Darmstadt, Techn. Universiẗat, Diss., 2004. / Dateien im PDF-Format.

Since the initial proposition that the archaebacteria form a primary kingdom as distinct as that of the eubacteria or the eukaryotes, sequence data generated from the ribosomal RNA genes have flooded the databases and periodicals. Phylogenetic trees based on these sequences have been constructed to map the finest details of topology and branching order within the archaebacteria. Yet, despite the plethora of sequence data, relatively little was discovered regarding rRNAgene regulation, transcript processing and requirements for mature ribosome function. The aim of this study is to analyze possible novel regulatory mechanisms in the rRNA genes of the extremely thermoacidophilic archaebacterium Sulfolobus acidoccddccrius. The three ribosomal RNA genes were cloned and sequenced. The gene organization was confirmed to differ from that of the halophilic archaebacteria and the eubacteria: the 5S gene was not linked to the 16S and 23S operon, and the operon lacked recognizable tRNA sequences. Southern hybridization unveiled, and sequence data confirmed a long-standing confusion regarding species identity. The previously published Sulfolobus acidocaldarius 5S sequence was shown to have been attributed to the wrong species. Mapping experiments showed that both transcripts initiated downstream of a previously defined archaebacteria! promoter sequence. While sequence data showed the 5S transcript start site and end site to be coincidental with the mature 5S termini, the 16S-23S transcript was shown to contain a 143 nucleotide transcribed leader sequence, a 138 nucleotide intergenic sequence, and a trailer sequence of at least 105 nucleotides. Inverted repeat sequences within these transcribed non-coding regions allow for the formation of numerous stem-loops conforming to a semi-conserved archaebacterial structure. While no processing took place within the 5S transcript, extensive processing of the 16S-23S transcript was observed. Of the 12 processing sites mapped, only 6 could be accounted for in the context of precursor processing and maturation events known directly or inferred by analogy from the halophilic archaebacteria and the eubacteria. Alignment of the remaining sites revealed a non-trivial sequence and structural similarity. If the novel processing indeed took place in the postulated context, it would mark a radical departure from the expected maturation mechanism thought to predate the speciation of archaebacteria and eubacteria. To examine this possibility, in vitro transcripts from judiciously selected DNA fragments were subjected to cell-free extract. Analysis of the resultant cleavage products confirmed the presence not only of a novel processing activity mediated by a ribonucleoprotein complex but also of a novel processing pathway. Based on the locations of the novel processing sites within the primary 16S-23S transcript, a model for transcriptional regulation independent of polycistronic linkage is presented. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

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Microvesicles secreted from the thermoacidophilic archaeon S. acidocaldarius (Sa-MVs) contain a membrane made exclusively of tetraether lipids and covered by crystalline surface layer proteins known as the S-layer. While tetraether lipids and S-layer proteins are known to be useful biomaterials, little has been done to exploit Sa-MVs for any scientific applications. In the present study, as the start point to explore this area, we isolated Sa-MVs and used dynamic light scattering, laser Doppler electrophoresis, and cryo-transmission electron microscopy (cryo-TEM) to characterize the particle size, size distribution, zeta potential, and morphology of Sa-MVs and tested their stabilities against temperature, pH, autoclaving, and the detergent Triton X-100. We found that, at the cell’s growth pH (~2.6) and growth temperature (75-80oC), Sa-MVs in the growth medium are ~180-183 nm in diameter with a polydispersity index (PDI) ≤ 0.15 and have a zeta potential of -0.5 mV. Sa-MVs in buffer exhibited long-term (at least 137 days) stability with no signs of vesicle disintegration or fusion. When the pH was decreased from 7.2 to 2.6, the average size of Sa-MVs was increased by ~40-45 nm, which probably came from conformational changes of S-layer proteins, in concomitant with vesicle aggregation, but not due to conformational changes in tetraether lipid headgroups. The isoelectric point (pI) for Sa-MVs in 1 mM KCl is 3.0 while that for the reconstituted liposomes (LUVMV) is estimated to be below 2.0. Sa-MVs dispersed in buffer at pH 2.6 change little in size over five autoclaving cycles, despite becoming slightly less spherical after autoclaving, while at this pH liposomes made of diester lipids cannot sustain multiple cycles of autoclaving. In