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.

The survival of three enteroviruses (polio 1, coxsackie B3 and echo 1) and a rotavirus (SA-11) was studied under laboratory conditions. The effects of temperature, dissolved oxygen, detention time, sludge source and virus type on virus inactivation were determined. Temperature was the single most important factor influencing the rate of virus inactivation. No significant differences were found for virus inactivation rates at dissolved oxygen levels between 0.9 and 5.8 mg/l. However, the inactivation rate of the viruses under aerobic conditions was found to be significantly greater than the inactivation rate under anaerobic conditions (−0.77log10/day vs −0.33 log10/day). Sludge source, detention time and virus type did not significantly influence the rate of virus inactivation.

As part of a large study aimed at searching for patterns of diversity in the genus Persephonella along the north to south geochemical gradient of the ELSC, ten novel strains of Alphaproteobacteria were isolated unexpectedly. Using defined media under microaerophilic conditions to enrich for Persephonella from chimney samples collected at the seven vent fields on the ELSC and the dilution to extinction by serial dilution method to purify cultures, a total of ten strains belonging to the Alphaproteobacteria were isolated. Two of these isolates, designate MN-5 and TC-2 were chosen for further characterization and are proposed as two new species of a novel genus to be namedThermopetrobacter. Both strains are aerobic, capable of chemoautotrophic growth on hydrogen and grow best at 55°C, pH 6 and 3.0% NaCl. Strain MN-5 is capable of heterotrophic growth on pyruvate and malate and TC-2 is only able to grow heterotrophically with pyruvate. The GC content of MN-5 is 69.1 and TC-2 is 67 mol%. GenBank BLAST results from the 16S rRNA gene reveal the most closely related sequence to MN-5 is 90% similar and the most closely related sequence to strain TC-2 is 89% similar. Sampling at a shallow marine vent on the coast of Vulcano Island, Italy in 2007 led to the isolation of a novel species of Hydrogenothermus, a genus within the Hydrogenothermaceae family. This isolate, designated NV1, represents the secondHydrogenothermusisolated from a shallow marine vent. NV1 cells are rod-shaped, approximately 1.5μm long and 0.7μm wide, motile by means of a polar flagellum and grow singularly or in short chains. Cells grow chemoautotrophically using hydrogen or thiosulfate as electron donors and oxygen as the sole electron acceptor. Growth was observed between 45 and 75°C with an optimum of 65°C (doubling time 140 min), pH 4.0-6.5 and requires NaCl (0.5-6.0% w/v). The G+C content of total DNA is 32 mol%.

Environmental perturbations are ubiquitous features of ecosystems and shape ecological structure and function. Climate change will alter the intensity and frequency of disturbances and expose ecosystems to novel combinations of useful inputs (subsidies) and harmful inputs (stressors). Coastal wetlands are particularly vulnerable to changing environmental conditions and are increasingly exposed to effects of interacting subsidies and stressors. In particular, the Florida Coastal Everglades, which has experienced accelerated change due to a history of water management practices, is vulnerable to new disturbances associated with climate change. The low-lying Florida Everglades faces multiple disturbances from storm surge, nutrient enrichment, and sea-level rise which will influence its responses to future environmental perturbations. Microbial communities are often used to characterize environmental change because of their high surface area to volume ratio, permeable membrane, and quick turnover rates. Therefore, assessing how microbial function changes can provide insights into how subsidies and stressors interact to alter biogeochemical cycles. I tested how nutrient enrichment can alter ecosystem responses to stress and found that it did not promote recovery in mangrove plants. I examined how long-term exposure to salinity and phosphorus (the limiting nutrient in the Everglades) affected microbial enzyme activity and found that salinity alone acts as a suppressor of enzyme activity but phosphorus addition can mitigate salinity stress in sawgrass soil. I tested how pulses of salinity can affect the microbially-mediated breakdown of organic material and found that the microbial community was functionally redundant and unaffected by saltwater pulses; however, microbial activity was consistently lower in the brackish marsh compared to the freshwater marsh. I investigated how gradients of salinity and phosphorus affected freshwater and brackish soils and determined previous exposure to saltwater intrusion dominates affects microbial function and soil composition. Across these experiments, I found that environmental perturbations alter the microbial-mediated processing of nutrients and carbon, and legacies of previous disturbances influence the microbial response to new disturbance regimes.

