Abstract The genetic diversity and identification of slow- and fast- growing soybean root nodule bacterial isolates from different agro-climatic regions in Mpumalanga, Limpopo and Gauteng Provinces of South Africa were evaluated. The 16S-rDNA-RFLP analysis of 100 rhizobial isolates and eight reference type strains placed the isolates into six major clusters, and revealed their site-dependent genomic diversity. Sequence analysis of single and concatenated housekeeping genes (atpD, glnII and gyrB), as well as the symbiotic gene nifH captured a considerably higher level of genetic diversity and indicated the dominance of Bradyrhizobium diazoefficiens and Bradyrhizobium japonicum in Mpumalanga, Limpopo and Gauteng Provinces. Gene sequence similarities of isolates with type strains of Bradyrhizobium ranged from 97.3 to 100% for the 16S rDNA, and 83.4 to 100% for the housekeeping genes. The glnII gene phylogeny showed discordance with the other genes, suggesting lateral gene transfer or recombination events. Concatenated gene sequence analysis showed that most of the isolates did not align with known type strains and might represent new species from South Africa. This underscores the high genetic variability associated with soybean Bradyrhizobium in South African soils, and the presence of an important reservoir of novel soybean-nodulating bradyrhizobia in the country. In this study, the grouping of isolates was influenced by site origin, with Group I isolates originating from Limpopo Province and Group II and III from Mpumlanga Province in the 16S rDNA-RFLP analysis.

