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Analysis of genome sequences is enabling identification of numerous novel characteristics that provide valuable means for genetic and biochemical studies. Of these characteristics, Conserved Signature Indels (CSIs) in proteins which are specific for a given group of organisms have proven particularly useful for evolutionary and biochemical studies. My research work focused on using comparative genomics techniques to identify a large number of CSIs which are distinctive characteristics of fungi and other important groups of organisms. These CSIs were utilized to understand the evolutionary relationships among different proteins (species), and also regarding their structural features and functional significance. Based on multiple CSIs that I have identified for the PIP4K/PIP5K family of proteins, different isozymes of these proteins and also their subfamilies can now be reliably distinguished in molecular terms. Further, the species distribution of CSIs in the PIP4K/PIP5K proteins and phylogenetic analyses of these protein sequences, my work provides important insights into the evolutionary history of this protein family. The functional significance of one of the CSI in the PIP5K proteins, specific for the Saccharomycetaceae family of fungi, was also investigated. The results from structural analysis and molecular dynamics (MD) simulation studies show that this 8 aa CSI plays an important role in facilitating the binding of fungal PIP5K protein to the membrane surface. In other work, we identified multiple highly-specific CSIs in the phosphoketolase (PK) proteins, which clearly distinguish the bifunctional form of PK found in bifidobacteria from its homologs (monofunctional) found in other organisms. Structural analyses and docking studies with these proteins indicate that the CSIs in bifidobacterial PK, which are located on the subunit interface, play a role in the formation/stabilization of the protein dimer. We have also identified 2 large CSIs in SecA proteins that are uniquely found in thermophilic species from two different phyla of bacteria. Detailed bioinformatics analyses on one of these CSIs show that a number of residues from this CSI, through their interaction with a conserved network of water molecules, play a role in stabilizing the binding of ADP/ATP to the SecA protein at high temperature. My work also involved developing an integrated software pipeline for homology modeling of proteins and analyzing the location of CSIs in protein structures. Overall, my thesis work establishes the usefulness of CSIs in protein sequences as valuable means for genetic, biochemical, structural and evolutionary studies. / Dissertation / Doctor of Philosophy (PhD)

Industry demand for local sources of grain for animal feed has increased sorghum production in the mid-Atlantic region of the U.S. Sorghum anthracnose (causal agent Colletotrichum sublineola) and the grain mold complex, which includes mycotoxin-producing Fusarium spp., limit the yield and quality of grain sorghum in humid climates worldwide. A majority of U.S. grain sorghum production is in arid regions, and management strategies have not been developed for the mid-Atlantic U.S. where warm, wet conditions favor disease. The specific objectives of this research were to: (1) determine the effectiveness of fungicides and their application timing for the management of sorghum foliar anthracnose, (2) compare five grain sorghum hybrids for their susceptibility to foliar anthracnose, grain mold and mycotoxin contamination under field conditions, (3) integrate host resistance and fungicide application to manage anthracnose and grain mold, and (4) identify Fusarium spp. associated with grain mold and mycotoxin contamination of sorghum in the mid-Atlantic U.S. For Objective 1, it was determined that a single application of pyraclostrobin-containing fungicide no later than flowering reduced anthrancose, protected yield and maximized farm income. Objective 2 focused on sorghum hybrid selection as a disease management tactic, and it was determined that hybrids with high yield potential and moderate disease resistance should be selected for mid-Atlantic sorghum production in order to maximize grain yield and quality while minimizing the need for fungicide inputs. Objective 3 focused on integrated management and demonstrated that under moderate disease pressure, a high-yielding susceptible hybrid required a single application of pyraclostrobin-based fungicide to minimize fungal diseases and maintain acceptable yields, whereas under high disease pressure it was necessary to integrate hybrid resistance and judicous applications of fungicides. The aim of Objective 4 was to characterize potential causal agents of mycotoxin contamination in mid-Atlantic sorghum, and thirteen phylogenetically distinct Fusarium species (F. lacertarum, F. graminearum. F. armeniacum, F. proliferatum, F. fujikuroi, F. verticillioides, F. thapsinum and several in Fusarium incarnatum-equiseti species complex) were found to be associated with grain mold and fumonisin and/or deoxynivalenol contamination of sorghum grain. This work has provided insights into the impacts of fungal diseases on grain sorghum yield and quality in the mid-Atlantic and has aided in development of best management practices for the region. / Doctor of Philosophy / Sorghum is grown in tropics, sub-tropics and semi-arid region worldwide for food, feed, forage and fuel. Sorghum acreage in the mid-Atlantic is increasing due the demand for locally grown grain by poultry and swine industries. During the growing season, warm and humid conditions are common in the southeastern and mid-Atlantic states favoring fungal diseases development that reduce the grain yield and quality. Anthracnose and grain mold, which includes toxic mycotoxin-producing Fusarium