The isolation of viable extremely halophilic archaea from 250-million-year-old rock salt suggests the possibility of their long-term survival under desiccation. Since halite has been found on Mars and in meteorites, haloarchaeal survival of martian surface conditions is being explored. Halococcus dombrowskii H4 DSM 14522T was exposed to UV doses over a wavelength range of 200–400 nm to simulate martian UV flux. Cells embedded in a thin layer of laboratory-grown halite were found to accumulate preferentially within fluid inclusions. Survival was assessed by staining with the LIVE/DEAD kit dyes, determining colony-forming units, and using growth tests. Halite-embedded cells showed no loss of viability after exposure to about 21 kJ/m2, and they resumed growth in liquid medium with lag phases of 12 days or more after exposure up to 148 kJ/m2. The estimated D37 (dose of 37 % survival) for Hcc. dombrowskii was ≥ 400 kJ/m2. However, exposure of cells to UV flux while in liquid culture reduced D37 by 2 orders of magnitude (to about 1 kJ/m2); similar results were obtained with Halobacterium salinarum NRC-1 and Haloarcula japonica. The absorption of incoming light of shorter wavelength by color centers resulting from defects in the halite crystal structure likely contributed to these results. Under natural conditions, haloarchaeal cells become embedded in salt upon evaporation; therefore, dispersal of potential microscopic life within small crystals, perhaps in dust, on the surface of Mars could resist damage by UV radiation.
Halococcus dombrowskii; Simulated martian UV radiation; LIVE/DEAD staining; Halite fluid inclusions; UV transmittance and reflectance; Desiccation
The recent engagement of Brazil in the construction and utilization of the International Space Station has motivated several Brazilian research institutions and universities to establish study centers related to Space Sciences. The Pontificia Universidade Catolica do Rio Grande do Sul (PUCRS) is no exception.
Method: The University initiated in 1993 the first degree course training students to operate commercial aircraft in South America (the School of Aeronautical Sciences. A further step was the decision to build the first Brazilian laboratory dedicated to the conduct of experiments in ground-based microgravity simulation. Established in 1998, the Microgravity Laboratory, which was located in the Instituto de Pesquisas Cientificas e Tecnologicas (IPCT), was supported by the Schools of Medicine, Aeronautical Sciences and Electrical Engineering/Biomedical Engineering. At the end of 2006, the Microgravity Laboratory became a Center and was transferred to the School of Engineering.
Results: The principal activities of the Microgravity Centre are the development of research projects related to human physiology before, during and after ground-based microgravity simulation and parabolic flights, to aviation medicine in the 21st century and to aerospace biomedical engineering.
Conclusion: The history of Brazilian, and why not say worldwide, space science should unquestionably go through PUCRS. As time passes, the pioneering spirit of our University in the aerospace area has become undeniable. This is due to the group of professionals, students, technicians and staff in general that have once worked or are still working in the Center of Microgravity, a group of faculty and students that excel in their undeniable technical-scientific qualifications.
Microgravity; space life sciences; research center; space biomedicine
Life on Earth evolved in the presence of gravity, and thus it is of interest from the perspective of space exploration to determine if diminished gravity affects biological processes. Cultivation of Escherichia coli under low-shear simulated microgravity (SMG) conditions resulted in enhanced stress resistance in both exponential- and stationary-phase cells, making the latter superresistant. Given that microgravity of space and SMG also compromise human immune response, this phenomenon constitutes a potential threat to astronauts. As low-shear environments are encountered by pathogens on Earth as well, SMG-conferred resistance is also relevant to controlling infectious disease on this planet. The SMG effect resembles the general stress response on Earth, which makes bacteria resistant to multiple stresses; this response is σs dependent, irrespective of the growth phase. However, SMG-induced increased resistance was dependent on σs only in stationary phase, being independent of this sigma factor in exponential phase. σs concentration was some 30% lower in exponential-phase SMG cells than in normal gravity cells but was twofold higher in stationary-phase SMG cells. While SMG affected σs synthesis at all levels of control, the main reasons for the differential effect of this gravity condition on σs levels were that it rendered the sigma protein less stable in exponential phase and increased rpoS mRNA translational efficiency. Since σs regulatory processes are influenced by mRNA and protein-folding patterns, the data suggest that SMG may affect these configurations.
