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

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

No description available.

No description available.

No description available.

In this thesis, the interactions of U(VI) with one representative each of the domains Bacteria (Paenibacillus sp. JG TB8) and Archaea (Sulfolobus acidocaldarius) are compared. We demonstrate that at highly acidic conditions (pH ≤ 3), U(VI) is bound to cells of the both strains exclusively via organic phosphate groups. In contrast to this, the U(VI) complexation modes differ between the studied strains at moderate acidic conditions. These differences are assigned to the different cell wall structures of both strains as well as to their different physiological characteristics. We also demonstrate that the aeration conditions can strongly influence the uranium accumulation of facultative anaerobic microorganisms at moderate acidic pH conditions. This finding could clearly be assigned to the dependency of the intrinsic phosphatase activity on the aeration conditions. The second part of this thesis deals with the outermost surface layer (SlaA-layer) of S. acidocaldarius. It was shown that this surface protein is not involved in the U(VI) complexation at highly acidic conditions, covering the physiological pH optimum of S. acidocaldarius. Hence the SlaA layer does not provide a protective function against U(VI) to the cells of this acidophilic archaeon. However, we demonstrated that purified SlaA-layer ghosts (i.e. empty cell sacculi) efficiently interact with gold ions and are a good macromolecular template for the formation of magnetic gold nanoparticles. / In dieser Doktorarbeit werden die Wechselwirkungen von U(VI) mit je einem Vertreter der Bakterien (Paenibacillus sp. JG TB8) und Archeen (Sulfolobus acidocaldarius) verglichen. Wir konnten zeigen, dass U(VI) im sehr sauren Milieu (pH ≤ 3) ausschließlich durch organische Phosphatgruppen an die Zellen beider Stämme gebunden ist. Im Gegensatz dazu unterscheiden sich die Mechanismen der U(VI)-Komplexierung beider untersuchter Stämme bei mäßig sauren Bedingungen voneinander. Diese Unterschiede basieren auf den unterschiedlichen Zellwandstrukturen und physiologischen Eigenschaften beider Stämme. Wir konnten außerdem zeigen, dass die atmosphärischen Bedingungen die Urankomplexierung durch fakultativ anaerobe Mikroorganismen bei mäßig sauren Bedingungen stark beeinflussen kann. Dieses Ergebnis konnte eindeutig auf die von den atmosphärischen Bedingungen-abhängige, enzymatische Aktivität der zelleigenen Phosphatase zurückgeführt werden. Der zweite Teil dieser Arbeit beschäftigt sich mit der äußeren Oberflächenschicht (SlaA-layer) von S. acidocaldarius. Es konnte gezeigt werden, dass dieses Oberflächenprotein nicht an der U(VI)-Komplexierung bei stark sauren pH, welcher dem physiologischen pH Optimum von S. acidocaldarius entspricht, beteiligt ist. Damit stellt der SlaA-layer keinen Schutz gegen Uran für die Zellen dieses azidothermophilen Archaeons dar. Allerdings konnten wir zeigen, dass isolierte „SlaA-layer ghosts“ (d.h. leere Zellhüllen) mit Goldionen interagieren und sich daher als makromolekulares Template für die Herstellung magnetischer Gold Nanopartikel eignen.

In this thesis, the interactions of U(VI) with one representative each of the domains Bacteria (Paenibacillus sp. JG TB8) and Archaea (Sulfolobus acidocaldarius) are compared. We demonstrate that at highly acidic conditions (pH ≤ 3), U(VI) is bound to cells of the both strains exclusively via organic phosphate groups. In contrast to this, the U(VI) complexation modes differ between the studied strains at moderate acidic conditions. These differences are assigned to the different cell wall structures of both strains as well as to their different physiological characteristics. We also demonstrate that the aeration conditions can strongly influence the uranium accumulation of facultative anaerobic microorganisms at moderate acidic pH conditions. This finding could clearly be assigned to the dependency of the intrinsic phosphatase activity on the aeration conditions. The second part of this thesis deals with the outermost surface layer (SlaA-layer) of S. acidocaldarius. It was shown that this surface protein is not involved in the U(VI) complexation at highly acidic conditions, covering the physiological pH optimum of S. acidocaldarius. Hence the SlaA layer does not provide a protective function against U(VI) to the cells of this acidophilic archaeon. However, we demonstrated that purified SlaA-layer ghosts (i.e. empty cell sacculi) efficiently interact with gold ions and are a good macromolecular template for the formation of magnetic gold nanoparticles. / In dieser Doktorarbeit werden die Wechselwirkungen von U(VI) mit je einem Vertreter der Bakterien (Paenibacillus sp. JG TB8) und Archeen (Sulfolobus acidocaldarius) verglichen. Wir konnten zeigen, dass U(VI) im sehr sauren Milieu (pH ≤ 3) ausschließlich durch organische Phosphatgruppen an die Zellen beider Stämme gebunden ist. Im Gegensatz dazu unterscheiden sich die Mechanismen der U(VI)-Komplexierung beider untersuchter Stämme bei mäßig sauren Bedingungen voneinander. Diese Unterschiede basieren auf den unterschiedlichen Zellwandstrukturen und physiologischen Eigenschaften beider Stämme. Wir konnten außerdem zeigen, dass die atmosphärischen Bedingungen die Urankomplexierung durch fakultativ anaerobe Mikroorganismen bei mäßig sauren Bedingungen stark beeinflussen kann. Dieses Ergebnis konnte eindeutig auf die von den atmosphärischen Bedingungen-abhängige, enzymatische Aktivität der zelleigenen Phosphatase zurückgeführt werden. Der zweite Teil dieser Arbeit beschäftigt sich mit der äußeren Oberflächenschicht (SlaA-layer) von S. acidocaldarius. Es konnte gezeigt werden, dass dieses Oberflächenprotein nicht an der U(VI)-Komplexierung bei stark sauren pH, welcher dem physiologischen pH Optimum von S. acidocaldarius entspricht, beteiligt ist. Damit stellt der SlaA-layer keinen Schutz gegen Uran für die Zellen dieses azidothermophilen Archaeons dar. Allerdings konnten wir zeigen, dass isolierte „SlaA-layer ghosts“ (d.h. leere Zellhüllen) mit Goldionen interagieren und sich daher als makromolekulares Template für die Herstellung magnetischer Gold Nanopartikel eignen.