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This innovation is a coupled fluorescence-activated cell sorting (FACS) and fluorescent staining technology for purifying (removing cells from sampling matrices), separating (based on size, density, morphology, and live versus dead), and concentrating cells (spores, prokaryotic, eukaryotic) from an environmental sample.
An environmental application using a microtiter culture assay to measure the metabolic sensitivity of microorganisms to petrochemical effluents will be tested. The Biomedical Operations and Research Branch at NASA JSC has recently developed a rapid and nondestructive method to measure cell growth and metabolism. Using a colorimetric procedure the uniquely modified assay allows the metabolic kinetics of prokaryotic and eukaryotic cells to be measured. Use of such an assay if adapted for the routine monitoring of waste products, process effluents, and environmentally hazardous substances may prove to be invaluable to the industrial community. The microtiter method as described will be tested using microorganisms isolated from the Galveston Bay aquatic habitat. The microbial isolates will be identified prior to testing using the automated systems available at JSC. Sodium dodecyl sulfate (SDS), cadmium, and lead will provide control toxic chemicals. The toxicity of industrial effluent from two industrial sites will be tested. An effort will be made to test the efficacy of this assay for measuring toxicity in a mixed culture community.
Assessing the biological risks associated with exposure to the high-energy charged particles encountered in space is essential for the success of long-term space exploration. Although prokaryotic and eukaryotic cell models developed in our laboratory and others have advanced our understanding of many aspects of genotoxicity, in vitro models are needed to assess the risk to humans from space radiation insults. Such models must be representative of the cellular interactions present in tissues and capable of quantifying I genotoxic damage. Toward this overall goal, the objectives of this study were to examine the effect of the localized microenvironment of cells, cultured as either 2-dimensional (2D) monolayers or 3-dimensional (3D) aggregates, on the rate and type of genotoxic damage resulting from exposure to iron charged particles, a significant portion of space radiation. We used rodent transgenic cell lines containing 50-70 copies of a LacI transgene to provide the enhanced sensitivity required to quantify mutational frequency and type in the 1,100-bp LacI target as well as assessment of DNA,damage to the entire 45-kbp construct. Cultured cells were exposed to high-ener~ir on charged particles at Brookhaven National Laboratory s Alternating Gradient Synchrotron facility for a total dose of 0, 0.1, 0.25,0.5, 1.0, or 2.0 Gy and allowed to recover for 0, 1, or 7 days, after which mutational type and frequency were evaluated. The mutational frequency was found to be higher in 3D samples than in 2D samples at all radiation doses. Mutational frequency also was higher at 7 days after irradiation than immediately after exposure. DNA sequencing of the mutant targets revealed that deletional mutations contributed an increasingly high percentage (up to 27%) of all mutations in cells as the dose was increased from 0.5 to 2 Gy. Several mutants also showed large and complex deletions in multiple locations within the Lac1 target. However, no differences in mutational type were found between the 2D and the 3D samples. These 3D tissue-like model systems can reduce the uncertainty involved in extrapolating risk between in vitro cellular and in vivo models.
Studies from the Skylab, SL-3 and D-1 missions have demonstrated that biological organisms grown in microgravity have changes in basic cellular functions such as DNA, mRNA and protein synthesis, cytoskeleton synthesis, glucose utilization, and cellular differentiation. Since microgravity could affect prokaryotic and eukaryotic cells at a subcellular and molecular level, space offers an opportunity to learn more about basic biological systems with one inmportant variable removed. The thin film bioreactor will facilitate the handling of fluids in microgravity, under constant temperature and will allow multiple samples of cells to be grown with variable conditions. Studies on cell cultures grown in microgravity would make it possible to identify and quantify changes in basic biological function in microgravity which are needed to develop new applications of orbital research and future biotechnology.