To investigate the morphological and growth characteristics of rabbit keratocytes when cultured on decellularized cornea under simulate microgravity (SMG) rotary cell culture system (RCCS) and static culture or in plastic culture supplemented with small molecules of valproic acid (VPA) and vitamin C (VC). Bovine corneas were firstly decellularized with Triton X-100 and NH4OH and through short-term freezing process. Then cell count kit-8 (CCK-8) and flow cytometry were used to test the effects of VPA and VC on the proliferation, cell cycle and apoptosis of rabbit keratocytes. Hematoxylin-eosin (H&E) staining and scanning electron microscopy (SEM) imaging showed that cells were eliminated in the decellularized bovine corneas. The proliferation of cultured keratocytes was promoted by VPA and VC in the cell proliferation assay. VPA and VC moderately decreased the number of apoptotic cells and obviously promoted cell-cycle entrance of keratocytes. Rabbit keratocytes in plastic displayed spindle shape and rare interconnected with or without VPA and VC. Cells revealed dendritic morphology and reticular cellular connections when cultured on the carriers of decellularized corneas supplemented with VPA and VC even in the presence of 10% fetal bovine serum (FBS). When cultured in RCCS supplemented with VPA, VC and 10% FBS, keratocytes displayed round shape with many prominences and were more prone to grow into the pores of carriers with aggregation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis proved that the keratocytes cultured on decellularized bovine cornea under SMG with VPA and VC expressed keratocan and lumican. Keratocytes cultured on plastic expressed lumican but not keratocan. Immunofluorescence identification revealed that cells in all groups were positively immunostained for vimentin. Keratocytes on decellularized bovine cornea under SMG or in static culture were positively immunostained for keratocan and lumican. Thus, we reasonably made a conclusion that the combination of VPA, VC, RCCS and decellularized corneal carriers provide a good condition for keratocytes to well grow. Keratocytes can be manipulated to be aggregates or physiological morphological growth in vitro, which are important for the research of corneal stem cells and corneal tissue engineering.
In the present study, we discovered that mouse oocyte maturation was inhibited by simulated microgravity via disturbing spindle organization. We cultured mouse oocytes under microgravity condition simulated by NASA's rotary cell culture system, examined the maturation rate and observed the spindle morphology (organization of cytoskeleton) during the mouse oocytes meiotic maturation. While the rate of germinal vesicle breakdown did not differ between 1 g gravity and simulated microgravity, rate of oocyte maturation decreased significantly in simulated microgravity. The rate of maturation was 8.94% in simulated microgravity and was 73.0% in 1 g gravity. The results show that the maturation of mouse oocytes in vitro was inhibited by the simulated microgravity. The spindle morphology observation shows that the microtubules and chromosomes can not form a complete spindle during oocyte meiotic maturation under simulated microgravity. And the disorder of γ-tubulin may partially result in disorganization of microtubules under simulated microgravity. These observations suggest that the meiotic spindle organization is gravity dependent. Although the spindle organization was disrupted by simulated microgravity, the function and organization of microfilaments were not pronouncedly affected by simulated microgravity. And we found that simulated microgravity induced oocytes cytoplasmic blebbing via an unknown mechanism. Transmission electron microscope detection showed that the components of the blebs were identified with the cytoplasm. Collectively, these results indicated that the simulated microgravity inhibits mouse oocyte maturation via disturbing spindle organization and inducing cytoplasmic blebbing.
The nitrogen cycle (N-cycle), principally supported by prokaryotes, involves different redox reactions mainly focused on assimilatory purposes or respiratory processes for energy conservation. As the N-cycle has important environmental implications, this biogeochemical cycle has become a major research topic during the last few years. However, although N-cycle metabolic pathways have been studied extensively in Bacteria or Eukarya, relatively little is known in the Archaea. Halophilic Archaea are the predominant microorganisms in hot and hypersaline environments such as salted lakes, hot springs or salted ponds. Consequently, the denitrifying haloarchaea that sustain the nitrogen cycle under these conditions have emerged as an important target for research aimed at understanding microbial life in these extreme environments.
The haloarchaeon Haloferax mediterranei was isolated 20 years ago from Santa Pola salted ponds (Alicante, Spain). It was described as a denitrifier and it is also able to grow using NO3-, NO2- or NH4+ as inorganic nitrogen sources. This review summarizes the advances that have been made in understanding the N-cycle in halophilic archaea using Hfx mediterranei as a haloarchaeal model. The results obtained show that this microorganism could be very attractive for bioremediation applications in those areas where high salt, nitrate and nitrite concentrations are found in ground waters and soils.
