Homing events in the gyrA gene of some mycobacteria.

The A subunit of DNA gyrase in Mycobacterium leprae, unlike its counterpart in Mycobacterium tuberculosis, is produced by protein splicing as its gene, gyrA, harbors a 1260-bp in-frame insertion encoding an intein, a putative homing endonuclease. Analysis of the gyrA locus from different mycobacterial species revealed the presence of inteins in Mycobacterium flavescens, Mycobacterium gordonae and Mycobacterium kansasii but not in 10 other pathogenic or saprophytic mycobacteria. In all four cases where intein coding sequences were found, they were localized in the same position in gyrA, immediately downstream of the codon for the key active-site residue Tyr-130. The intein products were similar, but not identical, in sequence and the splice junctions displayed all the features found in other polypeptides known to be produced by protein splicing from a precursor protein. Paired motifs, found in homing endonucleases encoded by some group I RNA introns, and inteins showing endonuclease activity, were present in the gyrA inteins as were other intein-specific signatures. Some strains of M. flavescens, M. gordonae, and M. kansasii were shown by PCR analysis to have inteinless gyrA genes, in contrast to the situation in M. leprae where all the isolates possessed insertions in gyrA. Sequencing of the corresponding regions revealed that, although the GyrA protein sequence was conserved, the nucleotide sequences differed in gyrA genes with and without inteins, suggesting that the homing endonuclease displays sequence specificity.

Relações filogenéticas das espécies de Crenicichla Heckel, 1840 e Teleocichla Kullander, 1988 (Teleostei: Cichlidae)

São reconhecidos 90 nomes específicos válidos de Crenicichla e oito de Teleocichla. Juntos, os dois gêneros-alvo do presente estudo compreendem quase 1/5 da diversidade total de Cichlinae, subfamília neotropical de Cichlidae. Apesar das espécies de Crenicichla e Teleocichla formarem um clado bem corroborado através de filogenias baseadas tanto em dados morfológicos quanto em dados moleculares, as relações entre as suas espécies foram ainda pouco estudadas. Os dois estudos filogenéticos de Crenicichla conhecidos são parcialmente discordantes entre si e incorporaram apenas uma parcela da diversidade do grupo. Baseados apenas em dados moleculares, não foram acompanhados por um estudo de caracteres morfológicos que indicariam as sinapomorfias ou características diagnósticas para identificação dos grupos monofiléticos delimitados. No presente estudo, os principais objetivos consistem em testar o monofiletismo do grupo formado pelas espécies de Crenicichla e Teleocichla e identificar e definir unidades monofiléticas dentro desse grupo, com base na análise cladística de caracteres morfológicos. Como objetivo secundário, são testadas as recentes hipóteses de relações filogenéticas dessas espécies com as demais espécies de ciclídeos neotropicais. Foram incluídas todas as espécies válidas de Teleocichla e 54 espécies válidas de Crenicichla (60% das espécies válidas), além de uma espécie nova de Teleocichla e cinco prováveis espécies novas de Crenicichla. 20 representantes de diferentes linhagens de Cichlinae foram incluídos, totalizando 88 táxons terminais. As análises cladísticas foram realizadas a partir de uma matriz com 211 caracteres provenientes do estudo comparado de morfologia externa, incluindo padrões de colorido e osteologia. Além da análise com pesagens igualitárias, foram explorados também os resultados das análises com pesagem implícita utilizando diferentes valores da variável k e com pesagem sucessiva. A partir da comparação e discussão dos resultados obtidos a partir das diferentes análises, a topologia obtida através da análise com pesagem implícita utilizando o valor de k=3 foi escolhida para obtenção das inferências filogenéticas. Duas classificações alternativas foram discutidas e, a fim de minimizar mudanças nomenclaturais, aquela baseada no reconhecimento de subgêneros de Crenicichla correspondendo aos grupos monofiléticos encontrados foi preferida em detrimento da proposta baseada no reconhecimento de vários gêneros. Isso porque o posicionamento de Crenicichla macrophthalma (espécie-tipo de Crenicichla) continua sendo considerado instável. O gênero Crenicichla é corroborado como um grande clado formado por todas as espécies de Crenicichla e Teleocichla incluídas e é sustentado por 40 sinapomorfias não-ambíguas. Um complexo cenário foi encontrado quanto às relações entre as espécies de Crenicichla, com várias linhagens dentro desse grande grupo, assim como era previsto de acordo com estudos filogenéticos prévios. As relações entre essas linhagens, por outro lado, são ainda instáveis, podendo variar de acordo com os diferentes tipos de pesagem aplicados e apresentam algumas divergências em relação aos estudos prévios, que também divergem entre si. Teleocichla é um grupo monofilético dentro de Crenicichla e foi considerado como um de seus oito subgêneros. O subgênero Crenicichla é constituído apenas por Crenicichla macrophthalma. Os seis subgêneros restantes (Wallaciia, Batrachops, Hemeraia, Saxatilia, Lugubria e Lacustria) correspondem totalmente ou parcialmente a grupos de espécies de Crenicichla previamente existentes na literatura. Em Lacustria, quatro complexos de espécies foram delimitados: C. missioneira, C. scotti, C. jaguarensis e C. lacustris sensu stricto. Foram listadas as espécies nominais de cada subgênero e uma diagnose para auxiliar a identificação dos mesmos foi elaborada. Uma nova hipótese de relações de Crenicichla em Cichlinae é inferida a partir da análise realizada, na qual Crenicichla é grupo-irmão de um clado formado por Chaetobranchus flavescens e todos os representantes de Cichlasomatini e Geophagini incluídos