species, are the two major constraints in sorghum production in the region. However, management alternatives have not been developed. The main goal of this research was to develop management strategies to protect yield and maximize farm profitability by controlling anthracnose and grain mold of sorghum using chemicals and/or host resistance. The specific objectives were to: (1) determine the effectiveness of fungicides and their application timing for the management of sorghum foliar anthracnose, (2) compare grain sorghum hybrids for their susceptibility to foliar anthracnose, grain mold and mycotoxin contamination under field conditions, (3) assess the value of integrating host resistance and judicious use of fungicides to manage sorghum anthracnose and grain mold, and (4) identify Fusarium spp. associated with grain mold and mycotoxin contamination of sorghum in the mid-Atlantic U.S. Results from this research indicate that a single application of pyraclostrobin-containing fungicides no later than flowering reduces anthrancose, protects yield, and increases farm income. Sorghum hybrids varied in susceptibility to anthrancnose and grain mold, and planting a moderately resistant hybrid and applying a fungicide under high disease risk conditions provided the greatest return on investment. Both fumonisin and deoxynivalenol were frequently detected from sorghum grain, and mycotoxin contamination was associated with 13 different Fusarium species from three distinct species complexes. Based on the results of this work, best management practices for minimizing sorghum disease losses were developed for the mid-Atlantic region.

The Nile tilapia Oreochromis niloticus is native to Africa and middle East, and is an important source of nutrition for many in sub-Saharan Africa. Understanding the genetic diversity within and differentiation among wild populations can help identify O. niloticus populations that are imperiled and require directed management, especially because of increasing threats to the species' long-term persistence in the wild, including habitat destruction, overfishing, climate change, and hybridization with farmed populations. Knowledge of the genetic variation among wild populations also can contribute to foundation and selection of genetically diverse populations for aquaculture. I assessed the genetic variation among tilapia populations using fin-clips collected between December 2014 and July 2017 from 14 farmed sources, mostly originating from cage farms on the Volta Lake, and 13 wild sources from nine river basins in Ghana. I also conducted a laboratory growth experiment in Ghana with two wild populations to evaluate the tolerance of different genotypes to high temperatures, to inform their development for aquaculture in West Africa. I found that pure O. niloticus populations persist in the wild but some have been extensively introgressed with the closely related species, O. aureus, which has not previously been documented in Ghana. Additionally, some wild populations appear to have recently declined significantly in numbers, likely due to overfishing and habitat modification, the latter primarily as a result of illegal alluvial mining ongoing in Ghana. Analysis of the farmed populations revealed that at least two farms were growing the unapproved genetically improved farmed tilapia (GIFT) and related strains, and that escaped individuals are admixed into some wild populations. The results of my laboratory experiment showed that O. niloticus populations occurring in northern Ghana already may be adapted to warmer temperatures and could be developed and used purposefully in aquaculture, taking advantage of their adaptation. To protect remnant pure O. niloticus populations in the wild, timely conservation decisions should be made and implemented. Protecting wild O. niloticus populations also would ensure that pure germplasms are available to develop aquaculture stocks from native populations. / Ph. D. / The Nile tilapia Oreochromis niloticus is an important food source for many people in Africa. However, many wild populations may be at risk of population decline and extinction because of increasing human activities such as overfishing and farming of non-native strains. Understanding the genetic differences among wild populations and comparing them with farmed strains can inform protection of wild populations and also help develop aquaculture strains using native populations as genetic resources. I assessed the genetic differences among tilapia populations using fin-clips I collected between December 2014 and July 2017 from 14 farmed sources, mostly originating from cage farms on the Volta Lake, and 13 wild sources from nine river basins in Ghana. I also conducted a laboratory study with two wild populations to test their tolerance to high water temperature. My research showed that pure O. niloticus populations still occur in Ghanaian rivers, but some have reproduced widely with a similar species, O. aureus, which is not known to occur in Ghanaian rivers. I also found that some wild populations may have reduced population sizes because of overfishing or because their environments have been impacted by illegal mining occurring in almost all Ghanaian rivers. My results indicated that at least two farms were growing the genetically improved farmed tilapia (GIFT) and related varieties, some of which have escaped the farms and mixed with wild populations. The results of my laboratory experiment showed that O. niloticus populations occurring in northern Ghana may be adapted to warmer water temperatures and could be selectively bred and used in aquaculture. The information generated from my research should help in making timely conservation decisions, which should help protect the remnant pure O. niloticus populations in the wild and contribute to developing aquaculture responsibly.