addition, compared to diester and PLFE liposomes, Sa-MVs and LUVMV exhibit unusual resistance against the surfactant Triton X-100. Although some man-made liposomes such as PLFE liposomes are also stable against temperature, pH, and other environmental stressors, Sa-MVs are unique in that they are naturally occurring nanoparticles with a native membrane environment suitable for inserting additional lipids and membrane-bound proteins as needed. With their great stability presented here and the lack of cytotoxicity known in the literature, Sa-MVs hold great promise for technological applications. In addition to these biophysical techniques employed to characterize these microvesicles, a series of fluorescence experiments have also been conducted to gain further insight into how the membrane packing of these vesicles compares to tetraether as well as diester liposomes. Intrinsic protein fluorescence of native microvesicles was examined to characterize the dynamics of the S. acidocaldarius MVs. We have used the probe 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) to monitor membrane packing and dynamics within the water-membrane interfacial region of the Sa-MVs. Specifically, we measured Laurdan’s generalized polarization (GP), which depends on the probe’s local polarity, probe location and nearby viscosity. We also measured Laurdan’s red edge excitation shift (REES), which depends on the dynamics of solvent relaxation around the fluorophore compared to the probe’s fluorescence lifetime. As temperature increased from 18 to 66.7 °C, GP decreased from 0.026 to -0.118. Comparing the GP values of reconstituted vesicles to that of the native Sa-MVs, it appears that the two curves are similar in both GP value and trend over increasing temperature range (values decrease from 0.112 to -0.215), which suggests that Laurdan in Sa-MVs resides in the lipid membrane, not in proteins and that Laurdan’s GP is not affected much by the presence of Sa-MV proteins. It is well known that, for liposomes made of diester lipids, the GP value of Laurdan fluorescence is high in the gel state and low in the liquid-crystalline state, with an abrupt change during the phase transition. However, Laurdan’s GP values obtained from liposomes comprised of tetraether lipids such as PLFE and Sa-MV lipids cannot be compared directly to those obtained from diester liposomes and cannot be interpreted simply based on membrane packing because probe location and chromophore orientation in tetraether liposomes could be distinctly different from those in diester liposomes. Our data show that the REES effect in PLFE LUVs is most pronounced among all the membranes examined showing a value of 10.58 nm at 24oC compared to 0.9 nm for DMPC LUVs at the same temperature. It is already known that membrane packing in PLFE liposomes is extraordinarily tight partly due to the strong hydrogen bond network in the polar head group regions where the phosphate and sugar moieties are abundant and partly due to the rigid and ordered dibiphytanyl hydrocarbon chains. Since the chromophore of Laurdan in PLFE liposomes is most likely located in the membrane-water interfacial region, it is not surprising that solvent tumbling around Laurdan in PLFE LUVs is much more restricted in PLFE LUVs than in DMPC LUVs, giving rise to a much higher REES value in PLFE LUVs than in DMPC LUVs. The REES values (10.3-14.1 nm) of Laurdan fluorescence in LUVs reconstituted from the extracted MV lipids are higher than those (9.3-10.6 nm) in PLFE LUVs, which suggests that the mobility of solvent molecules (including water and lipid polar groups) in the membrane-water interfacial regions of LUVMV is much less restricted than that in PLFE LUVs. Like PLFE, Sa-MV lipids are tetraethers. However, as mentioned earlier, PLFE lipids are different from Sa-MV lipids in their hydrophobic core composition. It is likely that their polar head groups are also different despite that the zeta potentials of LUVPLFE and LUVMV are virtually identical (-43 mV in 50 mM Tris buffer containing 10mM EDTA and 0.02% NaN3 at pH 7.2-7.6 and 25oC). The REES values (14.5-18.9 nm) of Laurdan fluorescence in Sa-MVs are higher still than those of both LUVPLFE and LUVMV, which is reasonable because tetraether lipids in Sa-MVs are covered with S-layer proteins. As a result, the mobility of solvent molecules around Laurdan’s chromophore in the lipid polar head group regions is more restricted than that in LUVPLFE or LUVMV. Free-standing planar membrane made of MV lipids built on a cellulose acetate partition and mounted onto a Teflon device sustained a nearly constant capacitance (~36-39 pF) for 8 h. Thereafter, the membrane collapsed as evidenced by a zero capacitance. In contrast, the planar membrane made of the diester lipid POPC had much lower stability, showing large fluctuations in capacitance before its collapse at 1.5 h, a very