Carbon and nitrogen limitation increase chitosan antifungal activity in Neurospora crassa and fungal human pathogens

Chitosan permeabilizes plasma membrane and kills sensitive filamentous fungi and yeast. Membrane fluidity and cell energy determine chitosan sensitivity in fungi. A five-fold reduction of both glucose (main carbon (C) source) and nitrogen (N) increased 2-fold Neurospora crassa sensitivity to chitosan. We linked this increase with production of intracellular reactive oxygen species (ROS) and plasma membrane permeabilization. Releasing N. crassa from nutrient limitation reduced chitosan antifungal activity in spite of high ROS intracellular levels. With lactate instead of glucose, C and N limitation increased N. crassa sensitivity to chitosan further (4-fold) than what glucose did. Nutrient limitation also increased sensitivity of filamentous fungi and yeast human pathogens to chitosan. For Fusarium proliferatum, lowering 100-fold C and N content in the growth medium, increased 16-fold chitosan sensitivity. Similar results were found for Candida spp. (including fluconazole resistant strains) and Cryptococcus spp. Severe C and N limitation increased chitosan antifungal activity for all pathogens tested. Chitosan at 100 μg ml-1 was lethal for most fungal human pathogens tested but non-toxic to HEK293 and COS7 mammalian cell lines. Besides, chitosan increased 90% survival of Galleria mellonella larvae infected with C. albicans. These results are of paramount for developing chitosan as antifungal.

Acanthamoeba polyphaga trophozoite binding of representative fungal single cell forms

Acanthamoeba polyphaga trophozoites bind yeast cells of Candida albicans isolates within a few hours, leaving few cells in suspension or still attached to trophozoite surfaces. The nature of yeast cell recognition, mediated by an acanthamoebal trophozoite mannose binding protein is confirmed by experiments utilizing concentration dependent mannose hapten blocking. Similarly, acapsulate cells of Cryptococcus neoformans are also bound within a relatively short timescale. However, even after protracted incubation many capsulate cells of Cryptococcus remain in suspension, suggesting that the capsulate cell form of this species is not predated by acanthamoebal trophozoites. Further aspects of the association of Acanthamoeba and fungi are apparent when studying their interaction with conidia of the biocontrol agent Coniothyrium minitans. Conidia which readily bind with increasing maturity of up to 42 days, were little endocytosed and even released. Cell and conidial surface mannose as determined by FITC-lectin binding, flow cytometry with associated ligand binding analysis and hapten blocking studies demonstrates the following phenomena. Candida isolates and acapsulate Cryptococcus expose most mannose, while capsulate Cryptococcus cells exhibit least exposure commensurate with yeast cellular binding or lack of trophozoites. Conidia of Coniothyrium, albeit in a localized fashion, also manifest surface mannose exposure but as shown by Bmax values, in decreasing amounts with increasing maturity. Contrastingly such conidia experience greater trophozoite binding with maturation, thereby questioning the primacy of a trophozoite mannose-binding-protein recognition model.