The transition to multicellularity has occurred numerous times in all domains of life, yet its initial steps are poorly understood. The volvocine green algae are a tractable system for understanding the genetic basis of multicellularity including the initial formation of cooperative cell groups. Here we report the genome sequence of the undifferentiated colonial alga, Gonium pectorale, where group formation evolved by co-option of the retinoblastoma cell cycle regulatory pathway. Significantly, expression of the Gonium retinoblastoma cell cycle regulator in unicellular Chlamydomonas causes it to become colonial. The presence of these changes in undifferentiated Gonium indicates extensive group-level adaptation during the initial step in the evolution of multicellularity. These results emphasize an early and formative step in the evolution of multicellularity, the evolution of cell cycle regulation, one that may shed light on the evolutionary history of other multicellular innovations and evolutionary transitions.

The butterflies in the genus Hamadryas are popular and noticeable representatives of the Neotropical Lepidoptera fauna. After a thorough taxonomic revision, 20 species were acknowledged within the genus, however no hypothesis of their phylogenetic relationship was proposed. The present dissertation provides a step further into the understanding of this fascinating group of butterflies not only by proposing the first phylogenetic hypothesis for the genus based on morphological and molecular data, but also by exploring for the first time in a group of butterflies the potential effect of venation associated with an specific behaviour on wing shape. Furthermore, this dissertation provides testable evolutionary hypotheses about the pattern of change for some of their most interesting natural history characters such as sound production and sexual dimorphism. The dissertation is organized in three chapters that can be visualized as manuscripts ready for publication; the first of these being already published (Garzón-Orduña, 2012).

Recentemente, o táxon Amblyomma cajennense sofreu uma divisão sistemática, através de estudos morfológicos, moleculares e biológicos. No Brasil, Amblyomma sculptum (pertencente ao complexo Amblyomma cajennense) tem sua importância em saúde pública relacionada à transmissão de Rickettsia rickettsii, agente etiológico da Febre Maculosa, doença esta considerada de maior importância dentre as enfermidades transmitidas por carrapatos na América Latina. A Febre Maculosa Brasileira (FMB) tem seu destaque de ocorrência na região sudeste do país, onde A. sculptum está bem estabelecido e quase sempre relacionado à presença de capivaras. Porém, nem todas as áreas da região sudeste com presença de carrapatos A. sculptum e capivaras tem notificações de transmissão de R. rickettsii para humanos. O intuito do presente estudo foi avaliar, em condições de laboratório, a susceptibilidade da infecção por R. rickettsii em seis populações geograficamente distintas de A. sculptum. Dessa forma, foi realizada a quantificação da perpetuação transestadial, da transmissão transovariana e de possíveis efeitos deletérios da infecção nos carrapatos. Além disso, tentou-se correlacionar a susceptibilidade à infecção das diferentes populações de carrapatos (de áreas endêmicas e não endêmicas para FMB) ao perfil genético das mesmas, através dos marcadores 16S rDNA mitocondrial e ITS2 nuclear. Com base nos resultados encontrados, pode-se constatar diferenças na susceptibilidade à infecção por R. rickettsii entre as seis populações de A. sculptum estudadas, muito embora todas elas se mostraram parcialmente refratárias à infecção por R. rickettsii. No entanto, não foi observado nenhum padrão específico de variabilidade nos parâmetros biológicos e reprodutivos de carrapatos infectados e não infectados. A partir dos resultados de caracterização molecular, não foi possível verificar divergências genéticas entre as diferentes populações para o marcador ITS2 nuclear. Da mesma forma, com a análise do marcador 16S rDNA mitocondrial não constatou-se divergências genéticas que pudessem ser atribuídas à maior susceptibilidade ou refratariedade dos carrapatos à infecção por R. rickettsii. Porém, vale ressaltar que variabilidade nucleotídica neste marcador foi observada entre algumas populações, que parece seguir padrões geográficos de origem. A exceção para este resultado foi a população de Belo Horizonte (Pampulha), que apresentou variabilidade