Organohalide-respiring Bacteria (OHRB) play key roles in the reductive dehalogenation of natural organohalides and anthropogenic chlorinated contaminants. Reductive dehalogenases (RDases) catalyze the cleavage of carbon-halogen bonds, enabling respiratory energy conservation and growth. Large numbers of RDase genes, a majority lacking experimental characterization of function, are found on the genomes of OHRB. In silico genomics tools were employed to identify shared sequence features among RDase genes and proteins, predict RDase functionality, and elucidate RDase evolutionary history. These analyses showed that the RDase superfamily could be divided into proteins exported to the membrane and cytoplasmic proteins, indicating that not all RDases function in respiration. Further, Hidden Markov models (HMMs) and multiple sequence alignments (MSAs) based upon biochemically characterized RDases identified previously uncharacterized members of an RDase superfamily, delineated protein domains and amino acid motifs serving to distinguish RDases from unrelated iron-sulfur proteins. Such conserved and discriminatory features among RDases may facilitate monitoring of organohalide-degrading microbial communities or improve accuracy of genome annotation. Phylogenetic analyses of RDase superfamily sequences provided evidence of convergent evolution and horizontal gene transfer (HGT) across distinct OHRB genera. Yet, the low frequency of RDase transfer outside the genus level and the absence of RDase transfer between phyla indicate that RDases evolve primarily by vertical evolution or HGT is restricted among related OHRB strains. Polyphyletic evolutionary lineages within the RDase superfamily comprise distantly-related RDases, some exhibiting activities towards the same substrates, suggesting a longstanding history of OHRB adaptation to natural organohalides. Similar functional and phylogenetic analyses provided evidence that nitrous oxide (N₂O, a potent greenhouse gas) reductase (nosZ) genes from versatile OHRB members of the Anaeromyxobacter and Desulfomonile genera comprised a nosZ sub-family evolutionarily distinct from nosZ found in non-OHRB denitrifiers. Hence, elucidation of RDase and NosZ sequence diversity may enhance the mitigation of anthropogenic organohalides and greenhouse gases (i.e., N₂O), respectively. The tetrachloroethene-respiring bacterium Geobacter lovleyi strain SZ exhibited genomic features distinguishing it from non-organohalide-respiring members of the Geobacter genus, including a conjugative pilus transfer gene cluster, a chromosomal genomic island harboring two RDase genes, and a diminished set of c-type cytochrome genes. The G. lovleyi strain SZ genome also harbored a 77 kbp plasmid carrying 15 out of the 24 genes involved in biosynthesis of corrinoid, likely related to this strains ability to degrade PCE to cis-DCE in the absence of supplied corrinoid (i.e., vitamin B₁₂). Although corrinoids are essential cofactors to RDases, the strictly organohalide-respiring Dehalococcoides mccartyi strains are corrinoid auxotrophs and depend upon uptake of extracellular corrinoids via Archaeal and Bacterial salvage pathways. A key corrinoid salvage gene in D. mccartyi, cbiZ, occurs at duplicated loci adjacent to RDase genes and appears to have been horizontally-acquired from Archaea. These comparative genome analyses highlight RDase dependencies upon corrinoids and also suggest mobile genomic elements (e.g., plasmids) are associated with organohalide respiration and corrinoid acquisition among OHRB. In summary, analyses of OHRB genomes promise to enable more complete modeling of metabolic and evolutionary processes associated with the turnover of organohalides in anoxic environments. These efforts also expand knowledge of biomarkers for monitoring OHRB activity in anoxic environments, and will improve our understanding of the fate of chlorinated contaminants.