Five new species of yeasts from fresh water and marine habitats in the Florida Everglades

Yeast populations in the Shark River Slough of the Florida Everglades, USA, were examined during a 3-year period (2002–2005) at six locations ranging from fresh water marshes to marine mangroves. Seventy-four described species (33 ascomycetes and 41 basidiomycetes) and an approximately equal number of undescribed species were isolated during the course of the investigation. Serious human pathogens, such as Candida tropicalis, were not observed, which indicates that their presence in coastal waters is due to sources of pollution. Some of the observed species were widespread throughout the fresh water and marine habitats, whereas others appeared to be habitat restricted. Species occurrence ranged from prevalent to rare. Five representative unknown species were selected for formal description. The five species comprise two ascomycetes: Candida sharkiensis sp. nov. (CBS 11368T) and Candida rhizophoriensis sp. nov. (CBS 11402T) (Saccharomycetales, Metschnikowiaceae), and three basidiomycetes: Rhodotorula cladiensis sp. nov. (CBS 10878T) in the Sakaguchia clade (Cystobasidiomycetes), Rhodotorula evergladiensis sp. nov. (CBS 10880T) in the Rhodosporidium toruloides clade (Microbotryomycetes, Sporidiobolales) and Cryptococcus mangaliensis sp. nov. (CBS 10870T) in the Bulleromyces clade (Agaricomycotina, Tremellales).

Compilation of maximum ingestion and maximum clearance rate data for marine pelagic organisms

The metabolic rate of organisms may either be viewed as a basic property from which other vital rates and many ecological patterns emerge and that follows a universal allometric mass scaling law; or it may be considered a property of the organism that emerges as a result of the organism's adaptation to the environment, with consequently less universal mass scaling properties. Data on body mass, maximum ingestion and clearance rates, respiration rates and maximum growth rates of animals living in the ocean epipelagic were compiled from the literature, mainly from original papers but also from previous compilations by other authors. Data were read from tables or digitized from graphs. Only measurements made on individuals of know size, or groups of individuals of similar and known size were included. We show that clearance and respiration rates have life-form-dependent allometries that have similar scaling but different elevations, such that the mass-specific rates converge on a rather narrow size-independent range. In contrast, ingestion and growth rates follow a near-universal taxa-independent ~3/4 mass scaling power law. We argue that the declining mass-specific clearance rates with size within taxa is related to the inherent decrease in feeding efficiency of any particular feeding mode. The transitions between feeding mode and simultaneous transitions in clearance and respiration rates may then represent adaptations to the food environment and be the result of the optimization of tradeoffs that allow sufficient feeding and growth rates to balance mortality.

Structural and Biochemical Dissection of the Trehalose Biosynthetic Complex in Pathogenic Fungi

Trehalose is a non-reducing disaccharide essential for pathogenic fungal survival and virulence. The biosynthesis of trehalose requires the trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. More importantly, the trehalose biosynthetic pathway is absent in mammals, conferring this pathway as an ideal target for antifungal drug design. However, lack of germane biochemical and structural information hinders antifungal drug design against these targets.

In this dissertation, macromolecular X-ray crystallography and biochemical assays were employed to understand the structures and functions of proteins involved in the trehalose biosynthetic pathway. I report here the first eukaryotic Tps1 structures from Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus) with substrates or substrate analogs. These structures reveal the key residues involved in substrate binding and catalysis. Subsequent enzymatic assays and cellular assays highlight the significance of these key Tps1 residues in enzyme function and fungal stress response. The Tps1 structure captured in its transition-state with a non-hydrolysable inhibitor demonstrates that Tps1 adopts an “internal return like” mechanism for catalysis. Furthermore, disruption of the trehalose biosynthetic complex formation through abolishing Tps1 dimerization reveals that complex formation has regulatory function in addition to trehalose production, providing additional targets for antifungal drug intervention.

