Bdellovibrio bacteriovorus, is a small mono-flagellate Gram-negative delta-proteobacterium, which has a bi-phasic lifecycle, consisting of a predatory phase; in which they invade on other Gram-negative bacteria and digest the prey cell’s content to grow and septate, or host independent phase; in which they can grow and septate in media rich in amino acids as well as vitamins and cofactors. As B. bacteriovorus can kill other Gram-negative bacteria including pathogens, they have potential to be used as a ‘living antibiotic’. I have been part of this field since 2004, a time at which the first B. bacteriovorus genome (HD100) had just been sequenced and made available, and only one study into making deletion mutants had been published. During my time in this field, the research has expanded almost exponentially, with the understanding of core pathways and systems that make B. bacteriovorus so novel being highlighted and greatly understood. In addition new techniques and methodologies never before attempted in B. bacteriovorus research have been made possible and I have been lucky to be a part of this and carried out some of the work myself. In particular I have worked on the mutation and phenotype testing of genes encoding pathways for motility, prey cell lysis, B. bacteriovorus intra-cellular signalling, and bi-phasic growth switching. These advances from my work including an animal trial into the predatory nature of B. bacteriovorus have laid the foundation for its use as a novel ‘living antibiotic’ in the future.

Abstract in HTML and technical report in PDF available on the Massachusetts Institute of Technology Joint Program on the Science and Policy of Global Change website (http://mit.edu/globalchange/www/) / Includes bibliographical references (p. 31). / Supported in part by the Univ. of Washington's Joint Institute for the Study of the Atmosphere and Ocean (JISAO) under NOAA Cooperative Agreement (NA67RJ0155 841). Also supported in part by MIT's Joint Program on the Science and Policy of Global Change, with support from the U.S. Dept. of Energy's Office of Biological and Environmental Research to (DE-FG02-93ER61677).

El trabajo realizado ha pretendido abordar la pinturadel mar y los conceptos generales que la representan. Seha intentado demostrar c´omo a trav´es de la imagen delmar entendida como trayecto fenomenol´ogico y los componentesque definen su materialidad est´etica se puede representarla sensaci´on de infinitud que la misma evocapartiendo de la consideraci´on de que su idea se encuentrainscrita en su misma imagen como realidad. En nuestrainterpretaci´on nos hemos dejado guiar por la metodolog´ýadel imaginario de Gilbert Durand y de la fenomenolog´ýade Gaston Bachelard y Merleau-Ponty demostrando que elmar como imagen no se centra tan s´olo en su realidad sinoque es libre de ser relacionado con diferentes valores quesubjetivizan su figura confiri´endole gran potencialidad comofigura. Sobre estas coordenadas hemos construido lasdiferentes propuestas analizadas. El estudio ha adoptadouna estructura tripartita y se ha movido dentro del campode la metaf´ýsica. En la primera parte se han determinadolos valores y las relaciones que dan personalidad est´eticaal mar singularizados en el color, el movimiento y el sonidoextrapolando su infinitud como concepto aut´onomoy como objeto de estudio individual de la segunda parte.Bas´andonos en el contexto pict´orico europeo del siglo XIXsu lectura se ha hecho desde el cruce de dos ejes ontol´ogicamenteopuestos que determinan el dinamismo de su idea:Su horizontalidad abordada desde su ilimitaci´on y desde la perspectiva c´ýclica de su temporalidad tomando la figuradel viaje y del barco como medios de interpretaci´on. Ysu verticalidad entendida desde la noci´on de profundidady enfocada desde la bidireccionalidad de su concepto quenos ha revelado la experiencia de trascendencia inmanentey existencial del mar a trav´es de la representaci´on delnaufragio. Esta propuesta nos ha guiado en nuestra ´ultimaparte hacia una interpretaci´on del sentimiento de infinituddel mar en la pintura valenciana de la segunda mitad delsiglo XIX estableciendo los valores y los medios de representaci´on utilizados en su captaci´on teniendo como representantesal grupo de los llamados marinistas puros encabezadosen primer lugar por Rafael Monle´on, Javier Juste,Salvador Abril, Enrique Saborit, Benito Lleonart y PedroFerrer y en segundo por Ignacio Pinazo, Joaqu´ýn Sorolla yAntonio Mu noz Degrain. Con nuestro estudio hemos constatadoque el mar como figura parad´ojica y contradictoriaincita la expresi´on de su infinitud desde perspectivas diversas.Hemos demostrado que la finalidad de los artistasseleccionados no ha sido s´olo modificar la realidad de laimagen del mar apoy´andose en la imaginaci´on para capturarla sensaci´on de infinito que la misma preludia sinoir en la b´usqueda del sentido de su existencia a trav´es delos conceptos y valores del mismo. / The present work addresses marine painting and thegeneral concepts underlying it. It sets out to demonstratehow the image of the sea as a phenomenologicalpath, and the components that define its aesthetic materiality,can represent the feeling of infinity that the imageevokes, starting from the consideration that the idea ofinfinity is written into the image of the sea as a reality.We were guided in our interpretation by the methodologyof the imaginary by Gilbert Durand and the phenomenologyof Gaston Bachelard and Merleau-Ponty in order todemonstrate that the sea, as image, is not only based onits reality, but it can also relate to different values whichsubjectivize its figure, giving it potential as a figure. Onthese principles we have constructed the various proposalsanalysed in this work. The work is structured in threeparts and is based on the field of metaphysics. In the firstpart we determined the values and relationships that conferaesthetic personality to the sea and that are identifiedthrough color, motion and sound, extrapolating its infinityas an independent concept that is the object of individualstudy in the second part. Set in the context of XIX centuryEuropean painting, its interpretation is based on theintersection of two ontologically opposed axes that determinethe dynamics of its idea: Its horizontality, seen fromits unlimitedness and from the cyclical perspective of itstemporality, that has the image of the journey and of the ship as interpretation keys; And its verticality, perceivedfrom the notion of depth and interpreted from the bidirectionalityof its concept, which revealed to us the experienceof the immanent and existential transcendence of the seathrough the representation of the shipwreck. This suggestionguided us in the last part towards an interpretationof the feeling of the infinity of the sea in the Valencianpainting of the second half of the XIX century, establishingthe values and the means of representation usedin understanding it, keeping as representatives the puremarine painters headed firstly by Rafael Monle´on, JavierJuste, Salvador Abril, Enrique Saborit, Benito Lleonartand Pedro Ferrer, and secondly by Ignacio Pinazo, Joaqu´ýnSorolla and Mu noz Degra´ýn . As a result of our analysis,we state that the sea, as a paradoxical and contradictoryfigure, creates the expression of its infinity from differentperspectives. We demonstrated that the purpose of theselected artists was not only to modify the reality of thesea image relying on the imagination to capture the feelingof infinity that it suggests, but also to pursue the meaningof its existence through its own concepts and values.