In eukaryotic cells, microtubules are 24-nm-diameter tubular structures composed of a class of conserved proteins called tubulin. They are involved in numerous cell functions including ciliary motility, nerve cell elongation, pigment migration, centrosome formation, and chromosome movement. Although cytoplasmic tubules and fibers have been observed in bacteria, some with diameters similar to those of eukaryotes, no homologies to eukaryotic microtubules have been established. Certain groups of bacteria including azotobacters, cyanobacteria, enteric bacteria, and spirochetes have been frequently observed to possess microtubule-like structures, and others, including archaebacteria, have been shown to be sensitive to drugs that inhibit the polymerization of microtubules. Although little biochemical or molecular biological information is available, the differences observed among these prokaryotic structures suggest that their composition generally differs among themselves as well as from that of eukaryotes. We review the distribution of cytoplasmic tubules in prokaryotes, even though, in all cases, their functions remain unknown. At least some tend to occur in cells that are large, elongate, and motile, suggesting that they may be involved in cytoskeletal functions, intracellular motility, or transport activities comparable to those performed by eukaryotic microtubules. In Escherichia coli, the FtsZ protein is associated with the formation of a ring in the division zone between the newly forming offspring cells. Like tubulin, FtsZ is a GTPase and shares with tubulin a 7-amino-acid motif, making it a promising candidate in which to seek the origin of tubulins.
In comparison with that known from later geologic time, the Archean fossil record is miniscule: although literally hundreds of Proterozoic formations, containing more that 2800 occurrences of bona fide microfossils are now known, fewer than 30 units containing some 43 categories of putative microfossils (the vast majority of which are of questionable authenticity) have been reported from the Archean. Among the oldest known fossils are Early Archean filaments reported from cherts of the Towers Formation and the Apex Basalt of the 3.3-3.6 Ga-old Warrawoona Group of Western Australia. The paleobiologic significance of the Towers Formation microstructures is open to question: thin aggregated filaments are properly regarded as dubiomicrofossils (perhaps biogenic, but perhaps not); therefore, they cannot be regarded as firm evidence of Archean life. Although authentic, filamentous microfossiles were reported from a second Towers Formation locality, because the precise layer containing the fossiliferous cherts was not relocated, this discovery can neither be reconfirmed by the original collector nor confirmed independently by other investigators. Discovery of microfossils in bedded cherts of the Apex Basalt, the stratigraphic unit immediately overlying the Towers Formation, obviates the difficulties stored above. The cellularly preserved filaments of the Apex Basalt meet all of the criteria required of a bona fide Archean microfossils. Recent studies indicate that the Apex assemblage includes at least six morphotypes of uniseriate filaments, composed of barrel-shaped, discoidal, or quadrate cells and exhibiting rounded or conical terminal cells and medial bifurcated and paired half-cells that reflect the occurrence of prokaryotic binary cell division. Interestingly, the majority of these morphotypes are morphologically more similar to extant cyanobacteria than to modern filamentous bacteria. Prokaryotes seem clearly to have been hypobradytelic, and the evidence suggests (but does not prove) that physiologically advanced oxygen-producing photosynthesizers may have been represented in the Early Archean biota.
This overview summarizes murine results on acute lethality responses, inactivation of marrow CFU-S and intestinal microcolonies, testes weight loss, life span shortening, and posterior lens opacification in mice irradiated with heavy charged particles. RBE-LET relationships for these mammalian responses are compared with results from in vitro studies. The trend is that the maximum RBE for in vivo responses tends to be lower and occurs at a lower LET than for inactivation of V79 and T-1 cells in culture. Based on inactivation cross sections, the response of CFU-S in vivo conforms to expectations from earlier studies with prokaryotic systems and mammalian cells in culture. Effects of heavy ions are compared with fission spectrum neutrons, and the results are consistent with the interpretation that RBEs are lower than for fission neutrons at about the same LET, probably due to differences in track structure.
Plant cell walls are glycan-rich extracellular matrices that fundamentally impact essential cellular processes, such as growth, adhesion, and cell shape acquisition. Understanding plant cell wall glycans requires the identification and characterization of the biosynthetic enzymes that produce these polymers. Most successful in vitro protein expression studies of plant cell wall glycosyltransferases have relied on insect, fungal/yeast, or human cell expression systems, whereas prokaryotic expression systems have been generally unsuccessful. Here, we show that Arabidopsis FRIABLE1 (FRB1)/rhamnogalacturonan-I rhamnosyltransferase 8 (RRT8) can be produced in Escherichia coli RosettaGami2 cells as N-terminal maltose-binding protein fusion proteins containing C-terminal 6X-His-tags. We also report the catalytic constants of FRB1/RRT8 with apparent K M and K cat values of 226 μM and 33 min -1 for UDP-Rhamnose and 117 μM and 28.7 min -1 for rhamnogalacturonan-I (RG-I), respectively. We examine the catalytic activities of mutated FRB1/RRT8 proteins based on an AlphaFold 3-generated FRB1/RRT8 protein structural model with a virtually docked UDP-Rha donor. Enzymatic characterization of the mutated and wildtype FRB1/RRT8 protein confirmed that mutation of predicted catalytic site amino acid residues resulted in a 20-fold reduction in RRT activity. FRB1 also robustly polymerizes RG-I in combination with RG-I galacturonosyltransferase 1. These results show how a robust E. coli expression system combined with artificial intelligence tools can be used to increase understanding of plant cell wall glycosyltransferase structure and function.