I also present here the structure of the Tps2 N-terminal domain (Tps2NTD) from C. albicans, which may be involved in the proper formation of the trehalose biosynthetic complex. Deletion of the Tps2NTD results in a temperature sensitive phenotype. Further, I describe in this dissertation the structures of the Tps2 phosphatase domain (Tps2PD) from C. albicans, A. fumigatus and Cryptococcus neoformans (C. neoformans) in multiple conformational states. The structures of the C. albicans Tps2PD -BeF3-trehalose complex and C. neoformans Tps2PD(D24N)-T6P complex reveal extensive interactions between both glucose moieties of the trehalose involving all eight hydroxyl groups and multiple residues of both the cap and core domains of Tps2PD. These structures also reveal that steric hindrance is a key underlying factor for the exquisite substrate specificity of Tps2PD. In addition, the structures of Tps2PD in the open conformation provide direct visualization of the conformational changes of this domain that are effected by substrate binding and product release.

Last, I present the structure of the C. albicans trehalose synthase regulatory protein (Tps3) pseudo-phosphatase domain (Tps3PPD) structure. Tps3PPD adopts a haloacid dehydrogenase superfamily (HADSF) phosphatase fold with a core Rossmann-fold domain and a α/β fold cap domain. Despite lack of phosphatase activity, the cleft between the Tps3PPD core domain and cap domain presents a binding pocket for a yet uncharacterized ligand. Identification of this ligand could reveal the cellular function of Tps3 and any interconnection of the trehalose biosynthetic pathway with other cellular metabolic pathways.

Combined, these structures together with significant biochemical analyses advance our understanding of the proteins responsible for trehalose biosynthesis. These structures are ready to be exploited to rationally design or optimize inhibitors of the trehalose biosynthetic pathway enzymes. Hence, the work described in this thesis has laid the groundwork for the design of Tps1 and Tps2 specific inhibitors, which ultimately could lead to novel therapeutics to treat fungal infections.

Collection of aboveground community and species-specific plant biomass from the Jena Experiment (time series since 2002).

This data set comprises time series of aboveground community plant biomass (Sown plant community, Weed plant community, Dead plant material, and Unidentified plant material; all measured in biomass as dry weight) and species-specific biomass from the sown species of several experiments at the field site of a large grassland biodiversity experiment (the Jena Experiment; see further details below). Aboveground community biomass was normally harvested twice a year just prior to mowing (during peak standing biomass twice a year, generally in May and August; in 2002 only once in September) on all experimental plots in the Jena Experiment. This was done by clipping the vegetation at 3 cm above ground in up to four rectangles of 0.2 x 0.5 m per large plot. The location of these rectangles was assigned by random selection of new coordinates every year within the core area of the plots. The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: individual species for the sown plant species, weed plant species (species not sown at the particular plot), detached dead plant material (i.e., dead plant material in the data file), and remaining plant material that could not be assigned to any category (i.e., unidentified plant material in the data file). All biomass was dried to constant weight (70°C, >= 48 h) and weighed. Sown plant community biomass was calculated as the sum of the biomass of the individual sown species. The data for individual samples and the mean over samples for the biomass measures on the community level are given. Overall, analyses of the community biomass data have identified species richness as well as functional group composition as important drivers of a positive biodiversity-productivity relationship. The following series of datasets are contained in this collection: 1. Plant biomass form the Main Experiment: In the Main Experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). 2. Plant biomass from the Dominance Experiment: In the Dominance Experiment, 206 grassland plots of 3.5 x 3.5 m were established from a pool of 9 species that can be dominant in semi-natural grassland communities of the study region. In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 3, 4, 6, and 9 species). 3. Plant biomass from the monoculture plots: In the monoculture plots the sown plant community contains only a single species per plot and this species is a different one for each plot. Which species has been sown in which plot is stated in the plot information table for monocultures (see further details below). The monoculture plots of 3.5 x 3.5 m were established for all of the 60 plant species of the Jena Experiment species pool with two replicates per species like the other experiments in May 2002. All plots were maintained by bi-annual weeding and mowing.