Sequence analysis of the entire 3.5 kb <i>Hind</i>III genomic DNA fragment previously isolated from <i>Nostoc commune</i> UTEX 584 (Defrancesco and Potts 1988), determined the exact locations of the <i>nifU, nifH</i>, and <i>nifD</i> genes and identified two potential stem loop structures, a direct repeat, and an ORF that codes for a protein with a predicted amino acid sequence similar to that of myoglobin from <i>Paramecium caudatum</i>. The <i>N. commune</i> UTEX 584 myoglobin-like protein has a predicted length of 118 amino acids and molecular mass of 12,906 Da. A PCR copy of the gene (<i>glbN</i>) was cloned for overexpression of the protein. The recombinant protein was purified and used for spectral analysis and for the production of polyclonal antisera. Treatment of the recombinant protein with dithionite and CO resulted in spectral shifts characteristic of hemoproteins that bind oxygen. While some of the spectral characteristics are unique to the protein, in general the spectra were more like those of globins than cytochromes. Based on these characteristics and the sequence similarity to the P. caudatum mnyoglobin, we proposed the name cyanoglobin, with the gene designation glbN and the protein designation GlbN. Western analysis of GlbN expression was performed on N. commune UTEX 584 and two species of Anabaena (Anabaena sp. PCC 7120 and Anabaena variabilis). In N. commune UTEX 584 a protein with a molecular mass similar to that predicted for GlbN was detected. This protein was produced in increased amounts under the same growth conditions that resulted in increased production of nitrogenase reductase (the nifH gene product). No proteins of similar size to GlbN were detected in Anabaena sp. PCC 7120 or A. variabilis. A possible function of GlbN may be for oxygen storage, transport, or protection of the nitrogenase system. These functions as well as those of the direct repeat and the potential stem loop structures and their relationship to nitrogen fixation or other physiological processes in N. commune UTEX 584 require further analysis. / Ph. D.

The products of nifE and nifN from Azotobacter vinelandii, which are involved in the biosynthesis of the iron-molybdenum cofactor (FeMo-co) co) from nitrogenase, have been analyzed using a variety of mutagenic techniques. NifE was the object of several site-specific, amino acid substitutions that were designed to elicit information regarding metal cluster ligands, subunit-subunit interactions, and the proposed transfer of FeMo-co.from a nifEN-products complex to the apo-MoFe protein. A model of metal cluster binding; regions within the nifEN-products is discussed insofar as it relates to the rationale for the targeting of particular amino acids for-substitution. A translational fusion between nifN and lacZ was constructed and used to study the regulation of nifEN. This gene fusion was regulated in the same manner as wild type nifN and produced a fusion protein which was enzymatically active with respect to substrates of β-galactosidase. Results from mutant strains which carry lesions in nifH or nifA in addition to the nifN / Master of Science

Two separate greenhouse esperiments were conducted to determine the effects of additions of a blue-green algae (Nostoc muscorum) to nutrient solution cultures with different rates of nitrogen fertilizer on the growth of lettuce (Lactuca sativa L. cv. Buttercrunch). Lettuce yields increased with increased rates of N. Yields were significantly greater in treatments containing 165 ppm N with N. muscorum culture added (at the rate of 33.3% by volume) than in treatments containing the same rate of N without N. muscorum. A similar growth increase was not noted at lower N. muscorum inoculation rates (16.7% by volume). These preliminary data indicate the potential for increased yields of lettuce grown under specific conditions in nutrient culture. Further research of the complex interactions within this biological equilibrium are required before specific application recommendations may be suggested. / Master of Science

Nostoc commune UTEX 584 is a photosynthetic desiccation-tolerant cyanobacterium capable of fixing nitrogen. Biotinylated nifH and nifD gene probes from Azotobacter vinelandii (a gift from Dr. Dennis Dean) hybridized to nifH and nifD sequences isolated from Nostoc commune UTEX 584. This result supports the view that the nitrogenase structural proteins and their genes are highly conserved in nitrogen-fixing organisms (Rice et al., 1980). Southern transfers of genomic DNA prepared from N- commune UTEX 584 were digested with Hind III and hybridized with nifH-specific and nifD-specific probes. Multiple copies of nifH (three) and nifD (two) were detected. Using colony hybridization, a recombinant N- commune UTEX 584 genomic DNA-pBR322 plasmid library was screened with the biotinylated nifH-specific probe and positive hybridizing nifH clones were isolated. A restriction map of the 3.5 kb Hind III insert of the recombinant plasmid (pND001) was produced. From partial sequencing data it was possible to determine the positions of the N. commune UTEX 584 nifH and nifD genes within the cloned fragment and compare the partial nucleotide sequence and deduced amino acid sequences of the E- commune UTEX 584 nifHD genes with other organisms. Isolation of the nifH and nifD genes from Nostoc commune UTEX 584 now permits a more detailed study of nif gene expression in this desiccation-tolerant photosynthetic microorganism. / M.S.