short lifetime typical for free-standing planar membranes made of diester lipids. The planar membrane made of the diester lipid DMPC also showed a short lifetime ~3h. In comparison, the planar membrane made of PLFE showed remarkable stability, exhibiting a constant capacitance for at least 11 days. Similar high stability of PLFE free-standing planar membranes over micro-pores on PDMS thin films in microchip platform was previously reported. Our data suggest that lipids extracted from S. acidocaldarius MVs are able to form fairly stable free-standing planar membranes across a pinhole on a solid support. However, even though both MV lipids and PLFE lipids are tetraethers, the planar membrane made of MV lipids is not as stable as that made of PLFE lipids. The molecular basis for the differential stability between planar membranes of MV lipids and PLFE lipids is not clearly understood at present, but the difference in stability is likely to originate from the chemical structure differences between PLFE lipids and MV lipids. As mentioned earlier, in terms of the hydrophobic cores, PLFE contains ~90% GDNT and ~10% GDGT, whereas MV lipids are mainly GDGT and GTGT, without any GDNT, and their headgroup structures are not known. We have also demonstrated the ability to observe channel activity in PLFE monolayers at a range of voltages from -200 to 200 mV. However, this was property was not replicated in lipids extracted from S. acidocaldarius microvesicles. In any case, our past and present data showed that archaeal tetraether lipids are excellent materials to make stable and yet biologically relevant free-standing planar membranes. / Biomedical Sciences

En este trabajo de tesis se estudió la influencia catalítica de Sulfolobus metallicus en la biolixiviación de calcopirita pura a 70ºC y pH 1,5. La lixiviación se realizó en medio basal Norris en matraces agitados con muestras de calcopirita sintética y natural molida entre -80 # + 120# de Andina. Las muestras fueron caracterizadas monitoreando la concentración de cobre, ferroso, hierro total, sulfato y la población de células en la solución. Se utilizó difracción de rayos x (DRX) para caracterizar los sólidos. Se realizaron análisis por microscopía electrónica de barrido acoplado a espectroscopía de energía dispersiva (SEM-EDS) para determinar la adherencia de las células al mineral. Para evaluar separadamente la influencia de las células planctónicas y las células adheridas en el proceso catalítico, en algunos experimentos los microorganismos fueron impedidos de estar en contacto con la calcopirita manteniéndolos en una cámara separada. Complementariamente, se estudiaron las diferentes especies de azufre disueltas en solución por cromatografía lìquida a alta presión (HPLC) en condiciones abióticas con aire o nitrógeno y en condiciones de inoculación con Sulfolobus metallicus. Finalmente, se estudió a través de espectrografía fotoelectrónica de rayos x (XPS) los cambios superficiales que ocurren sobre la superficie de calcopirita sintética a 60ºC en ausencia (N2) y presencia de aire. Los resultados indicaron que en ausencia de hierro inicial en la solución, la máxima recuperación de cobre se obtiene cuando parte los microorganismos son capaces de adherirse a la superficie de la calcopirita, seguido por el caso abiótico aireado y finalmente el caso abiótico anaeróbico (donde casi no se produce disolución). Se determinó además que es necesaria la adherencia de una parte de la población de microorganismos a la superficie del mineral para aumentar la velocidad de lixiviación de la calcopirita y para mantener la población de microorganismos. Esta adherencia comienza en las grietas sobre la superficie y luego ocurre de manera heterogénea sobre la superficie. Se estableció que el mecanismo de acción de los S. metallicus sobre calcopirita podría ser el siguiente: una parte de los microorganismos que se han adherido a la calcopirita producen un compuesto intermedio, tiosulfato, que los microorganismos que quedan libres en la solución oxidan a sulfito y bisulfito que finalmente pasa a sulfato. Además, estos microorganismos oxidan Fe+2 a Fe+3 producido por la disolución de la calcopirita. En el caso en que se agrega inicialmente 1 g/l de Fe +3 a la solución, la máxima recuperación de cobre se obtiene en el caso abiótico aireado seguido por los casos inoculados y finalmente el caso abiótico anaeróbico. El mecanismo de disolución de la calcopirita es principalmente química. En los casos inoculados, los microorganismos toman el azufre o algún compuesto intermedio de azufre producido durante el proceso férrico de disolución como fuente de energía y además regeneran el Fe+2 a Fe+3. En cambio en los casos abióticos el mecanismo de disolución de la calcopirita viene dado por la reducción del oxígeno sobre la superficie del mineral y el hierro presente en la solución como ferroso (Eh bajo).

