The primary contributor to Vibrio fischeri aminopeptidase activity is aminopeptidase N, PepN. Colonization assays revealed the pepN mutant strain to be deficient at forming dense aggregates and populating the host’s light organ compared to wildtype within the first 12 hours of colonization; however the mutant competed normally at 24 hours. To address the role of PepN in colonization initiation and establish additional phenotypes for the pepN mutant strain, stress response and other physiological assays were employed. Marked differences were found between pepN mutant and wildtype strain in response to salinity, acidity, and antibiotic tolerance. This study has provided a foundation for future work on identifying a putative role for V. fischeri PepN in regulating stress response.

As of January 2017, the U.S. poultry industry banned the use of antibiotics and now relies on alternatives such as probiotics to help protect animal health. Although probiotic use is not a new concept in the poultry industry, identifying the best combination of bacterial strains to generate an effective probiotic formula requires further investigation. This study aimed to detect a probiotic product of four bacterial strains (Pedioccoccus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, and Bacillus subtilis) in a feeding trial with broiler chickens. Birds given the probiotic were predicted to show an improved growth performance with the probiotics colonizing the gut. Ninety-six broiler chickens were equally divided into 3 treatment and 3 control pens. During the 25-day experiment, birds were fed a starter diet (days 0-11) and a grower diet (days 12-25). Experimental birds were administered the probiotic product via the drinking water at a concentration of 3.1×104 CFU/ml. Control birds had an equivalent amount of dextrose filler added to their water supply. Feces were collected hourly on day one and daily thereafter. On days 1, 22, and 25 of the experiment, 2 birds from each pen were euthanized for gut sampling. Lumen and mucosa samples were collected from the duodenum, jejunum, ileum, and ceca. Species-specific and strain specific PCR primers were employed for probiotic detection. Wild strains of P. acidilactici, P. pentosaceus, and L. plantarum were detected in the feeds, inhibiting detection of the probiotic strains when using species-specific PCR primers. Strain-specific primers were used to detect the probiotic Pedioccoccus acidilactici and Lactobacillus plantarum strains. B. subtilis was detected in feces within one hour of probiotic administration and was predominantly detected in experimental birds only. Both P. acidilactici and L. plantarum probiotic strains were initially detected in the feces of treated birds within two hours of probiotic administration and again ten days later. Both L. plantarum and B. subtilis were seen only in treated bird gut samples. L. plantarum was predominantly detected in the ceca near the end of the small intestine. P. pentosaceus was observed more often in treated gut samples and P. acidilactici was the least commonly detected probiotic strain. All administered bacteria were rarely seen in mucosa samples. Feed-endogenous P. acidilactici and L. plantarum strains became progressively more detectable in the mucosa along the gastrointestinal tract suggesting gut colonization, however, probiotic strains did not appear to colonize the mucosa of treated birds. Although probiotic strains were no longer detected after product removal, all probiotic strains were detected in feces and gut samples during probiotic administration, suggesting the bacteria can colonize the gut. Probiotic supplementation did not result in significant differences in body weight gain, feed intake, or feed conversion ratio. However, birds growing in a more stressful environment than the carefully controlled experimental set up used here may show probiotic-related effects. This study identified that the probiotic bacteria appeared to survive the gastrointestinal tract, exhibited a transit time of 1-2 hours, could possibly colonize chickens, and localized near the end of the chicken gut.

The unifying concept of endosymbiosis and the ‘holobiont’ is that the interaction of the microbial community and the host’s biology can affect myriad processes from speciation to physiology to behavior. This study explored the role of the microbiome as a potential facilitator of rapid evolution of social behavior in a socially polymorphic species of spider, Anelosimus studiosus. Adult females were collected from solitary and social colonies at two geographically distinct locations, and behaviorally assayed to assign individuals to ‘docile’ or ‘aggressive’ phenotypes. Microbiomes of each individual were analyzed by 16s rRNA sequencing. Correlations were found with external influences on the microbiome (colony type, local environmental microbiota, and among colony), and also between the microbiome and individual’s behavioral phenotype. While causation has not yet been established, these data suggest that demographics and ecology affect the microbiome, and that behavior may be affected by the microbiome.

