesins required for coaggregation. The expression of PpaC was highest at pH 6.5 - 7.0, the pH of a healthy oral cavity, indicating that PpaC may play an important part in dental plaque formation. Another differentially expressed protein was the heat-inducible transcription repressor (HrcA). Alterations in HrcA were consistent with its role as a negative repressor in regulating heat-shock proteins at low pH, even though no changes in the level of heat-shock proteins were observed as the pH declined. This result gave rise to the hypothesis that the possible reason cariogenic bacteria, such as Streptococcus mutans, can out compete S. gordonii at low pH might simply be due to their ability to manipulate their proteome in a complex manner for survival and persistence at low pH, unlike S. gordonii. This may imply some prevailing level of genetic regulation that is missing in S. gordonii.

Zugl.: Magdeburg, Univ., Diss., 2008

Ulm, Univ., Diss., 2003.

A produção de PHAMCL por Pseudomonas putida IPT 046 em cultivo contínuo sob limitação múltipla de nutrientes foi estudada, utilizando glicose e frutose como fontes de carbono. O estudo da limitação em nitrogênio, fósforo e simultânea de nitrogênio e fósforo para indução de acúmulo de polihidroxialcanoato, apontou que maiores valores de polímero foram acumulados quando fósforo foi o nutriente limitante (70%). Limitação em nitrogênio resultou no máximo em 40% de polímero. A limitação simultânea de nitrogênio e fósforo resultou em 68% de polímero, menor consumo de oxigênio para gerar células e maior fator de conversão de fonte de carbono em polímero (0,19 g/g) quando comparado com a limitação em fósforo, 0,16 g/g, ou à limitação em nitrogênio, 0,10 g/g. Oxigênio utilizado para manutenção das células não dependeu do tipo de limitação e dos valores das vazões específicas de alimentação. Um modelo metabólico foi proposto, ajustando aos dados experimentais na condição de limitação em nitrogênio. / Multiple nutrient limited growth of Pseudomonas putida IPT 046 was studied in chemostat culture from glucose and fructose as carbon source. Nitrogen, phosphorus and both nitrogen and phosphorus limitation was performed in order to accumulate medium-chain-length polhydroxyalkanoate. Phosphorus limitation resulted in higher polymer content accumulated (70%). Nitrogen limited assays achieved only 40% of polymer. Nitrogen and phosphorus simultaneous limitation resulted in 68 % polymer content, less oxygen demand to synthesize cells and best yields of carbon into polymer (0,19 g/g) when compared with 0,16 g/g on phosphorus limitation and 0,10 g/g on nitrogen limitation. Oxygen demand for maintenance is the same, independent of limitation strategy or dilution rate performed. A metabolic pathway model was proposed and fitted with nitrogen limited experimental data.

No description available.