The present study investigates the structural and functional roles of the metalloclusters present within the MoFe protein of nitrogenase from Azotobacter vinelandii. A gene replacement strategy was developed for oligonucleotide-directed mutagenesis of these proteins and the resulting biological and biochemical effects of these changes were examined. Identification of structurally important regions in the MoFe protein subunits and assignment of specific amino acid residues as potential metal cluster ligands were based upon several criteria: i. metallocluster extrusion requirements; spectroscopic properties of the MoFe protein; interspecies and intersubunit comparisons; iv. comparison of the MoFe protein subunit sequences to iron-molybdenum cofactor biosynthetic gene products. This mutagenesis strategy has permitted the construction of thirty-three mutant strains having specific amino acid substitutions within the MoFe protein subunits. Based on the diazotrophic growth characteristics and substrate reduction capabilities of these mutant strains, a model is presented in which potential metallocluster binding sites within the MoFe protein subunits are defined. In addition to analysis of the MoFe protein subunits, this site-directed mutagenesis and gene replacement strategy can be used to place specific mutations into any gene product encoded within the A. vinelandii nif gene cluster. Finally, nucleotide sequence analysis of the regions flanking the nifEN genes revealed the presence of three nif genes (nifT, nifY, and nifX) and four open reading frames (ORF1, ORF2, ORF3, and ORF4). Two of these genes, nifX and ORF3, were shown to be under nif control and synthesis of their products elevated in response to a demand for fixed nitrogen. Mutant strains with deletions in ORF3 appeared to accumulate an excess amount of MoFe protein when compared to wild type. The ORF3 gene product has been overproduced in E. coli. This provides an important step toward characterizing the protein and elucidating the molecular basis for its control of nifDK gene expression. / Ph. D.

In recent history, C. jamaicense has been displaced by another native monocot, T. domingensis, predominantly resulting from increased phosphorous enrichment in the Everglades. This study aimed to elucidate these two species responses to low and high [Pi] in terms of allocation, photosynthate partitioning and growth. C. jamaicense growth was independent of Pi, while T. domingensis growth increased with [Pi]. Under high [Pi], allocation to younger T. domingensis shoots occurred, while C. jamaicense shoots retained more [Pi], while low [Pi] resulted in homogeneous allocation patterns for both species. Additionally, Pi deficiencies induced carbohydrate levels in older shoots of T. domingensis, while [Pi] had no effect on photosynthate partitioning patterns in C. jamaicense. ACP activity was induced by Pi deficiency in all T. domingensis shoots and increased with shoot age, while no effect was observed in C. jamaicense. Results indicate these two species differ in allocation strategies when [Pi] is altered. / by Brian Hill. / Vita. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

As bromélias habitam os mais diferentes ambientes sendo que muitas são encontradas como epífitas. Essas últimas estão sujeitas a condições de disponibilidade de água e nutrientes com intermitência. Elas conseguem sobreviver a essas circunstâncias por serem dotadas de diversas adaptações morfológicas e fisiológicas. A bromélia tanque Guzmania monostachia tem sido bastante estudada por possuir uma grande plasticidade fotossintética, porém, pouco é conhecido a respeito de outras possíveis adaptações como a interação com micro-organismos. Sendo assim, o presente trabalho isolou bactérias fixadoras de nitrogênio (diazotróficas) tanto da parte externa (epifíticas) quanto da parte interna (endofíticas) de diferentes porções (ápice, mediana e base) de folhas coletadas tanto no ambiente natural quanto na casa de vegetação. As bactérias foram selecionadas por meio do ensaio de redução de acetileno (ARA) e também pelo uso de quatro diferentes meios de cultura que não contém fonte