Exposure to altered microgravity during space travel induces changes in the brain and these are reflected in many of the physical behavior seen in the astronauts. The vulnerability of the brain to microgravity stress has been reviewed and reported. Identifying microgravity-induced changes in the brain proteome may aid in understanding the impact of the microgravity environment on brain function. In our previous study we have reported changes in specific proteins under simulated microgravity in the hippocampus using proteomics approach. In the present study the profiling of the hypothalamus region in the brain was studied as a step towards exploring the effect of microgravity in this region of the brain. Hypothalamus is the critical region in the brain that strictly controls the pituitary gland that in turn is responsible for the secretion of important hormones. Here we report a 2-dimensional gel electrophoretic analysis of the mouse hypothalamus in response to simulated microgravity. Lowered glutathione and differences in abundance expression of seven proteins were detected in the hypothalamus of mice exposed to microgravity. These changes included decreased superoxide dismutase-2 (SOD-2) and increased malate dehydrogenase and peroxiredoxin-6, reflecting reduction of the antioxidant system in the hypothalamus. Taken together the results reported here indicate that oxidative imbalance occurred in the hypothalamus in response to simulated microgravity.
Brain; Hypothalamus; Microgravity
Manned space exploration has created a need to evaluate the effects of space-like stress (SLS) on pathogenic and opportunistic microbes. Interestingly, several Gram-negative enteric pathogens, e.g., Salmonella enterica serovar Typhimurium, have revealed a transient hyper-virulent phenotype following simulated microgravity (SMG) or actual space flight exposures. We have explored the virulence potential of Yersinia pestis KIM/D27 (YP) following exposure to mechanical low shear forces associated with SMG. Our experimental results demonstrated that SMG-grown YP was decreased in its induced HeLa cell cytotoxicity, suggesting that SMG somehow compromises T3SS functions. This was confirmed by an actual reduced amount of effector protein production and secretion through the T3SS injectisome. Also, SMG-grown YP proliferated less than their NG-grown counterparts did during an 8-h macrophage infection. Presently, we are evaluating the influence of SMG on various KIM/D27 mutant strains to further understanding of our initial phenomenology described above. Taken together, characterizing YP grown under the low shear forces of SMG can provide new insights into its pathogenesis and potentially uncover new targets that could be exploited for the development of novel antimicrobials as well as potential live-attenuated vaccines.
simulated microgravity; Yersinia pestis; type three secretion system; high aspect ratio vessel; low shear forces
The first International Caenorhabditis elegans Experiment (ICE-First) was carried out using a Russian Soyuz spacecraft from April 19-30, 2004. This experiment was a part of the program of the DELTA (Dutch Expedition for Life science Technology and Atmospheric research) mission, and the space agencies that participate in the International Space Station (ISS) program formed international research teams. A Japanese research team that conducted by Japan aerospace Exploration Agency (JAXA) investigated the following aspects of the organism: (1) whether meiotic chromosomal dynamics and apoptosis in the germ cells were normal under microgravity conditions, (2) the effect of the space flight on muscle cell development, and (3) the effect of the space flight on protein aggregation. In this article, we summarize the results of these biochemical and molecular biological analyses.
The microgravity environment during space flight imposes numerous adverse effects on animal and microbial physiology. It is unclear, however, how microgravity impacts those cellular interactions between mutualistic microbes and their hosts. Here, we used the symbiosis between the host squid Euprymna scolopes and its luminescent bacterium Vibrio fischeri as a model system. We examined the impact of simulated microgravity on the timeline of bacteria-induced development in the host light organ, the site of the symbiosis. To simulate the microgravity environment, host squid and symbiosis-competent bacteria were incubated together in high-aspect ratio rotating wall vessel bioreactors and examined throughout the early stages of the bacteria-induced morphogenesis. The host innate immune response was suppressed under simulated microgravity; however, there was an acceleration of bacteria-induced apoptosis and regression in the host tissues. These results suggest that the space flight environment may alter the cellular interactions between animal hosts and their natural healthy microbiome.
The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a “stimulatory” environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.
