<i>Pseudomonas fluorescens</i> BRG100 produces secondary metabolites with herbicidal activity to the grass weeds wild oat, Avena fatua, and green foxtail, Setaria viridis. The green fluorescence protein (gfp) gene was introduced into P. fluorescens BRG100 from Escherichia coli S17-1¥ë via a Tn5 mini transposon suicide vector system. Colony morphology, growth rate in liquid media, weed biocontrol efficacy (plant growth pouch), carbon utilization (Biolog GN) and root colonization of green foxtail by several P. fluorescens BRG100gfp transformants were determined to be the same as the wild type. <i>Pseudomonas fluorescens</i> BRGgfp-15 was found to be most similar to the wild-type in all of the above characteristics and was thus used in subsequent experiments. Note: all strains of Pseudomonas fluorescens will be referred to by only their strain throughout (ie. BRGgfp-15 and BRG100). <p>It was determined by population dynamics per section of root with spiral plating on culture medium, epi-fluorescence and confocal microscopy that BRGgfp-15 colonized all areas of the root, but showed a preference for the proximal 1/3 section and the seed. In the proximal section the mean number of viable cells per gram dry weight was log109.06 and log109.31, when applied as liquid inoculum and as the pesta granular formulation, respectively. With liquid inoculum there was only log107.53 viable cells/g in the middle 1/3 section and log107.01 viable cells/g in the distal 1/3 section. The number of viable cells/g with pesta granules was log107.61 and log107.34, for the middle and distal sections, respectively. The root hairs, root tip, and ventral portion of the seed were all areas of heavy colonization relative to the other areas of the root. <p>Survival of BRGgfp-15 in the pesta formulation was examined in 2 soil types, clay and clay loam, in a thermogradient plate apparatus by a factorial randomized design complete block experiment. The experiment included: 3-12 hour diurnal temperature regimes: 5-15¨¬C, 15-25¨¬C, and 25-35¨¬C and 3 moisture levels: 25, 50 and 75% of soil moisture holding capacity. Sampling was carried out after 0, 14, 28 and 42 days. The highest numbers of viable BRGgfp-15 cells/g were found in the pesta granules in soil subjected to the lowest diurnal temperature regime and moisture content. The lowest numbers of viable cells/g were found in the pesta granules incubated in the highest diurnal temperature and moisture. This suggests lower soil temperature and moisture enhances survival of BRGgfp-15 in pesta and/ or higher soil temperature and moisture enhances the release and dissemination of BRGgfp-15 from pesta granules. When subjected to a 5-15¨¬C-temperature regime the number of viable cells/g was log109.80. When subjected to 15-25¨¬C the viable cells/g was log108.96 and with 25-35¨¬C it was log107.33. The mean number of viable cells/g was log109.36, log108.86, and log107.87, for 25, 50, and 75% soil moisture holding capacity, respectively. There was also a significantly higher number of viable cells/g in the clay soil collected from Saskatoon, log109.00, as compared to the clay loam soil collected from Scott, which was log108.40. <p>These results suggest that Pseudomonas fluorescens BRG100 has considerable potential as a bioherbicide because of its successful root colonization of green foxtail and wheat. <i>Pseudomonas fluorescens</i> BRGgfp-15 survived well under various environmental conditions when formulated into pesta granules, proving the pesta formulation was an excellent formulation. In addition, gfp was shown to be an excellent conservative marker for monitoring the root colonization and survival of <i>P. fluorescens</i> BRG100.

Environmental heterogeneity has often been suggested as a general explanation for patterns of diversity at scales ranging from individuals within populations to communities within landscapes. I evaluate this proposition using laboratory experiments with two microbial species, the unicellular chlorophyte Chlamydomonas reinhardtii and the common bacterium Pseudomonas fluorescens. These experiments contrast the fate of diversity following selection in heterogeneous and homogeneous environments. Specifically, I show that (1) an individual's breadth of adaptation evolves to match the amount of environmental variation, specialists evolving in environments that remain constant through time and generalists evolving in environments that vary through time irrespective of the scale at which environmental variation occurs relative to the lifetime of an individual; (2) the maintenance of diversity in a spatially heterogeneous environment is context-dependent, diversity being more readily maintained when environmental conditions are very different and genotypes are widely divergent; (3) selection in heterogeneous environments represents a plausible mechanism for two well-known patterns of diversity at large spatial scales, namely that between species diversity and both productivity and disturbance. This thesis thus demonstrates that environmental heterogeneity is a plausible, and perhaps very general, factor responsible for the diversity of natural communities.