intrapopulacional, com genótipo semelhante à população do Parque Nacional Grande Sertão Veredas (GSV), ambas do estado de Minas Gerais e um segundo genótipo idêntico ao de Itu, no estado de São Paulo / Recently, the taxon Amblyomma cajennense taxon resulted in a systematic division, through morphological, molecular and biological studies. In Brazil, Amblyomma sculptum (belonging to the Amblyomma cajennense complex) has its importance in public health related to Rickettsia rickettsii transmission, the etiologic agent of spotted fever, disease that is considered the most important among the tick-borne diseases in Latin America. Brazilian Spotted Fever (BSF) has its occurrence in the southeastern region of the country where A. sculptum is well established and is almost always related to presence of capybaras. However, not all areas of southeastern of Brazil with A. sculptum ticks and capybaras have notifications of transmission of R. rickettsii to humans. This study aimed to evaluate, under laboratory conditions, the R. rickettsii susceptibility among six geographically distinct populations of A. sculptum. To this purpose, the transestadial perpetuation, transovarial transmission and possible deleterious effects of R. rickettsii infection were quantified in the six tick populations. In addition, we attempted to correlate the susceptibility to infection of different populations of ticks (from BSF-endemic and BSF-non-endemic areas) to the genetic profile of them through the markers mitochondrial 16S rDNA and nuclear ITS2. The results show differences in the susceptibility to infection with R. rickettsii among the six A. sculptum populations, although all populations were partially refractory to R. rickettsii infection. However, it was not observed any specific pattern of variation in biological and reproductive parameters of infected and uninfected ticks. Results of molecular characterization did not indicate genetic divergence between the populations for nuclear ITS2 marker. In the same way, analysis of mitochondrial 16S rDNA marker found no genetic differences that could be attributed to increased susceptibility or refractory of ticks to infection by R. rickettsii. However, it is note worth that nucleotide variability in this marker was observed among some populations, which seems to follow geographical patterns of origin. The exception to this result was the Belo Horizonte (Pampulha) population, which showed intrapopulation variability, having a similar genotype to the Grande Sertão Veredas population (GSV), both from the state of Minas Gerais and a second identical genotype to the Itu, from the state of São Paulo

O gênero Pilumnus apresenta um total de 143 espécies válidas sendo representado por caranguejos distribuídos em oceanos tropicais e temperados. Estudos anteriores acerca da sistemática do gênero evidenciam um alto grau de incertezas quanto à sua classificação, provavelmente decorrente do pouco conhecimento sobre as espécies, somada a abundância de seus representantes. A classificação inicial do gênero foi baseada em caracteres morfológicos, compartilhados por diversos gêneros de Brachyura, inclusive posicionados em famílias distintas. A utilização de ferramentas moleculares tem se mostrado bastante eficaz, principalmente quando em conjunto com estudos morfológicos, no auxílio de trabalhos taxonômicos e contribuindo na construção de hipóteses filogenéticas mais robustas para os crustáceos decápodos. Além de uma análise comparativa empírica das espécies de Pilumnus do Atlântico ocidental, foram realizadas análises moleculares baseadas nos genes mitocondriais 16S rRNA e o Citocromo Oxidase I (COI) (mesma sequência do barcoding) que resultaram em 17 espécies distintas, incluíndo duas espécies novas. Foi observado uma possível estruturação genética entre populações de P. reticulatus provenientes de diferentes regiões zoogeográficas (Caribe e Brasil), e a ausência de estruturação nas espécies P. caribaeus, P. dasypodus, P. floridanus e P. vinaceus que apresentam uma ampla distribuição. Os dados morfológicos e moleculares permitiu a avaliar o status taxonômico de 21 espécies de ocorrência no Atlântico ocidental em que foi corroborada a proposição de P. brasiliensis como sinônimo júnior de P. caribaeus. Ademais, os nomes P. dasypodus e P. vinaceus devem ser considerados válidos, sendo necessário a ressurreição do último, considerado anteriormente sinônimo júnior de P. dasypodus. Com base na nossa revisão, 20 espécies são consideradas válidas e a