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Ingeniera Civil Química / Ingeniera Civil en Biotecnología / VALE es la segunda compañía minera más grande en el mundo, lidera la producción de mineral de Hierro y pellets. La sede principal de la compañía se encuentra en Brasil y han requerido la asesoría del laboratorio de Biohidrometalurgia del Departamento de Ingeniería Química y Biotecnología de la Universidad de Chile, pues desean investigar la factibilidad técnica y económica de la recuperación de fósforo desde minerales fosfatados utilizando procesos relacionados con la biohidrometalurgia. Si bien el proyecto completo abarca muchas áreas, el objetivo principal de este trabajo fue estudiar experimentalmente el mecanismo de oxidación de azufre elemental con Sulfolobus metallicus a 67 ºC para la producción de ácido sulfúrico. Se realizaron tres experimentos. El experimento principal con el fin de estudiar la capacidad oxidativa de azufre elemental, por lo que se monitorearon variables como pH, acidez, concentración de sulfato y crecimiento celular durante 264 horas. El segundo experimento tenía por objetivo determinar la capacidad de adherencia inicial de las arqueas a las perlas de azufre elemental estériles, por lo que monitoreó la concentración celular por 2 horas. Luego, en el último experimento, se evaluó la influencia del pH inicial del medio sobre la capacidad oxidativa de las arqueas a través de la variación del pH. Los resultados indicaron que las células poseen una gran capacidad oxidativa, en comparación con la reacción química. También se observó que tanto la producción de ácido sulfúrico como la conversión presentaron una tendencia lineal entre las 48 y las 216 horas de cultivo con un máximo de 9,1 [g/L] de ácido y un 29% de conversión del azufre elemental alcanzado a las 216 horas. Mediante análisis de turbidimetría del ión sulfato se observó un comportamiento exponencial inicial para alcanzar un valor relativamente constante de 3600 [g/L]. Los datos obtenidos para el crecimiento presentaron una tendencia exponencial, para posteriormente decaer e incluso desaparecer en el caso de las células planctónicas, al alcanzar un pH inferior a 1. La reacción estuvo controlada por la reacción química con un τ de 1818 horas. El experimento de adherencia inicial demostró que no es tan alta (3,5x106 [células adheridas/cm2] equivalente al 53,2% a las 2 horas de cultivo), en comparación a la alcanzada con otros microorganismos, pero con el tiempo este valor aumenta considerablemente por la reproducción de las células. Finalmente en el tercer experimento se observó que la velocidad inicial de oxidación de las células aumentaba al disminuir el pH del medio inicial de cultivo, alcanzando 1,7x10-5 [h-1] con el medio de pH inicial 1. La biooxidación de azufre elemental ocurre mediante un mecanismo cooperativo, donde la concentración de células adheridas fue casi cinco veces mayor al de las células planctónicas. El proceso de biooxidación de azufre requirió de la presencia células para que ocurriera, pero su actividad catalítica se vio limitada por la presencia de intermediarios, provenientes de la oxidación parcial del azufre elemental realizada por las células, y por la alta acidez del medio, proveniente de la misma reacción. A pesar de esto, por la conversión alcanzada y la alta eficiencia del ácido sulfúrico como agente lixiviante de minerales fosfatados, este proceso debe ser estudiado, y con mayor información evaluar la factibilidad de su implementación en las operaciones de VALE.

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