Understanding the origins of biological diversity is a fundamental goal of evolutionary biology. A large body of theory attributes ecological and genetic diversification to divergent natural selection for resource specialization. This thesis examines adaptive radiation in response to selection for resource specialization in microcosm populations of the asexual bacterium Pseudomonas fluorescens. The general protocol for these experiments is to introduce a clonal population of Pseudomonas into a novel environment and to allow evolution to occur through the spontaneous appearance of novel genotypes carrying beneficial mutations. Adaptation can then be quantified through direct comparisons between evolved populations and their clonal ancestors. These experiments show that resource heterogeneity generates divergent natural selection for specialization on alternative resources, irrespective of the spatial structure of the environment. Adaptive radiation is possible in sympatry because of genetic trade-offs in the ability to exploit different resources, but these trade-offs are often not the result of antagonistic pleiotropy among loci that determine fitness on alternative resources. The rate of phenotypic diversification declines during adaptive radiation, apparently because the ecological opportunities required to support specialist lineages disappear as a consequence of initial diversification. The ultimate outcome of repeated instances of adaptive radiation is the evolution of a community of ecologically equivalent specialists that share similar adaptive traits, despite differences in the underlying genetic basis of specialization in replicate radiations. Comparisons with the literature on experimental evolution in microbial populations illustrate the results of this thesis are well-supported by experiments in a wide range of microbial microcosms.

Copies of author's previously published articles inserted. / Bibliography :leaves 65-80 / x, 80, [64] leaves, [24] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Ice nucleation active (INA) bacteria were detected in a pea field in South Australia. They were identified as strains of Pseudomonas syringae and Pseudomonas flourescens biotype 1. Some chemical agents were tested on the two ice nucleating species, as cryoprotectants. / Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1991

Thesis (Ph. D.)--University of Adelaide, Dept. of Crop Protection, 1991. / Copies of author's previously published articles inserted. Includes bibliographical references (leaves 65-80).