distribuição reportada de P. diomedeae, P. longleyi e P. spinosissimus é restrita. / The genus Pilumnus has 143 valid species being represented by crabs distributed in tropical and temperate oceans. Previous studies about the systematics of the genus evidenced a high degree of uncertainty regarding its classification, probably due to the lack of knowledge about the species, in addition to the abundance of its representatives. The initial classification of the genus was based on morphological characters, shared by several genera of Brachyura, even in different families. The use of molecular tools has been shown to be very effective, especially when combined with morphological studies, in the aid of taxonomic works and contributing to the construction of more robust phylogenetic hypotheses for decapod crustaceans. In addition to an empirical comparative analysis of Pilumnus species from the western Atlantic, molecular analyzes based on mitochondrial 16S rRNA and Cytochrome Oxidase I (COI) genes were performed, resulting in 17 distinct species, including two new species. It was observed a possible genetic structuring between P. reticulatus populations from different zoogeographic regions (Caribbean and Brazil), and the absence of genetic structuring in the species P. caribaeus, P. dasypodus, P. floridanus and P. vinaceus that present a wide distribution. The morphological and molecular data allowed evaluating the taxonomic status of 21 species with occurrence in the western Atlantic in which the proposition of P. brasiliensis as a junior synonym of P. caribaeus was corroborated. In addition, the names P. dasypodus and P. vinaceus should be considered valid, being necessary the resurrection of the last, previously considered junior synonym of P. dasypodus. Based on our review, 20 species are considered valid and the reported distribution of P. diomedeae, P. longleyi and P. spinosissimus is restricted.

Dados da Organização Mundial da Saúde (OMS) mostram que a raiva é um problema de saúde pública podendo acarretar sérios prejuízos ambientais e econômicos, a despeito da existência de vacinas eficazes de uso humano e veterinário. Segundo seu último informe, estima-se que no mundo em torno de 55.000 pessoas por ano morrem de raiva. O cão permanece como principal transmissor da raiva para o homem e também como principal vítima da doença. Nos países que conseguiram controlar a raiva em animais domésticos, o vírus se mantém circulante na natureza por meio dos animais silvestres, sendo os morcegos apontados como a segunda espécie transmissora da raiva a humanos. Os Lyssavirus têm sido detectados em morcegos, em diversos continentes, sendo identificados como transmissor em dez das onze espécies de Lyssavirus. Fósseis de morcego mostram sua presença há 50 milhões de anos. Mas somente em 1911, Carini relacionou pela primeira vez a raiva aos morcegos, levantando a hipótese destes serem os transmissores da doença a outros animais. Há registros de que o vírus da raiva foi isolado em pelo menos 41 das 167 espécies de morcegos brasileiras, sendo que a maioria dessas espécies está relacionada a atividades humanas com a presença destes animais próximos ao local de trabalho e moradia das pessoas. Os morcegos hematófagos Desmodus rotundus são encontrados do norte do México até a costa norte do Chile, região central da Argentina e costa do Uruguai e com exceção do Chile. Esta espécie de morcego tem sido apontada como reservatório natural do vírus da raiva nesta região. Alguns pesquisadores observaram que a raiva em morcegos não hematófagos precede a raiva bovina e em animais de estimação, sugerindo que os morcegos não hematófagos podem ser o elo entre a raiva silvestre e a raiva urbana e o fato de se detectar a variante mantida por morcegos hematófagos Desmodus rotundus em cães e gatos mostra que o papel deste morcego no ciclo da raiva não está limitado à raiva silvestre. As características dos Lyssavirus adaptados a morcegos têm mostrado diferenças quando comparadas à raiva relacionada aos carnívoros, confirmando a necessidade do desenvolvimento de metodologias que permitam estudos complementares mais precisos a respeito da biologia e epidemiologia da raiva em quirópteros. A escassez de dados na literatura, até o momento, a respeito do genoma completo da variante do vírus da raiva mantida por populações de morcegos hematófagos Desmodus rotundus, deixa uma lacuna no entendimento da epidemiologia molecular deste vírus. A importância epidemiológica desta espécie na transmissão