de nitrogênio reduzido. As linhagens isoladas foram identificadas por meio do gene 16sRNA. Dois isolados Pseudomonas sp. e Burkholderia sp. foram escolhidos para serem marcados com um gene de fluorescência verde (GFP) e foram então inoculados (separadamente) em plantas de G. monostachia cultivadas em casa de vegetação. A colonização dos tecidos foliares foi monitorada com auxílio de um microscópio confocal. Além disso, foram estimadas as densidades populacionais epifíticas e endofíticas em diferentes grupos foliares (jovens e intermediários) e as folhas do grupo intermediário por serem maiores e totalmente expandidas foram divididas em porções (apical, mediana e basal). Também foram pesquisadas as seguintes moléculas descritas como importantes na interação entre planta e micro-organismo: óxido nítrico (NO), ácido salicílico (SA), etileno (ET) e ácido-indol-3-acético (IAA). Como resultados, a maioria das linhagens bacterianas foram classificadas como pertencentes ao grupo Proteobacteria, mas também foram encontrados isolados gram positivos pertencentes aos grupos Actinobacteria e Firmicutes. As bactérias endofíticas foram isoladas somente da porção basal foliar (tanto das folhas originadas do meio ambiente quanto das folhas originadas da casa de vegetação). Cabe ressaltar que após a inoculação de ambas bactérias marcadas com GFP, foi observado elas no interior dos tricomas foliares (estruturas presentes principalmente na base foliar). Após 20 horas da inoculação, ambas bactérias já foram visualizadas no interior da epiderme das folhas. Após 5 dias as bactérias foram se espalhando para regiões mais distantes do tricoma e também foram observadas no parênquima. Após 10 dias a bactéria Pseudomonas sp. foi encontrada nas paredes dos vasos condutores. Foram re-isoladas bactérias epifíticas e endofíticas da mediana e da base foliar, mas não da porção apical. Após 10 dias as bactérias foram isoladas como endofíticas somente da base. Essa porção não apresentou diferenças nas populações epifíticas e endofíticas. O NO aumentou nas folhas jovens e na base das intermediárias em um curto período de tempo após a inoculação. Aparentemente, ambas as bactérias não dispararam a via do SA. De acordo com os resultados aqui presentes, ambas as bactérias não pareceram ser prejudiciais à G. monostachia. Além disso, o presente trabalho mostra fortes evidencias de que as bactérias entram nos tecidos foliares por meio dos tricomas na base foliar e permanecem nessa porção, que é precisamente a mais importante para absorção de nutrientes / Bromeliads inhabit different environments and many are found as epiphytes. These plants are often subjected to periods of water and nutrient shortage. For their survival, epiphytic bromeliads are endowed with different morphological and physiological adaptations. Guzmania monostachia is a tank-bromeliad that has been extensively studied because of its great photosynthetic plasticity. However, little is known about other possible survival adaptations, such as beneficial interactions with microorganisms. Here, we isolated nitrogen fixing (diazotrophc) bacteria from both the outside (epiphytic) and the inside (endophytic) of different leaf portions (apex, middle and base), collected in natural environment and in greenhouse-cultivated plants. The bacteria were selected using the acetylene reduction assay (ARA) and four different media that do not contain reduced nitrogen source. The strains were identified by 16S rRNA. Two isolates, Pseudomonas sp. and Burkholderia sp. has been chosen to be tagged with green fluorescent protein (GFP) and then inoculated in G. monostachia plants cultivated in greenhouse. The colonization of the leaf tissues was monitored with the aid of confocal microscopy and also we estimate the external and internal bacterial population densities in different leaf groups (younger and expanded) and portions (apex, middle and base). In addition, we studied some important molecules in plant-microbe interactions: nitric oxide (NO), salicylic acid (SA), ethylene (ET) and indol-3-acetic acid (IAA). As a result, most of the isolated strains belong to the Proteobacteria group, but