Thesis (Ph. D.)--Washington State University, December 2006. / Includes bibliographical references.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias. Programa de Pós-Graduação em Ciência dos Alimentos. / Made available in DSpace on 2012-10-17T00:17:10Z (GMT). No. of bitstreams: 1 264737.pdf: 1926692 bytes, checksum: 9ec6138b949476777fddfa783c19b12f (MD5) / O presente trabalho foi realizado em duas etapas. Na primeira etapa, avaliaram-se os condimentos vegetais (ervas aromáticas) na forma de erva processada (ERV-PRO); de erva in natura, (ERV-IN); de óleo resina, proveniente da erva processada (OR-PRO); de óleo essencial, proveniente da erva in natura (OE-IN), de diferentes marcas e fornecedores, adquiridos no comércio de Florianópolis-SC. Esta avaliação se deu em relação a sua atividade antimicrobiana frente a uma população padronizada de aproximadamente 106 UFC/mL de P. fluorescens ATCC 13525, a uma concentração de 1% P/V, pela técnica de difusão em ágar, durante o período de 24 horas, na temperatura de 30º C, para uma avaliação preliminar. A atividade antimicrobiana foi avaliada também através da técnica do gradiente de concentração em placa a uma concentração de 1% na temperatura de 30º C e 25º C e através da técnica em caldo com contagem de células a uma concentração de 0.5%, na temperatura de 25º e 4º C, e, somente com o óleo essencial de açafrão, a uma concentração de 0.2%, na temperatura de 4º C, monitorados durante o período de sete dias. Na segunda etapa, avaliou-se o óleo essencial de açafrão a concentração de 0.2% através de uma simulação em peito de frango picado (PFP) em relação a uma população de P. fluorescens inoculada e à microbiota natural do frango, sob a temperatura de refrigeração, monitorado pelo período de sete dias. Houve variações no poder de inibição dos condimentos vegetais (ervas aromáticas) testados, relacionadas com a temperatura de incubação, tipo de erva e forma de erva. Em uma concentração de 1% de ERV-PRO, em ordem decrescente de inibição, alecrim > açafrão > orégano > sálvia > tomilho. E na forma de ERV-IN, açafrão > sálvia > orégano, nas duas temperaturas (25º C e 4º C), e alecrim com inibição limitada somente na temperatura de 25º C. Na forma de óleo resina, alecrim > orégano > tomilho > sálvia > açafrão, sendo o alecrim e o açafrão bactericidas e, os demais, bacteriostáticos. Na forma de óleo essencial, açafrão > sálvia > orégano, na temperatura de 30º C. E na temperatura de 25º C, com exceção do açafrão, que continuou predominante, ocorreu mudança na ordem de inibição ficando orégano > sálvia > alecrim, que demonstraram atividade nessa temperatura. O alho porró demonstrou não possuir atividade inibidora, tanto na forma de erva in natura como na forma de erva processada frente à bactéria em estudo, na temperatura utilizada. E o alecrim, na forma in natura, apenas na temperatura de 30º C, não exibiu atividade inibidora. A sálvia (IN e PRO), o orégano (IN e PRO) e o alecrim (IN) foram bacteriostáticos, e o alecrim (PRO), e o açafrão (PRO) foram bactericidas. O óleo essencial de açafrão na concentração de 0.2% em uma simulação com uma população padronizada de aproximadamente 106 UFC/g de P. fluorescens em carne de frango picada (PFP) proporcionou uma redução acima de 2 Log durante um período de 7 dias sob temperatura de refrigeração (4º C), contudo, mostrou-se inferior quando comparado com a inibição, sob as mesmas condições, obtida em caldo BHI que foi superior a 5 Log. O óleo essencial de açafrão testado sob as mesmas condições frente à microbiota natural do frango, que se mostrou resistente, exibiu uma redução limitada, apenas no dia zero e no dia um diferentemente da amostra a qual se inoculou P. fluorescens, que, num contexto geral, durante o período de sete dias, foi caracterizada como uma redução de 2 Log em relação à amostra controle.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-03-31T16:23:50Z No. of bitstreams: 1 texto completo.pdf: 1093826 bytes, checksum: e4d82c39648cf8e6fb2d08b321516454 (MD5) / Made available in DSpace on 2017-03-31T16:23:50Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1093826 bytes, checksum: e4d82c39648cf8e6fb2d08b321516454 (MD5) Previous issue date: 2017-02-17 / As bactérias psicrotróficas contaminantes do leite produzem proteases termo- resistentes que hidrolisam as caseínas do leite, resultando em perda de qualidade de leite e rendimento de produtos lácteos. Estudos envolvendo a caracterização dessas enzimas e o conhecimento das condições que influenciam sua produção e atividade são essenciais para a prevenção destes problemas. Neste trabalho, uma protease extracelular produzida pela cepa Pseudomonas fluorescens 07A foi purificada para estudos visando o entendimento de sua atividade e produção em diferentes condições. A enzima é uma metaloprotease inibida por Cu 2+ , Ni 2+ , Zn 2+ , Hg 2+ , Fe 2+ e Mg 2+ , mas induzida por Mn 2+ e possui massa molar de 49.486 kDa. O seqüenciamento da enzima por espectrometria de massa permitiu identificar o gene que codifica esta protease extracelular no genoma da cepa P. fluorescens 07A. A enzima possui 477 aminoácidos e domínios altamente conservados de ligação a Ca 2+ e Zn 2+ , indicando que Ca 2+ , o principal íon no leite, é também um co-fator dessa enzima. Sua atividade é máxima a 37 oC e pH 7,5, mas ela mantém mais de 40% de atividade quando submetida a 100 oC por 5 minutos em meio ideal e apenas de 14 a 30% quando submetida a tratamentos térmicos mais brandos, podendo causar problemas significativos nas condições normalmente utilizadas para o processamento e armazenamento de leite e produtos lácteos. Foi observada baixa expressão relativa da protease após 12 h de incubação a 25 oC, quando a bactéria encontra-se na fase logarítmica de crescimento, comparado com as temperaturas de refrigeração de 4 e 10 oC, quando a bactéria está ainda em fase lag. A produção da protease aumentou significativamente (P <0.05) em 24 h a 25 oC e permaneceu constante por até 48 h, intervalo em que a bactéria permaneceu em fase estacionária, indicando que a enzima pode ser produzida como uma estratégia adaptativa da bactéria. As frações de caseína de leite desnatado reconstituído foram completamente degradadas pela P. fluorescens 07A, pela protease purificada e pelo extrato da bactéria em até sete dias de incubação a 25 oC, e em menor extensão a 10 °C para a amostra incubada com a bactéria. O tratamento térmico de 90 oC por 5 minutos inativou a enzima purificada e inibiu sua atividade no extrato da bactéria. Este trabalho permitiu compreender as características bioquímicas e biológicas de uma protease extracelular da cepa P. fluorescens 07A, bem como as condições que influenciam a produção de proteases por esta bactéria em leite e sua atividade. Esses resultados podem auxiliar as indústrias de lácteos na busca por alternativas durante o processamento de produtos lácteos visando o controle de sua produção e atividade. / The milk contaminating psychrotrophic bacteria produce thermo resistant proteases that hydrolyze milk caseins, resulting in loss of milk quality and yield of dairy products. Studies involving the characterization of these enzymes and the knowledge about the conditions that influence their production and activity are essential for preventing these problems. This work aimed to understand the activity and production of the extracellular protease produced by Pseudomonas fluorescens 07A strain. This enzyme showed to be a metalloprotease inhibited by Cu 2+ , Ni 2+ , Zn 2+ , Hg 2+ , Fe 2+ and Mg 2+ , but induced by Mn 2+ and has a molar mass of 49,486 kDa. Partial sequencing of the proteic structure of this enzyme by mass spectrometry allowed the identification of the gene that encodes this protein in the genome of P. fluorescens 07A. The enzyme has 477 amino acids and highly conserved Ca 2+ and Zn 2+ -binding domains, indicating that Ca 2+ , the main ion in milk, is also a cofactor of this enzyme. The enzyme activity is maximum at 37 °C and pH 7.5, b ut it maintains more than 40% activity when subjected to 100 °C for 5 min in ideal medi um and only 14 to 30% in milder heat treatments, which may cause significant problems in the conditions normally used for the processing and storage of milk and dairy products. Low relative expression of protease was observed after 12 h of incubation at 25 °C, when the bacteria is in logarithmic growth phase, compared to its expression in refrigeration temperatures of 4 and 10 °C, when the bacteria is still in lag p hase. Protease production significantly increased (P <0.05) after 24 h at 25 °C and remained constant up to 48 h, when the bacteria remained at stationary phase, indicating that this enzyme could be produced as an adaptive strategy of the bacteria. The casein fractions of reconstituted skim milk were completely degraded as by P. fluorescens 07A, the purified protease or the bacterial extract within seven days of incubation at 25 °C, and to a lesser extent at 10 °C for milk ino culated with the bacteria. Heat treatment at 90 °C for 5 min inactivated the purified enzym e and inhibited its activity in the bacterial extract. This work allowed understanding the biochemical and biological characteristics of the extracellular protease produced of P. fluorescens 07A strain, as well as the conditions that influence its production and activity in milk. These results can help the dairy industry in the search for alternatives for the processing of dairy products to control production and activity of these proteases in milk.