Summary: The responses of microorganisms (viruses, bacterial cells, bacterial and fungal spores, and lichens) to selected factors of space (microgravity, galactic cosmic radiation, solar UV radiation, and space vacuum) were determined in space and laboratory simulation experiments. In general, microorganisms tend to thrive in the space flight environment in terms of enhanced growth parameters and a demonstrated ability to proliferate in the presence of normally inhibitory levels of antibiotics. The mechanisms responsible for the observed biological responses, however, are not yet fully understood. A hypothesized interaction of microgravity with radiation-induced DNA repair processes was experimentally refuted. The survival of microorganisms in outer space was investigated to tackle questions on the upper boundary of the biosphere and on the likelihood of interplanetary transport of microorganisms. It was found that extraterrestrial solar UV radiation was the most deleterious factor of space. Among all organisms tested, only lichens (Rhizocarpon geographicum and Xanthoria elegans) maintained full viability after 2 weeks in outer space, whereas all other test systems were inactivated by orders of magnitude. Using optical filters and spores of Bacillus subtilis as a biological UV dosimeter, it was found that the current ozone layer reduces the biological effectiveness of solar UV by 3 orders of magnitude. If shielded against solar UV, spores of B. subtilis were capable of surviving in space for up to 6 years, especially if embedded in clay or meteorite powder (artificial meteorites). The data support the likelihood of interplanetary transfer of microorganisms within meteorites, the so-called lithopanspermia hypothesis.
It is generally known that bone loss is one of the most important complications for astronauts who are exposed to long-term microgravity in space. Changes in blood flow, systemic hormones, and locally produced factors were indicated as important elements contributing to the response of osteoblastic cells to loading, but research in this field still has many questions. Here, the possible biological involvement of thyroid C cells is being investigated. The paper is a comparison between a case of a wild type single mouse and a over-expressing pleiotrophin single mouse exposed to hypogravity conditions during the first animal experiment of long stay in International Space Station (91 days) and three similar mice exposed to hypergravity (2Gs) conditions. We provide evidence that both microgravity and hypergravity induce similar loss of C cells with reduction of calcitonin production. Pleiotrophin over-expression result in some protection against negative effects of gravity change. Potential implication of the gravity mechanic forces in the regulation of bone homeostasis via thyroid equilibrium is discussed.
Sustaining life beyond Earth either on space stations or on other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction. However, because of the difficulty of doing such experiments in mammals, most studies of reproduction in space have been carried out with other taxa, such as sea urchins, fish, amphibians or birds. Here, we studied the possibility of mammalian fertilization and preimplantation development under microgravity (µG) conditions using a three-dimensional (3D) clinostat, which faithfully simulates 10–3 G using 3D rotation. Fertilization occurred normally in vitro under µG. However, although we obtained 75 healthy offspring from µG-fertilized and -cultured embryos after transfer to recipient females, the birth rate was lower than among the 1G controls. Immunostaining demonstrated that in vitro culture under µG caused slower development and fewer trophectoderm cells than in 1G controls but did not affect polarization of the blastocyst. These results suggest for the first time that fertilization can occur normally under µG environment in a mammal, but normal preimplantation embryo development might require 1G.
Space travel induces many deleterious effects on the flight crew due to the ‘0’ g environment. The brain experiences a tremendous fluid shift, which is responsible for many of the detrimental changes in physical behavior seen in astronauts. It therefore indicates that the brain may undergo major changes in its protein levels in a ‘0’ g environment to counteract the stress. Analysis of these global changes in proteins may explain to better understand the functioning of brain in a ‘0’ g condition. Toward such an effort, we have screened proteins in the hippocampus of mice kept in simulated microgravity environment for 7 days and have observed a few changes in major proteins as compared to control mice. Essentially, the results show a major loss of proteins in the hippocampus of mice subjected to simulated microgravity. These changes occur in structural proteins such as tubulin, coupled with the loss of proteins involved in metabolism. This preliminary investigation leads to an understanding of the alteration of proteins in the hippocampus in response to the microgravity environment.
microgravity; hippocampus; two-dimensional gel electrophoresis
There have been many studies on the biological effects of simulated microgravity (SMG) on differentiated cells or adult stem cells. However, there has been no systematic study on the effects of SMG on embryonic stem (ES) cells. In this study, we investigated various effects (including cell proliferation, cell cycle distribution, cell differentiation, cell adhesion, apoptosis, genomic integrity and DNA damage repair) of SMG on mouse embryonic stem (mES) cells. Mouse ES cells cultured under SMG condition had a significantly reduced total cell number compared with cells cultured under 1 g gravity (1G) condition. However, there was no significant difference in cell cycle distribution between SMG and 1G culture conditions, indicating that cell proliferation was not impaired significantly by SMG and was not a major factor contributing to the total cell number reduction. In contrast, a lower adhesion rate cultured under SMG condition contributed to the lower cell number in SMG. Our results also revealed that SMG alone could not induce DNA damage in mES cells while it could affect the repair of radiation-induced DNA lesions of mES cells. Taken together, mES cells were sensitive to SMG and the major alterations in cellular events were cell number expansion, adhesion rate decrease, increased apoptosis and delayed DNA repair progression, which are distinct from the responses of other types of cells to SMG.