gram positive strains were also found belonging to the Firmicutes and Actinobacteria group. The endophytic bacteria were isolated only from the basal portion (both from leaves of natural environment and from leaves of greenhouse cultivated plants). Interestingly, after the inoculation of both bacteria tagged with GFP they were visualized entering by trichomes present mainly in the basal portion. Twenty hours after the inoculation, the bacteria were visualized inside the epidermis of the leaves. After five days, the bacteria were detected in the parenchyma and, ten days after the inoculation Pseudomonas sp. was found on the vessels walls. It was possible to re-isolate epiphytic and endophytic bacteria from the base and middle portions, but not from the apex. After 10 days the endophytic bacteria were found only in the base. The base did not show differences between epiphytic and endophytic populations. NO increased in a short time after the inoculation in the younger leaves and in the basal portion of intermediate leaves. Apparently, the SA pathway was not triggered by any of the bacteria used. According to these results, the bacteria tested do not seem to be harmful to the plant. Furthermore, we strongly suggest that they enter through the trichomes on the leaf base and remain in this portion, which is precisely the most important for the absorption of nutrients

O processo de fixação biológica de nitrogênio (FBN) é a principal forma de entrada de nitrogênio (N) em ecossistemas naturais e é intermediado por microorganismos diazotróficos simbióticos ou de vida livre. A produção de biofertilizantes com bactérias diazotróficas é a principal alternativa ao uso de fertilizantes nitrogenados solúveis. Apesar da simbiose Rhizobium-leguminosas ser eficiente em promover o crescimento das plantas, a inoculação de bactérias diazotróficas em gramíneas vem apresentando resultados questionáveis, principalmente devido à baixa eficiência e incompatibilidade entre os atuais inoculantes bacterianos e as plantas. Os biofertilizantes para gramíneas utilizam bactérias endofíticas as quais desenvolvem interações pouco conhecidas com as plantas. Uma possibilidade para melhorar a eficiência da FBN em gramíneas é a aplicação de bactérias epifíticas de menor seletividade em relação às bactérias endofíticas. Os objetivos deste trabalho foram isolar novos genótipos de bactérias diazotróficas da filosfera de bambu da Mata Atlântica, verificar seu potencial biotecnológico in vitro e avaliar sua eficiência agronômica como biofertilizante em cana-de-açúcar em casa-de-vegetação. Foram obtidos 120 isolados bacterianos os quais foram caracterizados morfológica e filogenéticamente. Com relação ao potencial biotecnológico, 48 isolados bacterianos apresentaram resposta positiva nos testes in vitro de redução de acetileno (ARA) e para produção in vitro de ácido indol-3-acético (AIA) na presença de L-triptofano. Desses 48 isolados, 50% apresentaram capacidade de solubilizar fosfato de cálcio, 8% de produzir quitinases, 16% de produzir ACC desaminase e 17% de produzir sideróforos. O sequenciamento do gene rRNA 16S indicou que 75% dos isolados da filosfera de bambu foram similares à classe Gammaproteobacteria (Proteobacteria) , e a família Enterobacteriaceae. Dentre eles, 32% dos isolados apresentaram sequências similares a Klebsiella sp. e 2% foram similares a Serratia e Enterobacter. Das 48 sequências analisadas, 37% delas não foram classificadas quanto ao gênero, podendo então representar novos gêneros ou espécies. Dez isolados contendo o gene nifH foram selecionados para experimentos de eficiência agronômica em casa-de-vegetação usando bactérias inoculadas via pulverização foliar ou encapsuladas em micro-esferas de alginato e associados a diferentes níveis de adubação nitrogenada. A inoculação por meio de pulverização nas folhas de cana-de-açúcar resultou em aumento significativo de massa seca de parte aérea e concentração de N na parte aérea das plantas na sua fase inicial de desenvolvimento. A maior taxa de fixação nas folhas inoculadas foi observada sete dias após a inoculação no tratamento sem adição de N mineral. A inoculação