da raiva é inquestionável. Neste estudo foi sequenciado e analisado, o genoma da variante do vírus da raiva mantido por populações de morcego hematófago Desmodus rotundus isolado de um morcego hematófago Desmodus rotundus. A amostra, procedente de área endêmica no Estado de São Paulo, foi filogeneticamente comparada com o genoma da amostra padrão para a espécie viral 1 - Rabies virus e outras amostras pertencentes ao ciclo aéreo ou terrestre de transmissão, disponíveis no GenBank, identificando possíveis padrões de diferenciação, próprios do ciclo aéreo, e em alguns casos relacionados somente à variante estudada. / Data from the World Health Organization (WHO) show that rabies is a public health problem which can cause serious environmental and economic damage, despite the existence of effective vaccines for human and veterinary use. According to WHO latest report, estimated that worldwide around 55,000 people per year died of rabies. The dog remains the main transmitter of rabies to humans as well as the main victim of the disease. In countries that were successful in controlling rabies in domestic animals, the virus is still circulating in nature by wild animals and the bats are seen as the second species transmitting rabies to humans. The Lyssavirus have been detected in bats in several continents and is identified as a transmitter in ten of eleven species of Lyssavirus. Bat fossils show their presence for 50 million years. But only in 1911, in the first time Carini related to rabies at bats, raising the possibility of these being the transmitters of the disease to other animals. Reports show that the Rabies virus was isolated in at least 41 of the 167 species of bats in Brazil, with the majority of these species is related to human activities with the animals living near the local job and houses of people. The vampire bat Desmodus rotundus is found from northern Mexico to northern Chile coast, central coast of Argentina and Uruguay and with the exception of Chile. This bat species has been identified as a natural reservoir of the Rabies virus in this region. Some researchers observed that rabies into non-hematophagous bats precedes the bovine rabies and in pets, suggesting that the non-hematophagous bats may be the link between wildlife rabies and urban rabies and the fact that detect the variant maintained by vampire bats Desmodus rotundus in dogs and cats shows that the role of bat rabies in the cycle is not limited to wildlife rabies. The characteristics of Lyssavirus bat adapted have been shown differences when compared to rabies related to the carnivores, confirming the need to develop methods that enable more accurate follow-up studies about the biology and epidemiology of rabies in bats. The paucity of data in the literature to date about the complete genome of the Rabies virus variant maintained by populations of vampire bats Desmodus rotundus leaves a gap in understanding the molecular epidemiology of this virus and the epidemiological importance of this species in the transmission of Rabies virus is unquestionable. In this study we sequenced and analyzed the genome of the Rabies virus variant maintained by populations of bat Desmodus rotundus isolated from a bat Desmodus rotundus. The sample, coming from an endemic area in São Paulo, was phylogenetically compared with the genome of the standard sample for spcies 1 - Rabies virus and other samples belonging to the Terrestrial and Aerial cycles of transmission, available in GenBank, to identify possible patterns of differentiating themselves Aerial cycle and in some cases linked only to variant studied.

Bdellovibrionaceae sind außergewöhnliche Bakterien, da sie als die kleinsten bekannten räuberischen Organismen gelten. Seit ihrer Entdeckung in Bodenproben im Jahr 1962 durch Stolp und Starr konnte eine weite Verbreitung in der Natur nachgewiesen werden. Besonderes Interesse erlangten Bdellovibrionaceae durch ihre Fähigkeit bakterielle Erreger wie Escherichia coli, Salmonella und Yersinia zu attackieren. Der bestuntersuchte Vertreter der Familie Bdellovibrionaceae ist Bdellovibrio bacteriovorus. Er durchläuft einen komplexen Lebenszyklus, der nur partiell verstanden ist. In der Attack-Phase werden durch einen noch nicht aufgeklärten Mechanismus potentielle Beutebakterien erkannt und der interzelluläre Kontakt hergestellt. Nachdem eine Pore in der Zellwand der ausschließlich Gram-negativen Beutebakterien erzeugt wurde, dringt B. bacteriovorus in den periplasmatischen Raum