Lambdoid phages suppress transcription termination to fully express their genes. Antitermination of early gene expression in most lambdoid phages is mediated by an interaction between the N protein and a number of host-encoded factors. Bacteriophage HK022 does not rely on a protein for antitermination. To promote full expression of early phage genes, the transcripts of the HK022 put sites interact directly with RNA polymerase to convert it to a termination resistant form. Bacteriophage HK639 also uses RNA-mediated antitermination. However, it only possesses a single put-like element in its left operon. Because most lambdoid phages, including HK022, have antiterminator elements in each of their early operons, the presence of a single antitermination site in HK639 was unexpected. We have shown that host genes involved in promoting protein-mediated antitermination are not required for HK639 growth. We have also shown that expression of the left operon is essential for lytic growth. Replacement of the left operon promoter, PL, and the putL antitermination sequence prevented HK639 phage release. A similar construct that only replaced putL also prevented phage release. These results suggest that antitermination is required for HK639 excision and/or lytic growth. To distinguish between a defect in phage excision versus a defect in lytic growth, the mutations were crossed onto lytically growing phage. Recombinant phages could not be recovered which suggests a defect in lytic growth is preventing phage release. Additional replacements of left operon sequences suggest that antitermination is not the only requirement for lytic growth. A 2,161bp deletion (HK639 genome coordinates 30,888-33,048) and a 1,736bp deletion (HK639 genome coordinates 29,152- 30,887) downstream of the HK639 putL site also prevented phage release, whereas a 1,746bp deletion (HK639 genome coordinates 29,151-27,406) did not. These results suggest that the deleted HK639 left operon sequences are required for lytic growth. BLAST analysis did not provide insight into the function of the deleted genes. Although the function of many of the HK639 left operon genes is unknown, their importance in phage growth can now be verified by complementation analysis. Our results suggest that HK639 may use a novel mechanism to control the expression of its early genes.

Loline alkaloids, found in many grass-Epichloë symbiota, are toxic or feeding deterrent to invertebrates. The loline alkaloids all share a saturated pyrrolizidine ring with a 1-amine group and an ether bridge linking C2 and C7. The steps in biosynthesis of loline alkaloids are catalyzed by enzymes encoded by a gene cluster, designated LOL, in the Epichloë genome. This dissertation addresses the enzymatic, genetic and evolutionary basis for diversification of these alkaloids, focusing on ether bridge formation and the subsequent modifications of the 1-amine to form different loline alkaloids. Through gene complementation of a natural lolO mutant and comparison of LOL clusters in strains with different loline alkaloid profiles, I found that lolO, predicted to encode a 2-oxoglutarate-dependent nonheme iron (2OG/Fe) dioxygenase, is required in formation of the ether bridge. Through application of isotopically labeled compound to Epichloë uncinata culture, I established that exo-1-acetamidopyrrolizidine (AcAP) and N-acetylnorloline (NANL) are true pathway intermediates. Application of AcAP to yeast expressing lolO resulted in production of NANL, establishing that LolO is sufficient to catalyze this unusual oxygenation reaction. After ether formation, modifications on the 1-amino group give loline, N-methylloline (NML), N-formylloline (NFL) and N-acetylloline (NAL). A double knockout of lolN, predicted to encode an acetamidase, and lolM, predicted to encode a methyltransferase, produced only NANL. Complementation of the double knockout with wild-type lolN and lolM restored the loline alkaloid profile. These results indicate that LolN is involved in deacetylating NANL to produce norloline, which is then modified to form the other lolines. Crude protein extract of a yeast transformant expressing LolM converted norloline to loline and NML, and loline to NML, supporting the hypothesis that LolM functions as a methyltransferase in the loline-alkaloid biosynthesis pathway. The alkaloid NAL was observed in some but not all plants symbiotic with Epichloë siegelii, and when provided with exogenous loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylloline (NAL), indicating that a plant acetyltransferase converts loline to NAL. I further analyzed the basis for loline alkaloid diversity by comparing the LOL clusters in the Epichloë and Atkinsonella species with different loline alkaloid profiles, and found that LOL clusters changed position, orientation and gene content over their evolutionary history. Frequent, independent losses of some or all late pathway genes, lolO, lolN, lolM and lolP, resulted in diverse loline alkaloid profiles. In addition, phylogenetic analyses demonstrated transspecies polymorphism of the LOL clusters. Based on my findings, I established that in Epichloë and Atkinsonella species the ether bridge is formed on acetamidopyrrolizidine. My study of the loline alkaloid profile of Adenocarpus decorticans (Fabaceae) suggests that these plants probably use a similar strategy at least with respect to ether-bridge formation. Further diversification steps of loline alkaloids in grass-Clavicipitaceae symbiota are carried out by enzymes of both Epichloë species and the host plant. Finally, I present evidence that LOL clusters have evolved by balancing selection for chemical diversity.