The rationale for looking for prokaryote fossils in Martian materials is based on our present understanding of the environmental evolution of that planet in comparison to the history of the terrestrial environments and the development and evolution of life on Earth. On Earth we have clear, albeit indirect, evidence of life in 3.8 b.y.-old rocks from Greenland and the first morphological fossils in 3.3-3.5 b.y.-old cherts from South Africa and Australia. In comparison, Mars, being smaller, probably cooled down after initial aggregation faster than the Earth. Consequently, there could have been liquid water on its surface earlier than on Earth. With a similar exogenous and endogenous input of organics and life-sustaining nutrients as is proposed for the Earth, life could have arisen on that planet, possibly slightly earlier dm it did on Earth. Whereas on Earth liquid water has remained at the surface of the planet since about 4.4 b.y. (with some possible interregnums caused by planet-sterilising impacts before 3.8. b.y. and perhaps a number of periods of a totally frozen Earth, this was not the case with Mars. Although it is not known exactly when surficial water disappeared from the surface, there would have been sufficient time for life to have developed into something similar to the terrestrial prokaryote stage. However, given the earlier environmental deterioration, it is unlikely that it evolved into the eukaryote stage and even evolution of oxygenic photosynthesis may not have been reached. Thus, the impetus of research is on single celled life simnilar to prokaryotes. We are investigating a number of methods of trace element analysis with respect to the Early Archaean microbial fossils. Preliminary neutron activation analysis of carbonaceous layers in the Early Archaean cherts from South Africa and Australia shows some partitioning of elements such as As, Sb, Cr with an especial enrichment of lanthanides in a carbonaceous-rich banded iron sediment . More significantly, preliminary TOF-SIMS investigations of organics in the cherts reveals the presence of a biomarker, which appears to be a derivative of bacterial polymer, in the carbonaceous parts of the rocks. We conclude that a combination of morphological, isotope and biogeochemical methods can be used to successfully identify signs of life in terrestrial material, and that these methods will be useful in searching for signs of life in extraterrestrial materials.
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Molecular methods of taxonomy and phylogeny have changed the way in which life on earth is viewed; they have allowed us to transition from a eukaryote-centric (five-kingdoms) view of the planet to one that is peculiarly prokarote-centric, containing three kingdoms, two of which are prokaryotic unicells. These prokaryotes are distinguished from their eukaryotic counterparts by their toughness, tenacity and metabolic diversity. Realization of these features has, in many ways, changed the way we feel about life on earth, about the nature of life past and about the possibility of finding life elsewhere. In essence, the limits of life on this planet have expanded to such a degree that our thoughts of both past and future life have been altered. The abilities of prokaryotes to withstand many extreme conditions has led to the term extremophiles, used to describe the organisms that thrive under conditions thought just a few years ago, to be inconsistent with life. Perhaps the most extensive adaptation to extreme conditions, however, is represented by the ability of many bacteria to survive nutrient conditions not compatible with eukaryotic life. Prokaryotes have evolved to use nearly every redox couple that is in abundance on earth, filling the metabolic niches left behind by the oxygen-using, carbon-eating eukaryotes. This metabolic plasticity leads to a common feature in physically stratified environments of layered microbial communities, chemical indicators of the metabolic diversity of the prokaryotes. Such 'metabolic extremophily' forms a backdrop by which we can view the energy flow of life on this planet, think about what the evolutionary past of the planet might have been, and plan ways to look for life elsewhere, using the knowledge of energy flow on earth.
Biological thiols are essential elements in most aspects of cell function but undergo rapid oxidation to disulfides in the presence of oxygen. The evolution of systems to protect against such oxygen toxicity was essential to the emergence of aerobic life. The protection system used by eukaryotes is based upon glutathione (GSH) and GSH-dependent enzymes but many bacteria lack GSH and apparently use other mechanisms. The objective of this research is to elaborate the thiol protective mechanisms employed by prokaryotes of widely divergent evolutionary origin and to understand why GSH became the central thiol employed in essentially all higher organisms. Thiol-selective fluorescent labeling and HPLC analysis has been used to determine key monothiol components.