ein. Nach etwa 30 Minuten ist der Invasionsprozess abgeschlossen und man kann die Ausbildung sphärischer Bdelloblasten beobachten. Im Beutebakterium verdaut B. bacteriovorus makromolekulare Bestandteile des Wirtes und wächst zu einem langen spiralförmigen Stäbchen aus. Es setzt schließlich die Zellteilung ein bei der 5 bis 30 Tochterzellen in Abhängigkeit zur Größe des Beutebakteriums freiwerden. Mit der Reproduktion ist der parasitäre Lebenszyklus nach etwa 3 Stunden beendet. Die komplexe Regulation der Expression von Proteinen konnte für die einzelnen Wachstumsphasen durch ein- sowie zweidimensionale Gelelektrophorese nachgewiesen werden. In der Vergangenheit haben verschiedene Studien die Komplexität der Wechselwirkung mit den Beutebakterien belegt. Hierbei wurde, neben dem offensichtlichen Abbau makromolekularer Bestandteile der Beutebakterien, ein Recycling und Einbau von Membranbestandteilen wie Lipopolysacchariden (LPS) und Outer Membrane Proteine (OmP) in das Membransystem von B. bacteriovorus diskutiert. Die biologische Interpretation dieses Phänomens ist eine erhöhte Effizienz in der Reproduktion von B. bacteriovorus wenn es Bestandteile des Wirtsbakteriums wiederverwertet im Gegensatz zur Eigensynthese. Trotz der morphologischen Ähnlichkeit und des ungewöhnlichen Lebensstils wurde festgestellt, dass die Familie der Bdellovibrionaceae phylogenetisch sehr heterogen ist. Es werden zur Zeit zwei Gattungen unterschieden Bdellovibrio und Bacteriovorax. Aufgrund von 16S rRNA Untersuchungen konnten auch innerhalb der Gattungen eine Vielzahl von phylogenetisch distinkten Arten nachgewiesen werden. In der Literatur wird eine regulierende Wirkung auf pathogene Gram-negative Bakterien für aquatische Systeme durch Bdellovibrionaceae beschrieben. Weiterhin konnten sie bei verschiedenen Nutztieren im Verdauungstrakt mikrobiologisch nachgewiesen werden. Ein positiver Einfluss auf die Gesundheit der Tiere wurde bei Vorkommen von B. bacteriovorus festgestellt. Für eine Charakterisierung von Umweltisolaten werden in der vorliegenden Arbeit genetische Methoden vorgestellt. Hierfür wurden Hybridisierungsmethoden und PCR-methoden entwickelt, die es ermöglichen aus der Umwelt isolierte Bdellovibrionaceae phylogenetisch einzuordnen. Es konnte gezeigt werden, das sowohl B. bacteriovorus als auch Bacteriovorax stolpii im Verdauungstrakt von Nutztieren vorkommen. Es ist weiterhin gelungen eine PCR-methode für Kotproben zu entwickeln, die einen direkten Nachweis ermöglicht ohne mikrobiologische Anzucht. B. bacteriovorus ist ein Gram-negatives Bakterium, allein dieser Sachverhalt spiegelt die Schwierigkeit wider, wenn der enzymattische Abbau von Zellwandbestandteilen der Beutebakterien Ziel der Untersuchung ist, da auch die Beutebakterien Gram-negativ sind. Es ergibt sich aufgrund eines ähnlichen Zellwandaufbaus ein immenses analytisches Problem die makromolekularen Bestandteile von Wirtsbakterium und Jäger zu trennen. Um dem Lebensstil angepasst Modifikationen von B. bacteriovorus zu untersuchen, wurde in dieser Arbeit LPS, charakteristischer Bestandteil der Äußeren Membran, von Wildtypstamm B. bacteriovorus HD100 und seiner wirtsunabhängigen Mutante HI100 isoliert und das Lipid A strukturell aufgeklärt. Die Isolation gelang durch die Ausnutzung unterschiedlicher Fällungseigenschaften des LPS von B. bacteriovorus HD100 gegenüber des E. coli K-12 LPS aus dem Extraktionsmittel. Weiterhin konnte nachgewiesen werden, das B. bacteriovorus S-Form LPS besitzt. Die außergewöhnliche Struktur des Lipid A von B. bacteriovorus wurde im Detail mit massenspektrometrischen Methoden, ein- und zweidimensionalen NMR Methoden sowie mikrochemischer Methoden in Kombination mit der GC/MS charakterisiert. Der Fettsäureanker besteht aus a-D-ManpII-(1(R)4)-ß-D-GlcpN3NII-(1(R)6)-ß-D-GlcpN3NI-(1(R)1)-a-D-ManpI. Dies stellt eine neuartige Struktur dar, da an allen bisher bekannten Lipid A´s sich Brönstedtsäuren am hydrophilen Fettsäureanker befinden, die in physiologischer Lösung durch Protonenabgabe negative Ladungen tragen. Im Lipid A von B. bacteriovorus sind diese Substituenten durch den Neutralzucker Mannose ersetzt. Weiterhin wurden als Fettsäuren nur 3-Hydroxyfettsäuren nachgewiesen, wobei sich auf die 6 gebunden Fettsäuren etwa 1,5 Doppelbindungen verteilen. Dieses Lipid A zeigt eine wesentlich reduzierte endotoxische Aktivität im Vergleich zu E. coli Lipid A und weist als biophysikalische Besonderheit eine erhöhte Fluidität über einen weiten Temperaturbereich auf. Die vorgestellte 