no solo com os mesmos 10 isolados encapsulados em matriz polimérica em conjunto com diferentes níveis de adubação nitrogenada mostrou aumentos significativos de massa seca da parte aérea, raízes e concentração de N na parte aérea da cana-de-açúcar em comparação aos controles, principalmente nas fases tardias do desenvolvimento das plantas. O solo que recebeu as bactérias encapsuladas apresentaram elevadas taxas de FBN, oscilando entre 0 e 4 g de N g-1 h-1, 7 dias após a inoculação. Os resultados sugerem que as bactérias selecionadas possuem alto potencial biotecnológico para promover o crescimento das plantas em momentos diferentes do seu ciclo de desenvolvimento, dependendo do tipo de abordagem para inoculação. / The process of biological nitrogen fixation (BNF) is the most important form of nitrogen (N) input in natural ecosystems and is mediated by symbiotic or free-living diazotrophic microorganisms. The production of biofertilizers containing diazotrophic bacteria is the main alternative to the use of soluble nitrogen fertilizers, improving plant growth by nitrogen fixation or other plant growth promoting mechanisms. Despite the efficiency of the symbioses Rhizobium-legumes in promoting plant growth, the inoculation of diazotrophic bacteria in grasses, such as sugarcane, has shown variable results, mainly due to the low efficiency and incompatibility between the current bacterial strains used in inoculants and plant genotypes. Most of the biofertilizers for grasses uses endophytic bacteria that develop complexes interactions with the host plant, which are not totally understood. A possibility to improve the efficiency of BNF in grasses is the application of epiphytic diazotrophic bacteria that are less selective as compared to endophytes. The aims of this work were to isolate new genotypes of diazotrophic bacteria from the phyllosphere of bamboo from Atlantic Forest, determine their biotechnological potential based on in vitro assays, and evaluate their agronomic efficiency as biofertilizer for sugarcane under greenhouse conditions. A total of 120 bacterial isolates were obtained and characterized morphologically and phylogenetically. Regarding the biotechnological potential, 48 isolates showed positive responses under in vitro the acetylene reduction assay (ARA) and indole-acetic-acid (IAA) production in vitro assay in the presence of L-tryptophan. Among the 48 isolates evaluated, 50% were able to solubilize calcium phosphate, 8% produced chitinases, 16% were able to produce ACC deaminase, and 17% produced siderophores. The sequencing of the rRNA 16S gene revealed that 75% of the isolates were phylogenetically related to the family Enterobacteriaceae (Gammaproteobacteria) . The genus Klebsiella accounted for 32% of the isolates, whereas Serratia and Enterobacter accounted for 2%. Aproximatelly 37% of the isolates were assembled unclassified Bacteria. Ten isolates containing the nifH gene were selected for agronomic efficiency test under greenhouse conditions, using bacteria inoculated via foliar spraying, or encapsulation in alginate beads and inoculation in the soil, associated with different doses of nitrogen fertilizer. The inoculation on the sugarcane leaf surfaces resulted in significant increases in root biomass and N concentration in the shoots at the early stage of plant development. The highest N fixation rates in inoculated leaves were observed 7 days after inoculation in the absence of mineral N. The soil inoculation with the same 10 isolates immobilized in polymeric matrix in addiction to different rates of nitrogen fertilization showed significant increases in shoot and root biomass and N concentration in the shoots of sugarcane, when compared to the controls, mostly at later stages of plant development. The soil inoculated with encapsulated bacteria showed high rates of BNF even when nitrogen fertilizer was applied, ranging between 0 and 4 g of N g-1 h-1 seven days after the inoculation. The results suggest that the selected bacteria have high biotechnological potential to promote sugarcane growth at different stages of development, depending on the inoculation approach.