Sulfate- and Fe(III)-reducing, and methanogenic prokaryotes (SRP, FRP, MGP) are key players in metabolic pathways involved in anaerobic biodegradation processes. Understanding the metabolic activity of these microbes in environments can enhance microbe-mediated processes such as oil spill bioremediation and methane biogas production. In this study, anaerobic microbial activities in Deepwater Horizon oil spill-impacted salt marsh sediments, and in methanogenic coal bed production water enrichment cultures amended with trace elements (TE), were elucidated by employing an approach combining methods in molecular biology and geochemistry. In situ metabolic activity of SRP, FRP and MGP were monitored seasonally and metabolically-active communities were identified in oil-impacted sediments using quantitative real time Reverse Transcription -PCR and clone library analysis of key functional genes: Dissimilatory (bi)sulfite reductase (dsrAB), Geobactereceae-specific citrate synthase (gltA), methyl coenzyme M reductase (mcrA), and benzyl succinate synthase (bssA). In situ application of montmorillonite clay was assessed for its potential at accelerating PHC degradation by stimulating microbial activities. Levels of dsrA, gltA and bssA transcripts suggested that PHC-oxidizing SRP are more active in summer while FRP are more active in winter, indicating their activities linked to the seasonal changes of redox potential and vegetation. BssA gene expression peaked in winter, and was highest at more highly oil-impacted sites. Expression of all genes was higher in clay-amended sites. bssA transcript level and Fe(II) production were highest in clay-amended microcosm. Total petroleum hydrocarbon (TPH) levels were lower in oil and clay-amended microcosm incubation than one with oil only amendment, suggesting enhanced TPH degradation by clay amendment. Pyrosequencing analysis 16S rRNA gene in clay-amended microcosms demonstrated the highest percentage abundance of groups closely related to known anaerobic aromatic degraders. Levels of mcrA transcripts correlated with methane production rates in TE-amended coal bed production water enrichments. The findings of the present study clearly support the advantage of gene expression analyses for estimating microbial activity. To the best of our knowledge, this is the first in situ study which employs key functional gene markers as molecular proxies for metabolic activity and diversity assessments in anaerobic oil-contaminated salt marsh sediment and also elucidates clay-enhanced in situ TPH degradation.

Planktothricoides raciborskii and Roseofilum reptotaenium are physiologically similar, yet ecologically distinct organisms found in a hot spring outflow and coral black band disease (BBD), respectively. The aim of this study was to elucidate the relationship between R. reptotaenium and sulfide in BBD, to compare microcystin (MC) production in response to environmental factors, and to determine chemotactic responses to MC and sulfide by the two organisms. Results showed that the pathogenicity of R. reptotaenium in BBD is dependent on sulfate-reducing bacteria as secondary pathogens. Roseofilum reptotaenium produced significantly more MC than P. raciborskii, as measured using ELISA. Roseofilum reptotaenium was negatively chemotactic to sulfide, determined using horizontal and vertical gradients in agar, while P. raciborskii was not affected. Neither cyanobacterium was chemotactic to MC in the agar assays. The ecophysiology of P. raciborskii and R. reptotaenium in relation to MC production and response to sulfide reflected their pathogenic versus non-pathogenic status.

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.