Orientadores: Gonçalo Amarante Guimarães Pereira, Juan Lucas Argueso / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T01:08:48Z (GMT). No. of bitstreams: 1 Rodrigues_Aline_D.pdf: 13944014 bytes, checksum: afa01e0dfca6ca3f789845a68d9a1018 (MD5) Previous issue date: 2013 / Resumo: A floculação é um processo de agregação celular observado em muitas linhagens de Saccharomyces cerevisiae que resulta em sedimentação, prejudicando o rendimento fermentativo e a operação de destilarias produtoras de bioetanol. A maioria das linhagens utilizadas por essa indústria, incluindo a linhagem PE-2/JAY270, foi selecionada pela incapacidade de flocular. O controle genético da floculação é bem caracterizado, vários genes foram associados ao fenômeno, incluindo principalmente a família de genes FLO. Previamente observamos que a linhagem JAY291 (haploide derivado de JAY270) não flocula, apesar de possuir o alelo funcional do regulador positivo de floculação FLO8, sugerindo que seu fenótipo envolve outro passo dessa via. A análise da progênie F2 do cruzamento de JAY291 com a linhagem de laboratório S288 (flo8-1) revelou uma proporção fenotípica de ~1:8 (floculantes/não floculantes), esperada para uma característica controlada por três genes não ligados. Sequenciamos os genomas de oito indivíduos F2 floculantes segregando ~40.000 SNPs existentes entre S288c e JAY291. As sequências foram comparadas entre si para localizarmos regiões que co-segregavam com a floculação. Esse mapeamento preliminar foi refinado por PCR-RFLP, revelando um segundo gene, FLO1, que está ausente em JAY291. Para identificar o terceiro gene, analisamos fenotipicamente novos haploides F2 segregantes de um cruzamento entre S288c (FLO8::KanMX4 FLO1::HphMX4) e JAY291 (FLO8 flo1?), dentre os quais os indivíduos FLO1:HphMX4 foram divididos em dois "bulks" (floculante e não floculante) e sequenciados. As frequências alélicas S288c ou JAY291 nos "bulks" foram interrogadas e validadas estatisticamente, revelando um terceiro gene fortemente associado ao fenótipo: MSS11 (fator de transcrição envolvido em floculação). Encontramos ainda outros doze loci contendo genes de efeito menor, mas que atuam como modificadores do fenótipo. Em experimentos independentes, observamos alteração na morfologia de colônia de JAY270 e troca de seu típico fenótipo liso para rugoso, também associado com um grau baixo de floculação. Tais colônias rugosas também são frequentemente encontradas na indústria. Curiosamente, o mesmo fenótipo foi observado cruzando-se haploides específicos derivados de JAY270. Os cruzamentos sugeriram que o fenótipo era controlado um único gene, porém específico de diploides. O mapeamento em genomas de haploides de JAY270 revelou o gene responsável pela alteração de fenótipo: ACE2, envolvido com separação celular. JAY270 é heterozigota para uma mutação frameshift em ACE2 (ACE2/ace2-7A). O fenótipo rugoso ocorre por recombinação mitótica associada com viii perda de heterozigosidade (LOH), produzindo células com duas cópias mutadas desse gene. Para evitar futuros problemas industriais causados pela sedimentação de linhagens rugosas resultantes de LOH na região de ACE2, o defeito detectado ace2-7A foi reparado no genoma da JAY270. O diploide FGY050 (ura3?/ura3?), derivado de JAY270 e auxotrófico para uracila, foi utilizado como plataforma. A linhagem desenvolvida passou a ter duas cópias do alelo selvagem de ACE2 (ACE2/ACE2) e portanto é incapaz de se tornar rugosa por recombinação. Finalmente, em um estudo não relacionado com floculação ou morfologia de colônia, testamos se LOH pode estar relacionada com alguma consequência fenotípica benéfica à JAY270. Dentre onze linhagens LOH testadas em ensaios fermentativos de competição direta com o parental, encontramos uma com ganho de competitividade durante a fermentação / Abstract: Flocculation is a process of cellular aggregation observed in several Saccharomyces cerevisiae strains which results in sedimentation, impairing the fermentation yield and the operation of Brazilian bioethanol distilleries. Most strains used in this industry, including the PE-2/JAY270 strain, were selected for their inability to flocculate. The genetic control of flocculation is well characterized and several genes were associated with this phenomenon, including the FLO family of genes. We previously observed that the JAY291 strain (derivative haploid of JAY270) does not flocculate, despite having the functional allele of the positive regulator of flocculation FLO8, suggesting that its phenotype involves another step of this pathway. Analysis of the F2 progeny from crossing JAY291 with the laboratory strain S288c (flo8-1) revealed the phenotypic ratio of ~1:8 (flocculent/non-flocculent), expected for a trait controlled by three unlinked genes. We sequenced the genomes of eight flocculent F2 individuals segregating ~40.000 SNPs that exist between S288c and JAY291. The sequences were compared to each other to localize regions that co-segregated with the flocculation. This preliminary mapping was refined by PCR-RFLP, revealing a second gene, FLO1, which is absent in JAY291. To identify the third gene, we phenotypically analyzed new F2 haploids segregating from a cross between S288c (FLO8::KanMX4 FLO1::HphMX4) and JAY291 (FLO8 flo1?), among which FLO1:HphMX4 individuals were separated into two bulks (flocculent and non-flocculent) and sequenced. Allele frequencies S288c or JAY291 in bulks were interrogated and statistically validated, revealing a third gene strongly associated with the phenotype: MSS11 (transcription factor involved in flocculation). We also found twelve other loci containing minor effect genes, but acting as modifiers of the phenotype. In independent experiments, we observed alterations in the colony morphology of JAY270 and a transition from its typical rough to smooth phenotype, also associated with a low degree of flocculation. This type of colony is often found in the industry. Interestingly the same phenotype was observed crossing specific haploids derivatives of JAY270. The crosses suggested that the phenotype was controlled by a single gene, but diploid specific. Mapping genomes of haploids of JAY270 revealed the gene responsible for the altered phenotype: ACE2, involved in cell separation. The JAY270 is heterozygous to a frameshift mutation in the ACE2 (ACE2/ace2-7A). The rough phenotype occurs by mitotic recombination associated with loss-of-heterozygosity (LOH), producing cells with two mutated copies of this gene. x To avoid future industrial problems caused by sedimentation of rough strains derived from LOH in the ACE2 region, the ace2-7A defect was repaired in the JAY270 genome. The diploid FGY050 (ura3?/ura3?), derived from JAY270 and auxotrophic to uracil was used as a platform. The developed strain has two copies of the ACE2 wild-type allele (ACE2/ACE2) and is therefore unable to become roughened by recombination. Finally, in a study unrelated to flocculation or colony morphology, we tested whether LOH may be associated with some phenotypic consequence beneficial to the JAY270. Out of eleven LOH strains tested in fermentative assays of direct competition against their parental strain, we found one with gain in competitiveness during fermentation / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular

Natural strains of the Saccharomyces cerevisiae growing on solid médium form structured, biofilm -like colonies. This ability is depended on the surface adhesin Flo11p. The expression of the FLO11 gene is upregulated by the RNA-binding protein Whi3p, which is likely to have a negative effect on the level of the transcription factor Yap6p. The aim of this study was to determine whether Yap6p affects colony morphology and FLO11 expression. Analysis of FLO11 expression using the fluorescent proteins pFlo11-GFP and Flo11p-DsRed in WHI3-deletion strains demonstrated a negative effect of Yap6p on FLO11 expression and confirmed changes in the effect of Yap6p on FLO11 expression in the presence of NaCl. In the strain overexpressing YAP6, the fluorescence values of pFlo11-GFP and Flo11p-DsRed were lower than in the strain with deletion of the YAP6 and in the presence of NaCl there was observed the largest increase in fluorescence. Although Yap6 protein is thought to have a negative effect on FLO11 expression under standard culture conditions, it seems to be responsible for a significant increase in FLO11 expression in the presence of mild osmotic stress. In WHI3-deletion strains, there was observed a significant increase in structuredness of colonies growing in the presence of NaCl. Analysis of structured...