Sclerotinia homoeocarpa (F.T. Bennett) is one of the most economically important pathogens on high amenity cool-season turfgrasses where it causes dollar spot. Due to decades of over-reliance and repeated chemical treatments, S. homoeocarpa has developed resistance and insensitivity to multiple classes of fungicides. To understand the genetic mechanisms of fungicide resistance, the whole genomes of two strains with varying resistance levels to fungicides, were sequenced. In unpublished data (Sang et al.), a RNA-sequencing analysis revealed three CYP450s that were validated to play a functional role in S. homoeocarpa’s resistance against different fungicide classes. We also identified CYP450 metabolic action on the multi-site mode of action fungicide chlorothalonil. Chlorothalonil is an extensively used contact fungicide and has been known to be persistent in soils. Yet, S. homoeocarpa resistance to chlorothalonil has not been reported in the field. High Performance Liquid Chromatography (HPLC) indicated faster rates of chlorothalonil biotransformation by CYP450 overexpression strains when compared to the wild-type. We show by GC-MS that the primary transformation intermediate found in soils, 4-hydroxy-2,5,6 trichloroisophthalonitrile is produced by CYP450s’ metabolism.

In the field of marine microbial ecology, many questions remain unanswered with regards to the physiological trade-offs made by phytoplankton to maximize growth (e.g., nutrient acquisition) and minimize loss (e.g., predation defenses). These tradeoffs, which occur at the cellular level, have wide reaching impacts on food web dynamics and global biogeochemical cycles. In the first chapter, we explored the use of a non-canonical amino acid (NCAA) technique, bioorthogonal non-canonical amino-acid tagging (BONCAT), in phytoplankton model systems. This technique has potential to work well in natural systems by enabling isolation of only newly synthesized proteins during an incubation period with the NCAA, reducing the complexity of natural proteomics and easing the elucidation of patterns. However, in testing BONCAT across several groups of cultured phytoplankton, we discovered that the NCAA molecule induced a stress response in the globally ubiquitous marine picocyanobacteria, Synechococcus sp. Therefore, in addition to confirming the uptake of modified amino acids by phytoplankton, chapter one investigated the implications of this stress response and limitations when using this technique to study marine microbial communities. In chapter two, we addressed our initial question by exploring tradeoffs at the protein level in a simplified culture system. This approach revealed insights into metabolic tradeoffs in response to predation pressure and nutrient stress. These insights into how phytoplankton negotiate these physiological tradeoffs at the protein level could ultimately allow for targeted proteomic studies in natural systems.

<p>C₄-dicarboxylic acids appear to be metabolized via the TCA cycle in N₂-fixing bacteria (bacteroids) within legume nodules. In <em>Sinorhizobium meliloti</em> bacteroids from alfalfa, NAD⁺-malic enzyme (DME) is required for symbiotic N₂-fixation and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont <em>Rhizobium leguminosarum</em> pyruvate synthesis can occur via either the DME pathway or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK), pyruvate kinase (PYK), and pyruvate dehydrogenase. Here we report that <em>dme</em> mutants of <em>Sin</em>or<em>hizobium sp</em>. NGR234 formed root nodules on a broad range of plants and that the level of N₂-fixation varied from 90% to 20% of wild type depending on the host plants inoculated. NGR234 bacteroids had significant PCK activity and while single <em>pckA</em> and single <em>dme</em> mutants fixed N₂ on <em>Macroptilium atropurpureum</em> and <em>Leucaena leucocephala</em> (albeit at a reduced rate), a <em>pckA</em> <em>dme</em> double mutant had no N₂-fixing activity (Fix^-). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix^- phenotype of <em>S. meliloti dme </em>mutants may be specific to the alfalfa-<em>S. meliloti </em>symbiosis. We therefore examined the ME-like genes <em>azc3656 </em>and <em>azc0119 </em>from <em>Azorhizobium caulinodans</em>, as <em>azc3656 </em>mutants were previously shown to form Fix^- nodules on the tropical legume <em>Sesbania rostrata</em>. We found that purified AZC3656 protein is an NAD (P)⁺-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N₂ fixation in <em>A. caulinodans </em>and <em>S. meliloti</em>, in other rhizobia this activity can be bypassed via another pathway(s).</p> <p>In <em>S. meliloti</em> both malic enzymes DME and TME share similar apparent <em>K_m</em>s for substrate and cofactors, but differ in their responses to TCA cycle intermediates, with DME activity inhibited by acetyl-CoA and induced by succinate and fumarate. Previous results in our laboratory indicated that DME is essential for symbiotic N₂ fixation, while TME fails to functionally replace DME. One possible reason for it is that a high ratio of NADPH/NADP⁺in<em> S. meliloti </em>bacteroids prevents TME from functioning in nodules. We sought to lower the<em> </em>NADPH/NADP⁺ratio by overexpressing a soluble pyridine nucleotide transhydrogenase (STH). However, metabolite measurements indicated that overproducing STH failed to lower the ratio of NADPH/NADP⁺ in<em> S. meliloti</em>.</p> <p>Previous studies assumed that DME and TME might play different roles in central carbon metabolism. To gain insight of their physiological functions, genome-wide microarray analysis was conducted in <em>S. meliloti</em> single<em> dme and</em> <em>tme</em> mutants grown on glucose or succinate. The most striking changes of gene expression were observed in <em>S. meliloti</em> <em>dme</em> mutants grown on succinate. The functions of upregulated genes suggested that DME might play an important role in regulating TCA cycle intermediates, important for the maintenance of metabolic flux through TCA cycle during C₄-dicarboxylate oxidation. However, changes of gene expression found in <em>tme </em>mutants were not significant enough to predict the physiological functions of TME protein in central carbon metabolism.</p> / Doctor of Philosophy (PhD)