Bdellovibrio bacteriovorus are small, predatory bacteria that grow within the periplasmic space of a host bacterium. Bdellovibrio has a biphasic life-cycle switching from a uni-nucleoid, growth-senescent ‘attack-phase’ to a novel, multi-nucleoid filamentous ‘growth-phase’, which elongates and divides, growing saprophytically within the periplasmic space of their prey. Little is known to date about Bdellovibrio developmental processes and cell division within this periplasmic niche. Recent publications have demonstrated that bacterial cytoplasms house highly organised matrices of protein structures, called the bacterial cytoskeleton. The Bdellovibrio processes of prey-cell entry, filamentous cell growth and division coordination brings cellular morphological changes and challenges that could be coordinated by cytoskeletal elements. Green Fluorescent protein (GFP)-tagging and gene knock out approaches were used to gain insights into the function of these elements including: an Intermediate filament like protein Ccrp, which has a role in the maintenance of cell morphology; two actin homologues, which appear to function at different points in the predatory cycle, MreB1 and MreB2; and a new type of cytoskeletal element designated ‘bactofilin’, which may have a role in cell division control. Recent advances in GFP technologies have led to the development of optimised GFP variants, such as mTFP1 and mCherry. These have been used to reveal previously unseen detail of Bdellovibrio development within prey. Bdellovibrio do not follow the familiar pattern of bacterial cell division by binary fission, instead divide synchronously at multiple sites along their length, once prey resources are depleted. This yields both odd and even numbers of progeny Bdellovibrio.