The Orgueil CII meteorite, which fell in southern France on the evening of May 14, 1864, has been one of the most extensively studied of all known carbonaceous meteorites. Field Emission Scanning Electron Microscopy (FESEM) studies of freshly fractured interior surfaces of the Orgueil meteorite have resulted in the detection of the fossilized remains of a large and diverse population of filamentous prokaryotic microorganisms. The taphonomy and the diverse modes of the preservation of these remains ,are diverse. Some of the remains exhibit carbonization of a hollow sheath and in other cases the remains are permineralized with water-soluble evaporite minerals, such as magnesium sulfate or ammonium salts. After the sample is fractured and the interior surfaces are exposed to the atmospheric moisture, some of these friable remains have been observed to exhibit significant alterations in appearance with time. Images are presented to document the changes that have been observed in some forms within the past two years. Images and EDS spectral data will also be presented to document the studies carried out on abiotic forms to search for possible nonbiological interpretations of the indigenous filamentous microstructures that have been found in the Orgueil meteorite. Images and EDS data will be presented showing the size, size range, morphology and chemical compositions of abiotic microstructures found in native crystalline and fibrous Epsomites from Poison Lake, Washington, USA and Catalayud, Zaragoza, Aragon, Spain. Many of these embedded forms are consistent in size and microstructure with cyanobacteria morphotypes. Some of the forms are exhibit known characteristics differentiation of cells, and reproductive structures of filamentous trichomic prokaryotes (bacteria and cyanobacteria) and the degraded remains of microfibrils associated with sheaths of cyanobacteria. In this paper, recently obtained comparative images and EDS data will be presented for the mineralized remains found in the Orgueil meteorite and these forms will be compared with known cyanobacteria of the family Oscillatoriaceae. Many of the indigenous filamentous microstructures found in Orgueil filaments are dramatically different from the abiotic microstructures found in the fibrous epsomites and these forms are interpreted as the molds, casts and the well preserved permineralized remains (microfossils) of filamentous prokaryotes that are embedded in and indigenous to the matrix of the Orgueil CI1 carbonaceous meteorite.
Arsenite (As III ) is toxic to all organisms due to its ability to tightly bind exposed thiols within cells. An important As III resistance mechanism in prokaryotes involves proteins encoded by thearsoperon. A central component of thearsoperon in many bacteria is the cytoplasmic ATPase, ArsA, which orchestrates a series of nucleotide-dependent handoffs, starting with the capture of As III by the ArsD metallochaperone and culminating in its removal from the cell by the ArsB efflux pump. Although the mechanism of ArsA has been widely studied, the molecular details of how nucleotide hydrolysis modulates these events remain unclear. ArsA is an archetypal member of the intradimeric Walker A (IWA) family of ATPases, implicated in a diversity of complex biological functions. Conformational changes typical of IWA ATPases have been postulated to drive these molecular events but have not been demonstrated. We report cryogenic electron microscopy (cryo-EM) structures of ArsA in MgADP-bound and MgATP-boundopenstates, as well as a distinctclosedMgATP-bound state liganded to As III . X-ray absorption spectroscopy (XAS) confirmed three-coordinate binding of As III to the conserved cysteines at the metalloid-binding site of the closed state. Coupled with biochemical characterization, our cryo-EM structures reveal key conformational changes in the ArsA catalytic cycle consistent with other IWA ATPases and provide the structural basis for allosteric activation of nucleotide hydrolysis by As III . This work establishes how the nucleotide state of ArsA transiently creates a high-affinity binding site that can sequester metalloid within the cell, followed by a nucleotide-driven handoff to ArsB for efflux.
The endosymbiotic hypothesis for the origin of the eukaryotic cell has been applied to the origin of the mitochondria and chloroplasts. However as has been pointed out by Mereschowsky in 1905, it should also be applied to the nucleus as well. If the nucleus, mitochondria and chloroplasts are endosymbionts, then it is likely that the organism that did the engulfing was not a DNA-based organism. In fact, it is useful to postulate that this organism was a primitive RNA-based organism. This hypothesis would explain the preponderance of RNA viruses found in eukaryotic cells. The centriole and basal body do not have a double membrane or DNA. Like all MTOCs (microtubule organising centres), they have a structural or morphic RNA implicated in their formation. This would argue for their origin in the early RNA-based organism rather than in an endosymbiotic event involving bacteria. Finally, the eukaryotic cell uses RNA in ways quite unlike bacteria, thus pointing to a greater emphasis of RNA in both control and structure in the cell. The origin of the eukaryotic cell may tell us why it rather than its prokaryotic relative evolved into the metazoans who are reading this paper.