The effects of managing soil with organic amendments were examined with respect to soil microbial community dynamics, macroaggregate formation, and plant physio-genetic responses. The objective was to examine the possibility of managing soil microbial communities via soil management, such that the microbial community would provide agronomic benefits. In part one of this research, effects of three amendments (hairy vetch residue, manure, compost) on soil chemical and microbial properties were examined relative to formation of large macroaggregates in three different soils. Vetch and manure promoted fungal proliferation (measured via two biomarkers: fatty acid methyl ester 18:2ω6c and ergosterol) and also stimulated the greatest macroaggregate formation. In part two of this research, effects of soil management (same amendments as above, inorganic N fertilization, organic production) on soil chemical and microbial properties were examined relative to the expression of nitrogen assimilation and defense response genes in tomato (Solanum lycopersicum L.). Soil management affected expression of a nitrogen assimilation gene (GS1, glutamine synthetase) and several defense-related genes. The GS1 gene was downregulated with inorganic N fertilization, expression of the pathogenesis-related PR1b gene (which codes for the pathogenesis-related PR1b protein) was increased in plants grown in soil amended with compost, vetch, and N fertilizer, and expression of three other defense-related genes coding for chitinase (ChiB), osmotin (Osm), and β-1,3-glucanase (GluA) were decreased in plants from soil amended with manure and in plants from the organically managed soil. Differential expression of defense-related genes was inversely related to the relative abundance of Gram-negative bacteria. The relative abundance of the 18:1ω7c Gram‑negative bacterial biomarker was greatest in manure treated soil and in organically managed soil (which recieves seasonal manure applications). These treatments also had the lowest expression of ChiB, Osm, and GluA, leading to speculation that manure, through increases in Gram-negative bacteria, may have suppressed populations of soil organisms that induce a defense response in plants, possibly allowing for less-stressed plants. Outcomes of this research may be useful for those interested in developing management strategies for maintaining or improving soil structure as well as those interested in understanding management effects plant physio-genetic responses.

As salas limpas são amplamente empregadas em indústrias farmacêuticas destinadas a fabricar medicamentos e dispositivos estéreis. Nós empregamos coloração de Gram e cromatografia gasosa de ésteres metílicos de ácidos graxos extraídos de células íntegras de microrganismos ambientais para caracterizar e identificar bactérias isoladas em 50 salas limpas diferentes projetadas para a fabricação de medicamentos estéreis e para fornecer um perfil de ácidos graxos das espécies mais comuns de bactérias isoladas. Uma análise estatística nos permitiu corroborar estudos anteriores e confirmar que cocos Gram positivos é o grupo mais relevante de microrganismos presentes nas salas limpas avaliadas. A espécie predominante é Micrococcus luteus, isolada de salas classe B e de pessoal, seguida de Staphylococcus cohnii em classe C, Bacillus subtilis em classe A e Staphylococcus hominis em classe D. Os perfis de ácidos graxos destas bactérias são, na maioria, consistentes com as bibliotecas padrão. Nós também tentamos estabelecer uma correlação entre a estação do ano e o nível de contaminação, embora a análise de variância tenha mostrado que não há diferença significativa entre o nível de contaminação no decorrer das estações. Além do mais, análises repetidas com um aumento gradual de massa celular nos permitiram concluir que a quantidade ótima de material celular necessário para extração de ácidos graxos varia com a espécie de bactéria. Finalmente, um estudo comparativo de algumas bactérias incubadas em diferentes temperaturas confirmou que o perfil de ácidos graxos é altamente influenciado pela temperatura. Portanto, nós acreditamos que este trabalho possa contribuir para identificar e compreender a comunidade bacteriana de algumas salas limpas farmacêuticas. / Clean rooms are largely employed in pharmaceutical companies whose purpose is to produce sterile drugs and devices. We employed Gram staining and gas chromatography of fatty acid methyl esters extracted from whole cells of environmental isolates to characterize and identify bacteria isolated in each of 50 different clean rooms designed for the manufacturing of sterile medicinal products and to provide a fatty acid profile of the most common species of isolated bacteria. Statistical analysis allowed us to corroborate previous studies and confirm that Gram-positive cocci are the most relevant group of microorganisms inside the studied clean rooms. The predominant species is Micrococcus luteus, isolated from Grade B zones and from personnel, followed by Staphylococcus cohnii in Grade C, Bacillus subtilis in Grade A and S. hominis in Grade D. Fatty acid profiles of these bacteria are, to a great extent, consistent with standard libraries. We also attempted to establish a correlation between season and level of contamination, although variance analysis showed that there is no significant difference on the level of contamination throughout seasons. Furthermore, repeated analysis with a gradual increase in cell mass allowed us to conclude that the optimal amount of cell material depends on the species of the bacteria studied. Finally, a comparative study with some bacteria incubated in different temperatures confirmed that fatty acid profile is highly influenced by temperature. Therefore, we believe that this work can contribute to identify and understand the bacterial community of some pharmaceutical clean rooms.

