Molecular characterisation of the mechanisms of compatible solute accumulation in Listeria monocytogenes

The ability of the Gram-positive foodborne pathogen Listeria monocytogenes to survive and grow in environments of elevated osmolarity can be attributed, at least in part, to the accumulation of a restricted range of low molecular mass solutes compatible with cellular function. Accumulated to high internal concentrations in hyper-saline environments, compatible solutes, either transported into the cell or synthesized de novo, play a dual role: helping to stabilize protein structure and function while also counterbalancing external osmotic strength, thus preventing water loss from the cell and plasmolysis. While previous physiological investigations identified glycine betaine, carnitine, and proline as the principal compatible solutes in the listerial osmostress response, genetic alanysis of the uptake/synthesis systems governing the accumulation of these compounds has, until now, remained largely unexplored. Representing the first genetic analysis of compatible solute accumulation in L. monocytogenes, this thesis describes the molecular characterization of BetL; a highly specific secondary glycine betaine transport system, OpuC; a multicomponent carnitine/glycine betaine transporter, and finally proBA; a two-gene operon encoding the first two enzymes of the listerial proline piosynthesis pathway. In addition to their role in osmotolerance, the potential of each system in contributing to listerial pathogenesis was investigated. While mutations in each gene cluster exhibited dramatic reductions in listerial osmotolerance, OpuC- mutants were additionally shown to exhibit reduced virulence when admisistered via the oral route. This represents the first direct link between the salt stress response and virulence in L. monocytogenes.

Osmotic stress tolerance mechanisms in Lactococcus lactis

The response of Lactococcus lactis subsp. cremoris NCDO 712 to low water activity (aw) was investigated, both in relation to growth following moderate reductions in the aw and in terms of survival following substantial reduction of the aw with NaCI. Lc.lactis NCDO 712 was capable of growth in the presence of ≤ 4% w/v NaCI and concentrations in excess of 4% w/v were lethal to the cells. The presence of magnesium ions significantly increased the resistance of NCDO 712 to challenge with NaCI and also to challenge with high temperature or low pH. Survival of Lc.lactis NCDO 712 exposed to high NaCI concentrations was growth phase dependent and cells were most sensitive in the early exponential phase of growth. Pre-exposure to 3% w/v NaCI induced limited protection against subsequent challenge with higher NaCI concentrations. The induction was inhibited by chloramphenicol and even when induced, the response did not protect against NaCI concentrations> 10% w/v. When growing at low aw, potassium was accumulated by Lc. lactis NCDO 712 growing at low aw, if the aw was reduced by glucose or fructose, but not by NaCI. Reducing the potassium concentration of chemically defined medium from 20 to 0.5 mM) produced a substantial reduction in the growth rate, if the aw was reduced with NaCI, but not with glucose or fructose. The reduction of the growth rate correlated strongly with a reduction in the cytoplasmic potassium concentration and in cell volume. Addition of the compatible solute glycine betaine, partially reversed the inhibition of growth rate and partially restored the cell volume. The potassium transport system was characterised in cells grown in medium at both high and low aw. It appeared that a single system was present, which was induced approximately two-fold by growth at low aw. Potassium transport was assayed in vitro using cells depleted of potassium; the assay was competitively inhibited by Na+ and by the other monovalent cations NH4+, Li+, and Cs+. There was a strong correlation between the ability of strains of Lc. lactis subsp. lactis and subsp. cremoris to grow at low aw and their ability to accumulate the compatible solute glycine betaine. The Lc. lactis subsp. cremoris strains incapable of growth at NaCI concentrations> 2% w/v did not accumulate glycine betaine when growing at low aw, whereas strains capable of growth at NaCI concentrations up to 4% w/v did. A mutant, extremely sensitive to low aw was isolated from the parent strain Lc. lactis subsp. cremoris MG 1363, a plasmid free derivative of NCDO 712. The parent strain tolerated up to 4% w/v NaCI and actively accumulated glycine betaine when challenged at low aw. The mutant had lost the ability to accumulate glycine betaine and was incapable of growth at NaCI concentrations >2% w/v or the equivalent concentration of glucose. As no other compatible solute seemed capable of substitution for glycine betaine, the data suggest that the traditional; phenotypic speciation of strains on the basis of tolerance to 4% w/v NaCI can be explained as possession or lack of a glycine betaine transport system.

Transport energy demand: techno-economic modelling and scenarios for Irish climate policy

The case for energy policy modelling is strong in Ireland, where stringent EU climate targets are projected to be overshot by 2015. Policy targets aiming to deliver greenhouse gas and renewable energy targets have been made, but it is unclear what savings are to be achieved and from which sectors. Concurrently, the growth of personal mobility has caused an astonishing increase in CO2 emissions from private cars in Ireland, a 37% rise between 2000 and 2008, and while there have been improvements in the efficiency of car technology, there was no decrease in the energy intensity of the car fleet in the same period. This thesis increases the capacity for evidenced-based policymaking in Ireland by developing techno-economic transport energy models and using them to analyse historical trends and to project possible future scenarios. A central focus of this thesis is to understand the effect of the car fleet‘s evolving technical characteristics on energy demand. A car stock model is developed to analyse this question from three angles: Firstly, analysis of car registration and activity data between 2000 and 2008 examines the trends which brought about the surge in energy demand. Secondly, the car stock is modelled into the future and is used to populate a baseline “no new policy” scenario, looking at the impact of recent (2008-2011) policy and purchasing developments on projected energy demand and emissions. Thirdly, a range of technology efficiency, fuel switching and behavioural scenarios are developed up to 2025 in order to indicate the emissions abatement and renewable energy penetration potential from alternative policy packages. In particular, an ambitious car fleet electrification target for Ireland is examined. The car stock model‘s functionality is extended by linking it with other models: LEAP-Ireland, a bottom-up energy demand model for all energy sectors in the country; Irish TIMES, a linear optimisation energy system model; and COPERT, a pollution model. The methodology is also adapted to analyse trends in freight energy demand in a similar way. Finally, this thesis addresses the gap in the representation of travel behaviour in linear energy systems models. A novel methodology is developed and case studies for Ireland and California are presented using the TIMES model. Transport Energy