Natural microbial communities are the ultimate drivers of change in any ecosystem. Through chemical contamination of natural environments, these communities are exposed to many different types of chemical stressors; however, research on whole genome responses to this contaminant stress is limited. This research examined the stress response of a common soil bacterium, <i>Pseudomonas aeruginosa</i>, to a common environmental pollutant, pentachlorophenol (PCP). In the first part of the research, it was revealed that nutrient-limited <i>P. aeruginosa</i> is able to respond to PCP with minimal physiological damage due to the upregulation of multidrug efflux pumps. Further study of this PCP-mediated induction of efflux pumps revealed a simultaneous increase in antibiotic resistance. It was discovered that the resistance nodulation-cell division (RND) efflux pump, MexAB-OprM, in particular is responsible for the PCP-induced increase in antibiotic resistance. Both whole cell physiological indicators and whole genome analysis were used to examine the stress response of <i>P. aeruginosa</i> to PCP. Cells were grown in a chemostat at a low growth rate to simulate nutrient-limiting growth in the natural environment. Whole cell acetate uptake rates (WAUR) and viable cell counts as colony forming units (CFU) were determined as cells were exposed to increasing concentration of PCP. At the same time, changes in gene expression were examined by Affymetrix microarray technology. Results showed little change in whole-cell physiology, with no difference in WAUR and only a slight reduction in CFU. However, the microarrays revealed that over 100 genes either increased or decreased expression greater than two-fold due to the PCP exposure. In particular, multiple multidrug efflux genes were upregulated in response to the PCP. The results were validated by real time reverse transcription polymerase chain reaction (RT-PCR) for one of these genes. Further analysis of the effects of MexAB-OprM showed that this particular efflux pump is essential for the response of <i>P. aeruginosa</i> to the toxin PCP. Induction of multidrug efflux pumps is responsible for the development of antibiotic resistance in strains of <i>P. aeruginosa</i>. Therefore, it was investigated whether PCP might induce resistance to a variety of antibiotics. The research was further extended to examine the effect of a variety of organic contaminants on MexAB-OprM efflux and antibiotic resistance development. PCP, 2,4-dinitrophenol, benzoate and Roundup® all induced antibiotic resistance. However, although MexAB-OprM is required for optimal growth in the presence of all chemicals, this particular efflux pump is only involved in increased resistance with PCP. This was confirmed using RT-PCR as <i>mexB</i> expression was induced by PCP, but not by the other three chemicals. A long term generational study on the effects of PCP did not result in a stable antibiotic-resistant phenotype; however, RT-PCR showed that <i>mexB</i> induction is a direct result of PCP exposure and can be reversed by removal of PCP. Together, these results demonstrate the necessity to understand functional responses to contaminant stress. Discovery of direct induction of multidrug efflux pumps and the resulting increase in antibiotic resistance has significant implications for environmental microbiology and public health. This research suggests that organic contamination may result in antibiotic resistance and that antibiotic resistant strains may have a survival advantage in contaminated environments. / Ph. D.

Our goal in this thesis is to study the dynamics of the classical predator-prey model and the predator-prey model in the chemostat when a discrete delay is introduced to model the time between the capture of the prey and its conversion to biomass. In both models we use Holling type I response functions so that no oscillatory behavior is possible in the associated system when there is no delay. In both models, we prove that as the parameter modelling the delay is varied Hopf bifurcation can occur. However, we show that there seem to be differences in the possible sequences of bifurcations. Numerical simulations demonstrate that in the classical predator-prey model period doubling bifurcation can occur, possibly leading to chaos while that is not observed in the chemostat model for the parameters we use. For a delay differential equation, a prerequisite for Hopf bifurcation is the existence of a pair of pure imaginary eigenvalues for the characteristic equation associated with the linerization of the system. In this case, the characteristic equation is a transcendental equation with delay dependent coefficients. For our models, we develop two different methods to show how to find values of the bifurcation parameter at which pure imaginary eigenvalues occur. The method used for the classical predator-prey model was developed first. However, it was necessary to develop a more robust, less complicated method to analyze the predator-prey model in the chemostat with a discrete delay. The latter method was then generalized so that it could be applied to any second order transcendental equation with delay dependent coefficients. / Thesis / Doctor of Philosophy (PhD)

It has been shown that the classical Rosenzweig-MacArthur predator-prey model is sensitive to the functional form of the predator response. To see if this sensitivity remains in the highly controlled environment of the chemostat, we use a predator-prey model with three trophic levels and a Holling type II predator response function. We first focus on the analysis of the model using an Ivlev functional response. Local and global dynamics are studied, with global stability of the coexistence equilibrium point obtained under certain conditions. Bifurcation analysis reveals the existence of a stable periodic orbit that appears via a super-critical Hopf bifurcation. The uniqueness of this periodic orbit is explored. Finally, we make comparisons between the dynamics of the model with Ivlev response and Monod response, both of which have nearly identical graphs. The same sensitivity to functional form is observed in the chemostat as in the classical model. / Thesis / Master of Science (MSc)