Yeast Saccharomyces cerevisiae as an unicellular organism is one of the best-studied experimental organisms. It is an important model organism for the study of intracellular processes of eukaryotic cells. Yeasts are also social organisms with cell-to-cell communication able to form organized multicellular structures (colonies and biofilms). Yeast and other microorganisms in nature prefer to form colonies on solid substrates rather than to grow as "planktonic" single cells (Palková, 2004; Wimpenny, 2009). The yeast S. cerevisiae typically forms colonies, biofilms were described only rarely. Yeast colonies exhibit an organized morphological pattern characteristic of each particular yeast strain (Kocková-Kratochvílová, 1982). This work is focusing on morphology and differentiation of the S. cerevisiae colonies of common laboratory strains forming less structured colonies, and strains of the Σ1278b genetic background forming highly structured "fluffy" colonies. It shows that polarized budding pattern and especially cell ability to form aggregates enable development of structured morphology. During development of "fluffy" colonies two differently regulated events of dimorphic switch from yeast form to filamentous growth occur. One of these events is dependent on the surface glycoprotein, Flo11p flocculin. This...

In the early 1900's the American chestnut (Castanea dentata (Marsh.) Borkh.) was nearly destroyed by the introduction of the orange-pigmented, chestnut blight fungus (Cryphonectria parasitica (Murr.) Barr). Chestnut blight is less severe in Europe, where hypovirulent (= reduced virulence) strains of the fungus are found to be associated with healing cankers. These European hypovirulent strains are infected with a dsRNA virus, Cryphonectria hypovirus 1 (CHV1), and have a white phenotype when grown in culture. Transmission of CHV1 in C. parasitica is limited by incompatibility between isolates in different vegetative compatibility (vc) types. In 1982-83, naturally formed blight cankers on American chestnut grafts, derived from large survivors, were inoculated with a mixture of four European (white) hypovirulent strains of C. parasitica. After 14 years the white strains were recovered throughout the inoculated grafts, which had low levels of blight damage. CHV1 had infected at least 45 new vc types, and was present in four different fungal colony morphology groups, including one type that had intermediate or partial pigmentation. However, CHV1 was unable to move throughout a single vc type within a natural canker. The objectives of this study were: 1) to determine the frequency and phenotypic diversity of CHV1-infected C. parasitica isolates recovered from stromata and canker tissue from natural cankers on the grafted American chestnut trees and artificially established cankers on forest American chestnuts; 2) to determine the presence or absence of CHV1 in intermediate-pigmented isolates recovered from the American chestnut research plots; 3) to investigate the roles of colony age, resistance to hypovirus infection, and functional mycelial units in the failure of CHV1 to move throughout a vc type of C. parasitica in vitro, and; 4) to examine the role of low temperatures and a high elevation topographic site on CHV1 survival within C. parasitica colonies in vivo and in vitro. The results indicated that there was no direct correlation between the amount of colony pigmentation and the presence of dsRNA. Within each of the three colony phenotype categories (pigmented, intermediate and white), several C. parasitica isolates tested positive for the presence of CHV1. This presence of CHV1 in intermediate isolates, coupled with the relatively large number of intermediate isolates collected from stromata on cankers, indicates that intermediate isolates may perform an important, and previously overlooked, function in biological control of chestnut blight. In this study, all CHV1 movement trials indicated that the age of the C. parasitica colony limited the movement of CHV1 throughout the colony. The majority of the CHV1 movement through a C. parasitica colony occurred between 0 and 7 days following challenge with an isogenic CHV1-infected strain. Isolation data using a lattice grid did not indicate a consistent pattern of CHV1 movement throughout a C. parasitica colony. Low temperatures associated with high altitude had no effect on hypovirus survival in vivo or in vitro. Additionally, no long-term C. parasitica resistance to CHV1 infection or movement was identified in this study. This research has identified new insights into CHV1 spread and survival that may be important in understanding the role of CHV1 in the biological control of chestnut blight. / Ph. D.

Biofilm formation is one of the most common bacterial survival strategies. Majority of bacterial species are able to form these three-dimensional structures, including pathogens like Mycobacterium tuberculosis. Representatives of Mycobacterium genus widely occur in the nature, although they can cause serious problems when they appear in medical equipment and artificial replacements of the human body. Non-pathogenic Mycobacterium smegmatis mc2 155 was used as a model organism in our experiments. We investigated morphology of the three- and six-day-old colonies (in fact biofilms) on agar and agar covered with cellophane using Stereo microscope and Scanning Electron Microscope. We found that a type of surface as well as a carbon source has a great influence on the morphology of the M. smegmatis colonies. We isolated proteomes from the agar and cellophane cultures and from planktonic culture. Two-dimensional electrophoresis was used as the main proteomic method. Proteomic data were analyzed using PDQuest software. Then the sets of proteins detected by qualitative and quantitative analyses were compared using Venn diagrams. As a result, we recognized 7 unique proteins that might be specific for recognition and adhesion of bacteria to the cellophane, no unique protein in agar proteome and 46 unique...