Novel methods of retroviral-mediated gene transfer for the in vivo corporation and stable expression of eukaryotic or prokaryotic foreign genes in tissues of living animals is described. More specifically, methods of incorporating foreign genes into mitotically active cells are disclosed. The constitutive and stable expression of E. coli .beta.-galactosidase gene under the promoter control of the Moloney murine leukemia virus long terminal repeat is employed as a particularly preferred embodiment, by way of example, establishes the model upon which the incorporation of a foreign gene into a mitotically-active living eukaryotic tissue is based. Use of the described methods in therapeutic treatments for genetic diseases, such as those muscular degenerative diseases, is also presented. In muscle tissue, the described processes result in genetically-altered satellite cells which proliferate daughter myoblasts which preferentially fuse to form a single undamaged muscle fiber replacing damaged muscle tissue in a treated animal. The retroviral vector, by way of example, includes a dystrophin gene construct for use in treating muscular dystrophy. The present invention also comprises an experimental model utilizable in the study of the physiological regulation of skeletal muscle gene expression in intact animals.
High-performance liquid chromatography (HPLC) with a gradient-elution technique was utilized to separate and quantify chlorophylls a and b as well as major carotenoid pigments present in freeze-dried preprations of prochloron-didemnid associations and in Prochloron cells separated from host colonies. Results confirm earlier spectrophotometric evidence for both chlorophylls a and b in this prokaryote. Chlorophyll a:b ratios range from 4.14 to 19.71; generally good agreement was found between ratios determined in isolated cell preprations and in symbiotic colonies (in hospite). These values are 1.5 to 5-fold higher than ratios determined in a variety of eukaryotic green plants. The carotenoids in Prochloron are quantitatively and qualitatively similar to those found in various freshwater and marine blue-green algae (cyanopbytes) from high-light environments. However, Prochloron differs from cyanophytes by the absence of myxoxanthophyll and related glycosidic carotenoids. It pigment characteristics are considered sufficiently different from those of cyanophytes to justify its assignment to a separate algal division.
ABSTRACT The nitrogenase gene cluster of unicellular diazotrophic cyanobacteria, such asCyanothece, is frequently selected by nature for nitrogen-fixing partnerships with eukaryotic phototrophs. The essential cluster components that confer an advantage in such partnerships remain underexplored. To use this cluster for the development of synthetic, phototrophic nitrogen-fixing systems, a thorough and systematic analysis of its constituent genes is necessary. An initial effort to assess the possibility of engineering this cluster into non-diazotrophic phototrophs led to the generation of aSynechocystis6803 strain with significant nitrogenase activity. In the current study, a refactoring approach was taken to determine the dispensability of the non-structural genes in the cluster and define a minimal gene set for constructing a functional nitrogenase for phototrophs. Using a bottom-up strategy, thenifgenes fromCyanothece51142 were re-organized to form new operons. The genes were then seamlessly removed to determine their essentiality in the nitrogen fixation process. We demonstrate that besides the structural genesnifHDK,nifBSUENPVZTXW, as well ashesAB, are important for optimal nitrogenase function in a phototroph. We also show that optimal expression of these genes is crucial for efficient nitrogenase activity. Our findings provide a solid foundation for generating synthetic systems that will facilitate solar-powered conversion of atmospheric nitrogen into nitrogen-rich compounds, a stride toward a greener world. IMPORTANCE Integrating nitrogen fixation genes into various photosynthetic organisms is an exciting strategy for converting atmospheric nitrogen into nitrogen-rich products in a green and energy-efficient way. In order to facilitate this process, it is essential that we understand the fundamentals of the functioning of a prokaryotic nitrogen-fixing machinery in a non-diazotrophic, photoautotrophic cell. This study examines a nitrogenase gene cluster that has been naturally selected on multiple occasions for a nitrogen-fixing partnership by eukaryotic photoautotrophs and provides a basic blueprint for designing a photosynthetic organism with nitrogen-fixing ability.