The halophilic archaeon Haloferax mediterranei is able to accumulate large amounts of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with high molar fraction of 3-hydroxyvalerate (3HV) from unrelated carbon sources. A Polyhydroxyalkanoate (PHA) synthase composed of two subunits, PhaCHme and PhaEHme, has been identified in this strain, and shown to account for the PHBV biosynthesis.
With the aid of the genome sequence of Hfx. mediterranei CGMCC 1.2087, three additional phaC genes (designated phaC1, phaC2, and phaC3) were identified, which encoded putative PhaCs. Like PhaCHme (54.8 kDa), PhaC1 (49.7 kDa) and PhaC3 (62.5 kDa) possessed the conserved motifs of type III PHA synthase, which was not observed in PhaC2 (40.4 kDa). Furthermore, the longer C terminus found in the other three PhaCs was also absent in PhaC2. Reverse transcription PCR (RT-PCR) revealed that, among the four genes, only phaCHme was transcribed under PHA-accumulating conditions in the wild-type strain. However, heterologous coexpression of phaEHme with each phaC gene in Haloarcula hispanica PHB-1 showed that all PhaCs, except PhaC2, could lead to PHBV accumulation with various 3HV fractions. The three kinds of copolymers were characterized using gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Their thermal properties changed with the variations in monomer composition as well as the different molecular weights (Mw), thus might meet various application requirements.
We discover three cryptic phaC genes in Hfx. mediterranei, and demonstrate that genetic engineering of these newly identified phaC genes has biotechnological potential for PHBV production with tailor-made material properties.
A critical step of spermatogenesis is the entry of mitotic spermatogonia into meiosis. Progresses on these topics are hampered by the lack of an in vitro culture system allowing mouse spermatogonia differentiation and entry into meiosis. Previous studies have shown that mouse pachytene spermatocytes cultured in simulated microgravity (SM) undergo a spontaneous meiotic progression. Here we report that mouse mitotic spermatogonia cultured under SM with a rotary cell culture system (RCCS) enter into meiosis in the absence of any added exogenous factor or contact with somatic cells. We found that isolated Kit-positive spermatogonia under the RCCS condition enter into the prophase of the first meiotic division (leptotene stage), as monitored by chromosomal organization of the synaptonemal complex 3 protein (Scp3) and up-regulation of several pro-meiotic genes. SM was found to activate the phosphatidyl inositol 3 kinase (PI3K) pathway and to induce in Kit-positive spermatogonia the last round of DNA replication, typical of the preleptotene stage. A PI3K inhibitor abolished Scp3 induction and meiotic entry stimulated by RCCS conditions. A positive effect of SM on germ cell differentiation was also observed in undifferentiated (Kit-negative) spermatogonia, in which RCCS conditions stimulate the expression of Kit and Stra8. In conclusion, SM is an artificial environmental condition which promotes postnatal male germ cell differentiation and might provide a tool to study the molecular mechanisms underlying the switch from mitosis to meiosis in mammals.
Viable extremely halophilic archaea (haloarchaea) have been isolated from million-year-old salt deposits around the world; however, an explanation of their supposed longevity remains a fundamental challenge. Recently small roundish particles in fluid inclusions of 22 000- to 34 000-year-old halite were identified as haloarchaea capable of proliferation (Schubert BA, Lowenstein TK, Timofeeff MN, Parker MA, 2010, Environmental Microbiology, 12, 440–454). Searching for a method to produce such particles in the laboratory, we exposed rod-shaped cells of Halobacterium species to reduced external water activity (aw). Gradual formation of spheres of about 0.4 μm diameter occurred in 4 m NaCl buffer of aw ≤ 0.75, but exposure to buffered 4 m LiCl (aw ≤ 0.73) split cells into spheres within seconds, with concomitant release of several proteins. From one rod, three or four spheres emerged, which re-grew to normal rods in nutrient media. Biochemical properties of rods and spheres were similar, except for a markedly reduced ATP content (about 50-fold) and an increased lag phase of spheres, as is known from dormant bacteria. The presence of viable particles of similar sizes in ancient fluid inclusions suggested that spheres might represent dormant states of haloarchaea. The easy production of spheres by lowering aw should facilitate their investigation and could help to understand the mechanisms for microbial survival over geological times.