To investigate the effectiveness of physical forces in destroying microorganisms a heat resistant (D7Soc=20 min) Gram-positive vegetative bacterium, Microbacterium lacticum, a Gram-negative vegetative bacterium, Escherichia coli, and vegetative cells and spores of Gram-positive bacterium, Bacillus subtilis, were subjected to high mechanical energies using gelatin and maize grits as carriers. A twin screw extruder, a piston capillary rheometer and a rotational rheometer were used. When extruded with gelatin there was a strong correlation between the destruction of M. lacticum and the die wall shear stress and specific mechanical energy (SME). Within the limit of detection, no surviving M. lacticum could be detected in gelatin at the highest die wall shear stress of 409 kPa and a SME of 390 kJ -kg" , giving at least 5.3 decimal reductions. There was no surviving M. lacticum in maize grits at 289 kPa die wall shear stress and 294 kl-kg" SME, giving a 4.6 decimal reduction. The temperature at the extruder die remained below 61°C for all the extrusion experiments indicating that the bacterial destruction was due to combined shear and thermal forces rather than thermal forces alone. It was suggested that the thermal energy supplied during extrusion weakened the bacterial cell wall, making the cells susceptible to shear forces. A maximum 3.2 decimal reduction in the number of spores of B. subtilis in maize grits was obtained at 595 kPa die wall shear stress and 844 kJ.kg-1 SME, below 43°C extruder die temperature. There was no statistically significant difference in the survival of B. subtilis PS346 and B. subtilis PS361, which is a heat sensitive strain due to lack of (l- and P-SASP proteins, under the same extrusion conditions, suggesting that the main destruction mechanism was not heat. High reduction in the number of the viable spores suggested a possible "mechanical germination" inside the dynamic environment of the extruder. A 4.2 logarithmic reduction in the number of M lacticum in 30% (wwb) moisture content gelatin was observed in an unsheared sample in the piston capillary rheometer at 192 MPa and 60°C, showing that pressure could cause major destruction at high temperatures (60-75°C). No survival of M. lacticum was detected beyond 695 kPa shear stress and 64 MPa at 60°C, suggesting that an optimum combination of shear, thermal and pressure forces can cause an important reduction in the numbers of vegetative cells. Shearing of the microorganisms in the rotational rheometer in gelatin showed that the shear resistance of the microorganisms were different. Although only three species of bacteria were tested, it appeared that Gram-positive bacteria were more resistant to shear forces than Gram-negative bacteria. The results suggested that the destruction of the microorganisms at low shear forces (-3 kPa shear stress) was due to weakening of the bacterial cell wall at temperatures above 60°C. A maximum 1.4 logarithmic reduction in the number of M. lacticum was achieved after 4 min of shearing at 804 S-1 shear rate and 75°C. Based on the heat resistance data, thermal forces were not enough to cause significant destruction in the numbers of the microorganism, however, the temperature played a significant role by weakening the bacterial cell wall making it susceptible to shear forces. In this context, it is possible that there was a synergistic relationship between the shear and thermal forces. A shear D-value concept was introduced which was used to evaluate the shear resistance of microorganisms at different temperatures. Starch conversion (determined by differential scanning calorimeter) due to low temperature extrusion of maize grit inoculated with M. lacticum and spores of B.subtilis showed that there is a positive correlation between the bacterial destruction and the starch conversion. Up to 94% starch conversion was obtained during low temperature extrusion of maize grit where the estimated degree of starch conversion due to heat alone was 3.8%. The results suggested that if the shear forces can be optimally combined with thermal forces, an acceptable sterility can be achieved at significantly lower temperatures which would help to keep the quality of food products high.

Bacteria have been isolated from virtually every environment on Earth. The Antarctic continent is no exception. In this extremely cold and dry environment bacteria have colonised various refugia and have evolved a number of strategies for coping with the extreme physico-chemical fluctuations they are exposed to within the environment. These psychrophilic adaptations include cold adapted proteins and lipids which are interest for biotechnology in areas such as frozen foods, agriculture and cryogenic storage. One type of cold adapted protein of particular interest is the antifreeze protein (AFP) for its recrystalisation inhibition and thermal hysteresis activity. It was first isolated from Antarctic fish in the 1970, but has since been found in plants, fungi, insects and bacteria. Over 800 bacterial isolates were cultured from lakes of the, Vestfold Hills, Larsemann Hills and MacRobertson Land, Antarctica. Approximately 87% were Gram negative rods. A novel AFP assay designed for high-throughput analysis in Antarctica, demonstrated putative activity in 187 isolates. Subsequent SPLAT analysis (qualification assessment of recrystalisation inhibition activity) of the putative positive isolates showed 19 isolates with significant recrystalisation inhibition activity. These 19 isolates were cultured from five separate lakes with substantial physico-chemical differences. The 19 AFP active isolates were characterised, using amplified ribosomal DNA restriction analysis (ARDRA) and 16S rDNA sequencing, as predominantly belonging to genera from the a- and y-Proteobacteria, although they were more prominent in the gamma subdivision. One of these isolates (213, Halomonas sp.) was shown as dominant within its community by DGGE analysis, indicating a possible selective advantage for AFP active bacteria. This is the first report of the phylogenetic distribution of AFP activity within bacteria, thus providing information which could enable future bacterial AFP assessments to be aimed at specific taxonomic groups.