Estudou-se a influência do fosfato na multiplicação de Saccharomyces cerevisiae em uma fermentação contínua em mini-fermentador. Determinou-se a massa seca, a concentração dos substratos, a velocidade específica de consumo dos substratos, a velocidade específica de formação de gás carbônico, velocidade específica de consumo de oxigênio e o quociente respiratório bem como, o fósforo intracelular em diversas vazões específicas de alimentação, em cultivo contínuo de levedura de panificação, em condições de substratos limitantes. Controlando-se a quantidade de fosfato no meio de alimentação de tal modo que o fosfato residual no meio de fermentação mal pudesse ser detectado, o valor da vazão específica de alimentação crítica era aparentemente aumentado de 0,23 h-1 para 0,32 h-1. Isto sugere uma possível influência do fosfato nas funções anaeróbicas e aeróbicas da levedura de panificação. / The influence of phosphate in a continuous culture was studied using mini-fermentor on the Saccharomyces cerevisiae multiplication. Dry matter, substrate concentration, specific substrate comsumption, specific carbon dioxide release, specific oxygen uptake rates and respiration quotient , as well as phosphorous content of the cells were measured in dependence on the dilutionrate. In continuous culture glicose-limited, of baker\'s yeast if the supply of phosphorous were restricted to a extent that residual phosphate in the medium could hardly be observed, the value of critical dilution rate was apparently enhanced from 0,23 h-1 to 0,32 h-1. This observation suggests a possible mediation by phosphate between anaerobic and aerobic functions of the baker\'s yeast.