The extremely halophilic archaea are present worldwide in saline environments and have important biotechnological applications. Ten complete genomes of haloarchaea are now available, providing an opportunity for comparative analysis.
We report here the comparative analysis of five newly sequenced haloarchaeal genomes with five previously published ones. Whole genome trees based on protein sequences provide strong support for deep relationships between the ten organisms. Using a soft clustering approach, we identified 887 protein clusters present in all halophiles. Of these core clusters, 112 are not found in any other archaea and therefore constitute the haloarchaeal signature. Four of the halophiles were isolated from water, and four were isolated from soil or sediment. Although there are few habitat-specific clusters, the soil/sediment halophiles tend to have greater capacity for polysaccharide degradation, siderophore synthesis, and cell wall modification. Halorhabdus utahensis and Haloterrigena turkmenica encode over forty glycosyl hydrolases each, and may be capable of breaking down naturally occurring complex carbohydrates. H. utahensis is specialized for growth on carbohydrates and has few amino acid degradation pathways. It uses the non-oxidative pentose phosphate pathway instead of the oxidative pathway, giving it more flexibility in the metabolism of pentoses.
These new genomes expand our understanding of haloarchaeal catabolic pathways, providing a basis for further experimental analysis, especially with regard to carbohydrate metabolism. Halophilic glycosyl hydrolases for use in biofuel production are more likely to be found in halophiles isolated from soil or sediment.
The physiological responses and transcription profiling of Pichia pastoris GS115 to simulated microgravity (SMG) were substantially changed compared with normal gravity (NG) control. We previously reported that the recombinant P. pastoris grew faster under SMG than NG during methanol induction phase and the efficiencies of recombinant enzyme production and secretion were enhanced under SMG, which was considered as the consequence of changed transcriptional levels of some key genes. In this work, transcriptiome profiling of P. pastoris cultured under SMG and NG conditions at exponential and stationary phases were determined using next-generation sequencing (NGS) technologies. Four categories of 141 genes function as methanol utilization, protein chaperone, RNA polymerase and protein transportation or secretion classified according to Gene Ontology (GO) were chosen to be analyzed on the basis of NGS results. And 80 significantly changed genes were weighted and estimated by Cluster 3.0. It was found that most genes of methanol metabolism (85% of 20 genes) and protein transportation or secretion (82.2% of 45 genes) were significantly up-regulated under SMG. Furthermore the quantity and fold change of up-regulated genes in exponential phase of each category were higher than those of stationary phase. The results indicate that the up-regulated genes of methanol metabolism and protein transportation or secretion mainly contribute to enhanced production and secretion of the recombinant protein under SMG.
Recent space-flight experiments performed by Tabony's team provided further evidence that a microgravity environment strongly affects the spatio-temporal organization of microtubule assemblies. Characteristic time and length scales were found that govern the organization of oriented bundles under Earth's gravitational field (GF). No such organization has been observed in a microgravity environment. This paper discusses physical mechanisms resulting in pattern formation under gravity and its disappearance in microgravity. The subtle interplay between chemical kinetics, diffusion, gravitational drift, thermal fluctuations, electrostatic interactions and liquid crystalline characteristics provides a plausible scenario.
microtubules; microgravity; tubulin; assembly; pattern formation
Evidence for the widespread occurrence of extraterrestrial halite, particularly on Mars, has led to speculations on the possibility of halophilic microbial forms of life; these ideas have been strengthened by reports of viable haloarchaea from sediments of geological age (millions of years). Raman spectroscopy, being a sensitive detection method for future astrobiological investigations onsite, has been used in the current study for the detection of nine different extremely halophilic archaeal strains which had been embedded in laboratory-made halite crystals in order to simulate evaporitic conditions. The cells accumulated preferentially in tiny fluid inclusions, in simulation of the precipitation of salt in natural brines. FT-Raman spectroscopy using laser excitation at 1064 nm and dispersive micro Raman spectroscopy at 514.5 nm were applied. The spectra showed prominent peaks at 1507, 1152 and 1002 cm−1 which are attributed to haloarchaeal C50 carotenoid compounds (mainly bacterioruberins). Their intensity varied from strain to strain at 1064-nm laser excitation. Other distinguishable features were peaks due to peptide bonds (amide I, amide III) and to nucleic acids. No evidence for fatty acids was detected, consistent with their general absence in all archaea.