Ten overlapping fragments covering the entire Clostridium difficile toxin A gene were cloned and expressed in Escherichia coli. Eight fragments (a', a2, b, c, d, e, f and g) represented the first 5.55kb of the gene whereas two fragments (hl and h2) each spanned the entire C-terminal repeat region of the molecule. All activities relating to binding to carbohydrates (i. e. cold haemagglutination of rabbit erythrocytes), binding to bovine thyroglobulin and non-specific binding to a murine monoclonal antibody were restricted solely to peptides H1 (amino acids [aa] 1834-2683) and H2 (aa 1832-2683). Peptide H2 alone also displayed the ability to bind to cells and to be internalised into endosome-like compartments within the cells. Taken together with the observation that peptide H2 caused a cytopathic effect on Vero cells which was atypical of the holotoxin, these results may indicate that the repeat region of toxin A stimulates intracellular signalling pathways prior to Rho glucosylation. Peptide A2 (aa 1-536) glucosylated recombinant RhoA (rRhoA) in vitro, whereas peptides A'(aa 1-205), B (aa 542-859), C (aa 114-859), D (aa 869-1330), E (aa 542- 1161), G (aa 869-1830) and H2 (aa 1832-2683) did not. The results obtained for peptides A', A2 and C indicate that the first 536 as encompass the catalytic domain for this activity, that more than the first 205 as alone are needed for expression of enzymic activity, and that for a peptide to be active it must not lack the first 113 aa. The first 113 as of the holotoxin are probably essential for the correct folding of the catalytic domain and expression of its activity. These studies were also the first to locate the toxin A ATP binding site to a peptide spanning as 542-859 (peptide B) of the holotoxin. Antibody reaction profiles of antiserum to holotoxin A against toxin A peptides and of antiserum to the peptides against holotoxin A indicate that this region is unexposed in the native state. Also of interest was the observation that the only peptides, which contained the nucleotidebinding site (B and E), lacked the ability to glucosylate rRhoA. Further peptide A2, which possessed glucosyltransferase activity, lacked the nucleotide-binding site. These studies therefore, suggest that a nucleotide-binding site is not required for in vitro glucosylation of rRhoA by toxin A, and fail to identify a role for the toxin A nucleotide binding site. An engineered truncated form of toxin A, consisting of the first 539 as of the holotoxin (encompassing glucosyltransferase activity) fused to the 852 as C-terminal peptide H2 (repeat end binding portion) caused a conventional cytopathic effect (CPE), but was 1,400 fold less cytotoxic to Vero cells than the holotoxin. Peptide A2 (aa 1-536) alone had no effect on Vero cells or in rabbit ileal loops suggesting that peptide H2 aided delivery of the glucosyltransferase molecule into cells leading to a CPE. The truncated toxin lacked the nucleotide binding site and the putative membrane-translocating domain (internal hydrophobic region). The reduced activity of the truncated toxin suggests that although not essential for cytotoxic activity, the nucleotide-binding site and the internal hydrophobic region are important for stability and/or efficient translocation of the holotoxin into the cytosol.

In this study several components responsible for the formation and function of the unidirectional flagellum of R. sphaeroides WS8 were identified via the characterisation of motility impaired TnphoA mutants. The role of the alternative sigma factor sigma 54 in flagellar gene regulation was also examined. Mutant M18 was defective in a fliI homologue, characterisation of this mutant revealed that FliI is not essential for flagellar formation in R. sphaeroides. This differs from that reported in the literature for S. typhimurium and so highlights the importance of studying R. sphaeroides as a model for flagellar motility. Analysis of another mutant Nm7 revealed that it was defective in FliF, a rotor component around which other flagellar components assemble. Overexpression of a FliF fusion protein allowed the production of anti FliF antiserum. DNA sequencing upstream and downstream of the fliF gene, revealed several other genes encoding flagellar components and a potential flagellar gene regulator (Torf). fliE, encoding a component of the basal body of unknown function, was identified upstream of fliF, an interposon mutant was created and was unable to be complemented by the wild type gene in trans suggesting a dominant effect. This is the first dominant mutation to be isolated in any fliE . The gene encoding the motor component FliG was also identified downstream of fliF and its C-terminal motility domain was found to contain regions that are conserved between FliG proteins from unidirectional and bidirectional motors, these may play a role in motor rotation and not switching. An overexpressed poly histidine FliG fusion protein was found to form a complex with the FliF-GST fusion protein ill vitro. The torf gene encodes a protein with homology to sigma 54 enhancer binding proteins. The Torf protein lacks any obvious DNA binding motif and may represent a novel member of the sigma 54 enhancer binding protein family.

Phyllis A. Wallace. / Includes bibliographical references.

Stuart E. Madnick. / Bibliography: leaves 20-22.