[en] This PhD work was conducted in three different interrelated areas of knowledge. The first strategy followed consisted in the isolation of bacteria from the genus Micromonospora located inside nodules formed by Lupinus angustifolius. 61 strains were obtained from these experiments and grouped in 5 different groups with the following techniques: BOX-PCR, TP-RAPD and microsatelites. From the 5 groups mentioned, 22 strains were chosen for sequencing 16s rRNA gene. With these sequences a phylogenetic tree was made, including also Micromonospora strains that are already described. The strains isolated were a 98 to 99% similar to the following type strains: Micromonospora fulviviridis DSM 43906T, Micromonospora echinospora DSM 43816T, Micromonospora chaiyaphumensis JCM 12873T y Micromonospora olivasterospora DSM 43868T Micromonospora. This indicates the possible presence of new species within these strains.In the second part of the work, the fact that nitrogen fixating bacteria different that Bradyrrhizobium can develop nodules and fix nitrogen in Lupinus albus was demonstrated. 23 strains were isolated from nodules, and received the name Lupsa . One of the strains, Lupsa 11, was chosen by means of nodulation analysis as the specimen for the rest of the experiments. With the analysis of the 16s rRNA gene from the strain Lupsa 11, it was concluded that it belonged to the genus Paenibacillus. Said strain was used in the Lupinus albus nodulation experiments, and after 6 weeks, plants were found to have effective nodules in their roots that could reduce acetylene. Further experiments demonstrated that Paenibacillus Lupsa 11 could grow in media lacking nitrogen, and had the gene nifH, which had a 99% of similarity with the gene nifH from Frankia alni ACN14a.The third group of experiments were conducted to find Micromonospora and Paenibacillus bacteria inside nodules from Lupinus, using the fluorescent in situ hybridization technique (FISH). Probes tagged with Cy5.5 dye were highly specific and hybridized specifically with bacteria present in the inner cortex of the nodule, but not with any other structure, such as the intercellular tissue.

[es] Este trabajo de tesis se realiz? en tres ?reas diferentes que se interrelacionan entre s?. En la primera estrategia se realiz? el aislamiento de bacterias del g?nero Micromonospora a partir de n?dulos de Lupinus angustifolius. De este ensayo se obtuvieron 61 cepas que fueron reunidas en cinco grupos mediante las t?cnicas moleculares de BOX-PCR, TP-RADP y Microsat?lites. A partir de los grupos formados se escogieron 22 cepas a las cuales les secuenci? el gen ribos?mico 16S. Con las secuencias obtenidas se construy? el ?rbol filogen?tico teniendo en cuenta las cepas tipo de Micromonospora descritas. Se encontr? que las cepas aisladas presentaban porcentajes de similitud entre el 98% y 99% con las cepas tipo: Micromonospora fulviviridis DSM 43906T, Micromonospora echinospora DSM 43816T, Micromonospora chaiyaphumensis JCM 12873T y Micromonospora olivasterospora DSM 43868T, lo cual indica que algunas cepas puedan representar nuevas especies. En la segunda parte del trabajo, se demostr? que bacterias fijadoras de nitr?geno diferentes de Bradyrhizobium, forman n?dulos y fijan nitr?geno en la planta Lupinus albus. Inicialmente a partir de los n?dulos se aislaron 23 cepas denominadas Lupsa y mediante ensayos de nodulaci?n se eligi? a la cepa Lupsa 11 como modelo para los dem?s experimentos. El an?lisis del gen ARNr 16S de la cepa Lupsa 11 mostr? que pertenec?a al g?nero Paenibacillus. Dicha cepa se utiliz? en los ensayos de nodulaci?n de Lupinus albus y despu?s de 6 semanas de cultivo, se encontr? que las plantas presentaban n?dulos efectivos en las ra?ces y que fueron capaces de reducir el acetileno. En ensayos adicionales se encontr? que la cepa Paenibacillus Lupsa11 crec?a en medios de cultivo sin nitr?geno y adem?s, que conten?a el gen nifH el cual mostr? un 99% de similitud con respecto al gen nifH de la cepa Frankia alni ACN14a.En la tercera parte del trabajo se detect? la colonizaci?n de las bacterias Micromonospora y Paenibacillus en el interior de los n?dulos de Lupinus empleando la t?cnica de hibridaci?n in situ con fluorescencia (FISH). Las sondas marcadas con Cy5.5 fueron altamente selectivas e hibridaron de manera espec?fica con las bacterias presentes en el cortex interno del n?dulo y no se unieron a otros sitios como es el tejido intercelular.