O metamidofós é um inseticida organofosforado altamente tóxico por ser um forte inibidor da acetilcolinesterase, sendo utilizado em diversas culturas para o controle de pragas. Há poucas informações sobre a biodegradação deste composto disponíveis na literatura. Foram isoladas e caracterizadas 12 bactérias a partir de amostras de solo e água de uma área contaminada com metamidofós, as quais mostraram inicialmente capacidade de degradar o pesticida, utilizando-o como fonte de enxofre/nitrogênio e fósforo/enxofre. Estes isolados foram identificados por métodos de biologia molecular e pela caracterização do perfil lipídico da célula como pertencentes aos gêneros, Serratia, Pseudomonas, Stenotrophomonas e Curtobacterium. Ensaios preliminares da cinética de degradação do metamidofós por GC/MS evidenciaram o consumo do pesticida pelas bactérias isoladas. A retomada destes ensaios após alguns meses de armazenamento em glicerol a -80ºC resultou na perda da capacidade de biodegradação do composto-alvo por causas não-identificadas. Fatores que podem ter contribuído para este resultado negativo incluem eventual perda de plasmídeos com partes das vias de biodegradação ou interferentes utilizados na estabilização do metamidofós. / Methamidophos is a strong acetylcholinesterase inhibitor and, therefore, a very toxic organophosphorus insecticide. This product has been widely employed for pest control in a variety of cultures, but little information is available about its biodegradation. 12 bacteria were isolated and characterized, from water and soil samples obtained from a site contaminated with methamidophos, which in preliminary tests showed the ability to degrade methamidophos, using it as a combined source of sulfur/nitrogen and/or phosphorus/sulfur. These isolates were identified by molecular biology methods and by characterization of its fatty acids profile as members of the genus Serratia, Pseudomonas, Stenotrophomonas and Curtobacterium. The ability to biodegrade the compound was lost after prolonged storage at -80ºC for unknown reasons. It was hypothesized that this negative result may have occurred due to loss of plasmids or by interference of products used in the stabilization of commercial methamidophos formulations.

Aplicou-se duas metodologias para isolamento de bactérias ainda \"não-cultivadas\" em amostras de água do mar de Ubatuba (SP, Brasil) e da Baía do Almirantado (Antártica). Pela adaptação do método de cultivo de alto desempenho (HTC), foi possível isolar 4 culturas bacterianas, nas quais duas podem ser consideradas ainda não-cultivadas e foram identificadas através das análises filogenéticas do gene rRNA 16S. Ao aplicar o método de inóculo em meio de cultura diluído 1/10, nas amostras de água do mar antártico, 81 isolados bacterianos foram identificados (gene rRNA 16S), sendo que um deles, é candidato a um isolado \"não-cultivado\". Outras abordagens fenotípicas e genômicas serão necessárias para definir a caracterização taxonômica destas bactérias \"não-cultivadas\" obtidas. A presença dos genes alk foi detectada em 11 isolados bacterianos antárticos, destes, um representante apresentou similaridade com uma sequência de gene alkM, recém-descrita em estudo prévio, em um dos clones de bibliotecas metagenômicas de sedimentos, na mesma região de amostragem. / Two different methods were applied for culturing marine bacteria in order to isolate uncultured representatives from Ubatuba seawater (SP, Brazil) and from Admiralty Bay (Antarctica). The adapted high-throughput culturing (HTC) procedures allowed to obtain four isolates. Two of them are uncultured bacteria candidates from coastal seawater studied and they were identified by phylogenetic analysis for 16S rRNA genes. The use of traditional plating on 1/10 dilution of agar media for the Antarctica seawater samples, allowed to isolate 81 cultures that were identified (16S rRNA gene), and one of these isolates is most likely to be an uncultured micro-organism. For taxonomic purposes, several others phenotypic and genomic methods must be applied for the further characterization of these uncultured bacteria. The alk genes were detected in eleven bacterial isolates from Antarctic. One of them, showed similarity to the sequence of an alkM gene, described in previous work in environmental clones libraries od sediments, from the same sampling area.