Tropical rainforests have an unparalleled capacity to sequester carbon, harbor biodiversity, and cycle water and nutrients due to their high rates of primary production. The large biomass stocks and rapid regeneration rates of these forests are often attributed to ample soil nitrogen and quick recovery of the nitrogen cycle in tropical soils following disturbance. Symbiotic nitrogen-fixing trees, which are relatively abundant at tropical latitudes, have the greatest capacity to provide tropical rainforests with new nitrogen, yet the ecological drivers of tropical symbiotic nitrogen fixers and their effects on the forests they inhabit are not well understood. This dissertation consists of four chapters that examine the patterns, environmental controls, and ecological consequences of symbiotic nitrogen-fixing trees in regenerating and intact rainforests in the Caribbean lowlands of Costa Rica. In chapter 1, I use field sampling in a chronosequence of rainforest plots to show that symbiotic nitrogen fixation declines through succession despite increases in the basal area of nitrogen-fixing trees. Chapters 2 and 3 describe results from a controlled shadehouse experiment assessing the effects of light, soil nitrogen, and plant competition on nitrogen fixation rates and the growth and biomass allocation of nitrogen fixers and non-fixers. In chapter 2, I demonstrate that light regulates nitrogen fixation more strongly than soil nitrogen availability. This is a departure from the historical focus on soil nitrogen as the primary regulator of nitrogen fixation and has the potential to resolve longstanding paradoxes of tropical nitrogen cycling. In chapter 3, I show that nitrogen fixation provides some resistance to competitive effects from neighboring plants in nitrogen-limited conditions, and that nitrogen fixers in these conditions downregulate their fixation rates in the presence of a competitor. This chapter also demonstrates that nitrogen fixation does not represent a significant structural cost to the plant, as reduced root biomass of nitrogen fixers more than compensates for allocation to nodule production. Finally, in Chapter 4, I demonstrate that nitrogen-fixing trees in our chronosequence plots do not promote forest growth, as expected given their capacity to fertilize their neighbors, but rather inhibit forest growth because they are strong competitors. These chapters describe several unexpected findings – i.e. that light primarily drives nitrogen fixation and that nitrogen fixers slow forest growth – which provide new and important insight into the role that nitrogen-fixing trees play in the growth of Costa Rican rainforests.

As bromélias habitam os mais diferentes ambientes sendo que muitas são encontradas como epífitas. Essas últimas estão sujeitas a condições de disponibilidade de água e nutrientes com intermitência. Elas conseguem sobreviver a essas circunstâncias por serem dotadas de diversas adaptações morfológicas e fisiológicas. A bromélia tanque Guzmania monostachia tem sido bastante estudada por possuir uma grande plasticidade fotossintética, porém, pouco é conhecido a respeito de outras possíveis adaptações como a interação com micro-organismos. Sendo assim, o presente trabalho isolou bactérias fixadoras de nitrogênio (diazotróficas) tanto da parte externa (epifíticas) quanto da parte interna (endofíticas) de diferentes porções (ápice, mediana e base) de folhas coletadas tanto no ambiente natural quanto na casa de vegetação. As bactérias foram selecionadas por meio do ensaio de redução de acetileno (ARA) e também pelo uso de quatro diferentes meios de cultura que não contém fonte de nitrogênio reduzido. As linhagens isoladas foram identificadas por meio do gene 16sRNA. Dois isolados Pseudomonas sp. e Burkholderia sp. foram escolhidos para serem marcados com um gene de fluorescência verde (GFP) e foram então inoculados (separadamente) em plantas de G. monostachia cultivadas em casa de vegetação. A colonização dos tecidos foliares foi monitorada com auxílio de um microscópio confocal. Além disso, foram estimadas as densidades populacionais epifíticas e endofíticas em diferentes grupos foliares (jovens e intermediários) e as folhas do grupo intermediário por serem maiores e totalmente expandidas foram divididas em porções (apical, mediana e basal). Também foram pesquisadas as seguintes moléculas descritas como importantes na interação entre planta e micro-organismo: óxido nítrico (NO), ácido salicílico (SA), etileno (ET) e ácido-indol-3-acético (IAA). Como resultados, a maioria das linhagens bacterianas foram