Nature changes continuously and is only seemingly at equilibrium. Environmental parameters like temperature, humidity or insolation may strongly fluctuate on scales ranging from seconds to millions of years. Being part of an ecosystem, species have to cope with these environmental changes. For ecologists, it is of special interest how individual responses to environmental changes affect the dynamics of an entire population – and, if this behavior is predictable. In this context, the demographic structure of a population plays a decisive role since it originates from processes of growth and mortality. These processes are fundamentally influenced by the environment. But, how exactly does the environment influence the behavior of populations? And what does the transient behavior look like? As a result from environmental influences on demography, so called cohorts form. They are age or size classes that are disproportionally represented in the demographic distribution of a population. For instance, if most old and young individuals die due to a cold spell, the population finally consists of mainly middle-aged individuals. Hence, the population got synchronized. Such a population tends to show regular fluctuations in numbers (denoted as oscillations) since the alternating phases of individual growth and population growth (due to reproduction) are now performed synchronously by the majority of the population.That is, one time the population growths, and the other time it declines due to mortality. Synchronous behavior is one of the most pervasive phenomena in nature. Gravitational synchrony in the solar system; fireflies flashing in unison; coordinate firing of pacemaker cells in the heart; electrons in a superconductor marching in lockstep. Whatever scale one looks at, in animate as well as inanimate systems, one is likely to encounter synchrony. In experiments with phytoplankton populations, I could show that this principle of synchrony (as used by physicists) could well-explain the oscillations observed in the experiments, too. The size of the fluctuations depended on the strength by which environmental parameters changed as well as on the demographic state of a population prior to this change. That is, two population living in different habitats can be equally influenced by an environmental change, however, the resulting population dynamics may be significantly different when both populations differed in their demographic state before. Moreover, specific mechanisms relevant for the dynamic behavior of populations, appear only when the environmental conditions change. In my experiments, the population density declined by 50% after ressource supply was doubled. This counter-intuitive behavior can be explained by increasing ressource consumption. The phytoplankton cells grew larger and enhanced their individual constitution. But at the same time, reproduction was delayed and the population density declined due to the losses by mortality. Environmental influences can also synchronize two or more populations over large distances, which is denoted as Moran effect. Assume two populations living on two distant islands. Although there is no exchange of individuals between them, both populations show a high similarity when comparing their time series. This is because the globally acting climate synchronizes the regionally acting weather on both island. Since the weather fluctuations influence the population dynamics, the Moran effect states that the synchrony between the environment equals the one between the populations. My experiments support this theory and also explain deviations arising when accounting for differences in the populations and the habitats they are living in. Moreover, model simulations and experiments astonishingly show that the synchrony between the populations can be higher than between the environment, when accounting for differences in the environmental fluctuations (“noise color”). / Die Natur unterliegt ständigen Veränderungen und befindet sich nur vermeintlich in einem Gleichgewicht. Umweltparameter wie Temperatur, Luftfeuchtigkeit oder Sonneneinstrahlung schwanken auf einer Zeitskala von Sekunden bis Jahrmillionen und beinhalten teils beträchtliche Unterschiede. Mit diesen Umweltveränderungen müssen sich Arten als Teil eines Ökosystems auseinandersetzen. Für Ökologen ist interessant, wie sich individuelle Reaktionen auf die Umweltveränderungen im dynamischen Verhalten einer ganzen Population bemerkbar machen und ob deren Verhalten vorhersagbar ist. Der Demografie einer Population kommt hierbei eine entscheidende Rolle zu, da sie das Resultat von Wachstums- und Sterbeprozessen darstellt. Eben jene Prozesse werden von der Umwelt maßgeblich beeinflusst. Doch wie genau beeinflussen Umweltveränderungen das Verhalten ganzer Populationen? Wie sieht das vorübergehende, transiente Verhalten aus? Als Resultat von Umwelteinflüssen bilden sich in Populationen sogenannte Kohorten, hinsichtlich der Zahl an Individuen überproportional stark vertretene Alters- oder Größenklassen. Sterben z.B. aufgrund eines außergewöhnlich harten Winters, die alten und jungen Individuen einer Population, so besteht diese anschließend hauptsächlich aus Individuen mittleren Alters. Sie wurde sozusagen synchronisiert. Eine solche Populationen neigt zu regelmäßigen Schwankungen (Oszillationen) in ihrer Dichte, da die sich abwechselnden Phasen der individuellen Entwicklung und der Reproduktion nun von einem Großteil der Individuen synchron durchschritten werden. D.h., mal wächst die Population und mal nimmt sie entsprechend der Sterblichkeit ab. In Experimenten mit Phytoplankton-Populationen konnte ich zeigen, dass dieses oszillierende Verhalten mit dem in der Physik gebräuchlichen Konzept der Synchronisation beschrieben werden kann. Synchrones Verhalten ist eines der verbreitetsten Phänomene in der Natur und kann z.B. in synchron schwingenden Brücken, als auch bei der Erzeugung von Lasern oder in Form von rhythmischem Applaus auf einem Konzert beobachtet werden. Wie stark die Schwankungen sind, hängt dabei sowohl von der Stärke der Umweltveränderung als auch vom demografischen Zustand der Population vor der Veränderung ab. Zwei Populationen, die sich in verschiedenen Habitaten aufhalten, können zwar gleich stark von einer Umweltveränderung beeinflusst werden. Die Reaktionen im anschließenden Verhalten können jedoch äußerst unterschiedlich ausfallen, wenn sich die Populationen zuvor in stark unterschiedlichen demografischen Zuständen befanden. Darüber hinaus treten bestimmte, für das Verhalten einer Population relevante Mechanismen überhaupt erst in Erscheinung, wenn sich die Umweltbedingungen ändern. So fiel in Experimenten beispielsweise die Populationsdichte um rund 50 Prozent ab nachdem sich die Ressourcenverfügbarkeit verdoppelte. Der Grund für dieses gegenintuitive Verhalten konnte mit der erhöhten Aufnahme von Ressourcen erklärt werden. Damit verbessert eine Algenzelle zwar die eigene Konstitution, jedoch verzögert sich dadurch die auch die Reproduktion und die Populationsdichte nimmt gemäß ihrer Verluste bzw. Sterblichkeit ab. Zwei oder mehr räumlich getrennte Populationen können darüber hinaus durch Umwelteinflüsse synchronisiert werden. Dies wird als Moran-Effekt bezeichnet. Angenommen auf zwei weit voneinander entfernten Inseln lebt jeweils eine Population. Zwischen beiden findet kein Austausch statt – und doch zeigt sich beim Vergleich ihrer Zeitreihen eine große Ähnlichkeit. Das überregionale Klima synchronisiert hierbei die lokalen Umwelteinflüsse. Diese wiederum bestimmen das Verhalten der jeweiligen Population. Der Moran-Effekt besagt nun, dass die Ähnlichkeit zwischen den Populationen jener zwischen den Umwelteinflüssen entspricht, oder geringer ist. Meine Ergebnisse bestätigen dies und zeigen darüber hinaus, dass sich die Populationen sogar ähnlicher sein können als die Umwelteinflüsse, wenn man von unterschiedlich stark schwankenden Einflüssen ausgeht.