These results contribute to a growing database on Raman spectra of terrestrial microorganisms from hypersaline environments and highlight the influence of the different macromolecular composition of diverse strains on these spectra.
Raman spectroscopy; extremely halophilic archaea; halite; astrobiology; fluid inclusions; carotenoids; bacterioruberins; Martian subsurface
Like eukarya and bacteria, archaea also perform N-glycosylation. However, the N-linked glycans of archaeal glycoproteins present a variety not seen elsewhere. Archaea accordingly rely on N-glycosylation pathways likely involving a broad range of species-specific enzymes. To harness the enormous applied potential of such diversity for the generation of glycoproteins bearing tailored N-linked glycans, the development of an appropriate archaeal glycoengineering platform is required. With a sequenced genome, a relatively well-defined N-glycosylation pathway, and molecular tools for gene manipulation, the haloarchaeon Haloferax volcanii (Hfx. volcanii) represents a promising candidate. Accordingly, cells lacking AglD, a glycosyltransferase involved in adding the final hexose of a pentasaccharide N-linked to the surface (S)-layer glycoprotein, were transformed to express AglD homologues from other haloarchaea. The introduction of nonnative versions of AglD led to the appearance of an S-layer glycoprotein similar to the protein from the native strain. Indeed, mass spectrometry confirmed that AglD and its homologues introduce the final hexose to the N-linked S-layer glycoprotein pentasaccharide. Heterologously expressed haloarchaeal AglD homologues contributed to N-glycosylation in Hfx. volcanii despite an apparent lack of AglD function in those haloarchaea from where the introduced homologues came. For example, although functional in Hfx. volcanii, no transcription of the Halobacterium salinarum aglD homologue, OE1482, was detected in cells of the native host grown under various conditions. Thus, at least one AglD homologue works more readily in Hfx. volcanii than in the native host. These results warrant the continued assessment of Hfx. volcanii as a glycosylation “workshop.”
Polyhydroxyalkanoates (PHAs) are accumulated in many prokaryotes. Several members of the Halobacteriaceae produce poly-3-hydroxybutyrate (PHB), but it is not known if this is a general property of the family. We evaluated identification methods for PHAs with 20 haloarchaeal species, three of them isolates from Permian salt. Staining with Sudan Black B, Nile Blue A, or Nile Red was applied to screen for the presence of PHAs. Transmission electron microscopy and 1H-nuclear magnetic resonance spectroscopy were used for visualization of PHB granules and chemical confirmation of PHAs in cell extracts, respectively. We report for the first time the production of PHAs by Halococcus sp. (Halococcus morrhuae DSM 1307T, Halococcus saccharolyticus DSM 5350T, Halococcus salifodinae DSM 8989T, Halococcus dombrowskii DSM 14522T, Halococcus hamelinensis JCM 12892T, Halococcus qingdaonensis JCM 13587T), Halorubrum sp. (Hrr. coriense DSM 10284T, Halorubrum chaoviator DSM 19316T, Hrr. chaoviator strains NaxosII and AUS-1), haloalkaliphiles (Natronobacterium gregoryi NCMB 2189T, Natronococcus occultus DSM 3396T) and Halobacterium noricense DSM 9758T. No PHB was detected in Halobacterium salinarum NRC-1 ATCC 700922, Hbt. salinarum R1 and Haloferax volcanii DSM 3757T. Most species synthesized PHAs when growing in synthetic as well as in complex medium. The polyesters were generally composed of PHB and poly-ß-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). Available genomic data suggest the absence of PHA synthesis in some haloarchaea and in all other Euryarchaeota and Crenarchaeota. Homologies between haloarchaeal and bacterial PHA synthesizing enzymes had indicated to some authors probable horizontal gene transfer, which, considering the data obtained in this study, may have occurred already before Permian times.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-010-2611-6) contains supplementary material, which is available to authorized users.
Polyhydroxybutyrate; Haloarchaea; Halococcus; Halobacterium; Haloalkaliphile