Utilization of cyanide as the sole nitrogen source by Pseudomonas fluorescens NCIMB 11764 (Pf11764) occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated oxygenolytically by an enzyme referred to as cyanide oxygenase (CNO), which exhibits properties of a pterin-dependent hydroxylase. The pterin requirement for Pf11764 CNO was satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 µM). These compounds included, for example, biopterin, monapterin and neopterin, all of which were also identified in cell extracts. A related CNO-mediated mechanism of cyanide utilization was identified in cyanide-degrading P. putida BCN3. This conclusion was based on (i) the recovery of CO2 and NH3 as enzymatic reaction products, (ii) the dependency of substrate conversion on both O2 and NADH, and (iiii) utilization of cyanide, O2 and NADH in a 1:1:1 reaction stoichiometry. In contrast to findings reported for Pf11764, it was not possible to demonstrate a need for exogenously added pterin as a cofactor for the PpBCN3 enzyme system. However, results which showed that cells of PpBCN3 contained approximately seven times the amount of pterin as Pf11764 (of which a significant portion was protein-bound) were interpreted as indicating that sufficient bound CNO-cofactor exists, thus eliminating any need for a supplemental source.

Pseudomonas fluorescens NCIMB 11764 was capable of utilizing cyanide as a sole nitrogen source for growth. Cyanate (OCN") and S-cyanoalanine could also serve as nitrogenous substrates, but do not appear to play a role as intermediates in cyanide metabolism. Growth of this strain on cyanate as the sole nitrogen source led to the induction of an enzyme characterized as a cyanase (EC 3.5.5.3) based on its stoichiometric conversion of cyanate to ammonia, and dependence on bicarbonate for maximal activity. However, since cyanase activity was not elevated in cyanide-grown cells it was concluded that it serves no role in cyanide metabolism. Related studies aimed at examining a possible role for S-cyanoalanine as a cyanide-assimilation intermediate showed that while this compound also serves as a nitrogen source, it also is not important in cyanide metabolism. Studies focused on the utilization of free cyanide as a growth substrate led to the development of a fed-batch cultivation procedure greatly facilitating further experimentation aimed at the identification of cyanide metabolites. In addition to CO_2 and NH_3 as described earlier, two additional metabolites including formamide and formate were detected by using nC-NMR, HPLC, radioisotrapping methods and other analytical means. The formation of metabolites was shown to be induced after growth on cyanide with the relative product yields dependent on the availability of oxygen. These findings support earlier work in which an oxygen-dependent mechanism was proposed for the formation of C02 and NH3. However, at least two additional oxygen-independent pathways of cyanide conversion can be elaborated by this organism. One of these involves conversion to formate and ammonia while the other leads to the formation of formamide, which is not further degraded. Thus, growth on cyanide appears to occur by several mechanisms of chemical transformation presumably serving both detoxification and nutritional roles. Since two of these mechanisms generate ammonia, which is readily assimilated, growth is presumed to proceed via ammonia as a provisionary nitrogenous substrate.