16S rRNA Analyse und die Strukturanalyse des Lipid A von B. bacteriovorus belegen seine besondere Stellung in der Welt der Bakterien. / Bdellovibrionaceae are extraordinary bacteria known as the smallest predatory organism so far. Since their discovery in soil samples by Stolp and Starr 1963 they have been detected in a wide range of other natural habitats. Bdellovibrionaceae became the focus of attention concerning their ability to attack pathogens like Escherichia coli, Salmonella and Yersinia. For Bdellovibrio bacteriovorus the most detailed studies are available. The up to now only partially understood lifecycle consists of several complex phases. In the attack phase B. bacteriovorus is motile possessing flagella and the preys are recognized by an unknown mechanism. After attachment on the cell wall within 15 to 30 minutes a pore is formed which is used as entrance to the periplasmatic space of the Gram-negative prey bacteria. The completion of the invasion process can be observed by the change of the prey s shape to spherical bdelloblasts. Inside of the prey B. bacteriovorus degrades macromolecular compounds and transforms into a long spirally shaped rod. At the end of the lifecycle the rod divides yielding 5 up to 30 daughter cells depending on the size of the prey bacteria. This reproduction phase is completed within 3 hours. The complex regulation of expression of a certain number of proteins was observed by one and two dimensional gelelectrophoresis. The complexity of the interaction between predator and prey were examined in several studies. Besides the obvious degradation of macromolecular compounds of the prey, reutilising of lipopolysaccharides (LPS) and Outer Membrane Proteins (OmP) into the membrane system of B. bacteriovorus was discussed. The biological interpretation of such behaviour was that it is more efficient for reproduction to recycle components of the prey than to perform de novo synthesis. Unexpectedly, despite the unique predatory lifecycle and common morphological features, Bdellovibrionaceae show a great phylogenetical diversity based on 16S rRNA analyses. Bdellovibrionaceae are divided into the three species Bdellovibrio bacteriovorus, Bacteriovorax stolpii, Bacteriovorax starrii and some strains yet to be assigned. In two studies Bdellovibrionaceae were found as part of regulation processes for decreasing the number of pathogenic Gram-negative bacteria in aquatic systems. Furthermore, B. bacteriovorus were found in the intestinal tract of several domestic animals showing a positive influence on the state of health. For the phylogenetic characterization of environmental isolates techniques were developed based on hybridisation methods and the PCR. In this work we detected B. bacteriovorus and B. stolpii strains in the gut of animals. Furthermore, a PCR method for direct detection of Bdellovibrionaceae in fecal samples was developed. B. bacteriovorus are Gram-negative bacteria. This fact complicates the study of the degradation of the prey s cell wall as it possesses the architecture of Gram-negative bacteria also. Furthermore, the search for important modifications of the cell wall of B. bacteriovorus concerning the predatory lifestyle becomes an analytical problem. In this study we isolated LPS of the wild type strain B. bacteriovorus HD100 and its host independent mutant strain HI100. For the isolation of pure B. bacteriovorus HD100 S-form LPS we took advantage of different precipitation properties in the extraction solvent of E. coli K-12 LPS and the predator s LPS. The structure of the lipid A was examined in detail by mass spectrometric methods, one- and two-dimensional NMR and chemical analytical techniques. The novel structure consists of backbone built of a-D-ManpII-(1(R)4)-ß-D-GlcpN3NII-(1(R)6)-ß-D-GlcpN3NI-(1(R)1)-a-D-ManpI. This is the first known natural lipid A without negatively charged substituents in physiological solution. The lipid A of B. bacteriovorus carries the neutral sugar mannose instead of Brönstedt acids. Furthermore, the lipid A exclusively consists of 3-hydroxy fatty acids with approximately 1.5 double bounds distributed on six bounded fatty acids. This lipid A shows a significant decreased endotoxic activity in comparison to E. coli lipid A and revealed increased fluidity over broad temperature range as further remarkable biophysical property. The 16S rRNA analysis and the structural analysis of the lipid A of B. bacteriovorus document the unique position in the world of bacteria.