classificadas como pertencentes ao grupo Proteobacteria, mas também foram encontrados isolados gram positivos pertencentes aos grupos Actinobacteria e Firmicutes. As bactérias endofíticas foram isoladas somente da porção basal foliar (tanto das folhas originadas do meio ambiente quanto das folhas originadas da casa de vegetação). Cabe ressaltar que após a inoculação de ambas bactérias marcadas com GFP, foi observado elas no interior dos tricomas foliares (estruturas presentes principalmente na base foliar). Após 20 horas da inoculação, ambas bactérias já foram visualizadas no interior da epiderme das folhas. Após 5 dias as bactérias foram se espalhando para regiões mais distantes do tricoma e também foram observadas no parênquima. Após 10 dias a bactéria Pseudomonas sp. foi encontrada nas paredes dos vasos condutores. Foram re-isoladas bactérias epifíticas e endofíticas da mediana e da base foliar, mas não da porção apical. Após 10 dias as bactérias foram isoladas como endofíticas somente da base. Essa porção não apresentou diferenças nas populações epifíticas e endofíticas. O NO aumentou nas folhas jovens e na base das intermediárias em um curto período de tempo após a inoculação. Aparentemente, ambas as bactérias não dispararam a via do SA. De acordo com os resultados aqui presentes, ambas as bactérias não pareceram ser prejudiciais à G. monostachia. Além disso, o presente trabalho mostra fortes evidencias de que as bactérias entram nos tecidos foliares por meio dos tricomas na base foliar e permanecem nessa porção, que é precisamente a mais importante para absorção de nutrientes / Bromeliads inhabit different environments and many are found as epiphytes. These plants are often subjected to periods of water and nutrient shortage. For their survival, epiphytic bromeliads are endowed with different morphological and physiological adaptations. Guzmania monostachia is a tank-bromeliad that has been extensively studied because of its great photosynthetic plasticity. However, little is known about other possible survival adaptations, such as beneficial interactions with microorganisms. Here, we isolated nitrogen fixing (diazotrophc) bacteria from both the outside (epiphytic) and the inside (endophytic) of different leaf portions (apex, middle and base), collected in natural environment and in greenhouse-cultivated plants. The bacteria were selected using the acetylene reduction assay (ARA) and four different media that do not contain reduced nitrogen source. The strains were identified by 16S rRNA. Two isolates, Pseudomonas sp. and Burkholderia sp. has been chosen to be tagged with green fluorescent protein (GFP) and then inoculated in G. monostachia plants cultivated in greenhouse. The colonization of the leaf tissues was monitored with the aid of confocal microscopy and also we estimate the external and internal bacterial population densities in different leaf groups (younger and expanded) and portions (apex, middle and base). In addition, we studied some important molecules in plant-microbe interactions: nitric oxide (NO), salicylic acid (SA), ethylene (ET) and indol-3-acetic acid (IAA). As a result, most of the isolated strains belong to the Proteobacteria group, but gram positive strains were also found belonging to the Firmicutes and Actinobacteria group. The endophytic bacteria were isolated only from the basal portion (both from leaves of natural environment and from leaves of greenhouse cultivated plants). Interestingly, after the inoculation of both bacteria tagged with GFP they were visualized entering by trichomes present mainly in the basal portion. Twenty hours after the inoculation, the bacteria were visualized inside the epidermis of the leaves. After five days, the bacteria were detected in the parenchyma and, ten days after the inoculation Pseudomonas sp. was found on the vessels walls. It was possible to re-isolate epiphytic and endophytic bacteria from the base and middle portions, but not from the apex. After 10 days the endophytic bacteria were found only in the base. The base did not show differences between epiphytic and endophytic populations. NO increased in a short time after the inoculation in the younger leaves and in the basal portion of intermediate leaves. Apparently, the SA pathway was not triggered by any of the bacteria used. According to these results, the bacteria tested do not seem to be harmful to the plant. Furthermore, we strongly suggest that they enter through the trichomes on the leaf base and remain in this portion, which is precisely the most important for the absorption of nutrients