Jonathan D. Trent

The NASA Astrobiology Roadmap.

The NASA Astrobiology Roadmap provides guidance for research and technology development across the NASA enterprises that encompass the space, Earth, and biological sciences. The ongoing development of astrobiology roadmaps embodies the contributions of diverse scientists and technologists from government, universities, and private institutions. The Roadmap addresses three basic questions: How does life begin and evolve, does life exist elsewhere in the universe, and what is the future of life on Earth and beyond? Seven Science Goals outline the following key domains of investigation: understanding the nature and distribution of habitable environments in the universe, exploring for habitable environments and life in our own solar system, understanding the emergence of life, determining how early life on Earth interacted and evolved with its changing environment, understanding the evolutionary mechanisms and environmental limits of life, determining the principles that will shape life in the future, and recognizing signatures of life on other worlds and on early Earth. For each of these goals, Science Objectives outline more specific high-priority efforts for the next 3-5 years. These 18 objectives are being integrated with NASA strategic planning.

Impact of low-temperature plasmas on Deinococcus radiodurans and biomolecules

The effects of cold plasma on Deinococcus radiodurans, plasmid DNA, and model proteins were assessed using microbiological, spectrometric, and biochemical techniques. In low power O2 plasma (∼25 W, ∼45 mTorr, 90 min), D. radiodurans, a radiation‐resistant bacterium, showed a 99.999% reduction in bioburden. In higher power O2 plasma (100 W and 500 mTorr), the reduction rate increased about 10‐fold and observation by atomic force microscopy showed significant damage to the cell. Damage to cellular lipids, proteins, and chromosome was indicated by losses of infrared spectroscopic peaks at 2930, 1651, 1538, and 1245 cm‐1, respectively. In vitro experiments show that O2 plasmas induce DNA strand scissions and cross‐linking as well as reduction of enzyme activity. The observed degradation and removal of biomolecules was power‐dependent. Exposures to 200 W at 500 mTorr removed biomolecules to below detection limits in 60 s. Emission spectroscopy indicated that D. radiodurans cells were volatilized into CO2, CO, N2, and H2O, confirming that these plasmas were removing complex biological matter from surfaces. A CO2 plasma was not as effective as the O2 plasma, indicating the importance of plasma composition and the dominant role of chemical degradation. Together, these findings have implications for NASA planetary protection schemes and for the contamination of Mars.

Microbial diversity in chronic open wounds

Chronic wounds expose the dermal matrix and underlying tissue to a diversity of microbes from the body and surrounding environment. We determined the microbial diversity of 19 chronic wounds using both molecular methods (sequence analysis of rRNA genes) and routine clinical culturing methods using swab samples. We identified 93 phylotypes in 2,653 rRNA clone sequences and found that compared with other environments, the microbial diversity of chronic wounds is relatively well characterized, i.e., 95% of sequences have ≥97% identity with known human commensals. In total, 75% of sequences belonged to four well‐known wound‐associated phylotypes: Staphylococcus (25%), Corynebacterium (20%), Clostridiales (18%), and Pseudomonas (12%). Approximately 0.5% of sequences (seven phylotypes) belonged to potentially new species. Individual wound samples contained four to 22 phylotypes, but in all wounds only a few (one to three) phylotypes were dominant. In more than half the wound specimens, polymerase chain reaction and culturing methods gave different diversity and dominance information about the microbes present. This exploratory study suggests that combining molecular and culturing methods provides a more complete characterization of the microbial diversity of chronic wounds, and can thereby expand our understanding of how microbiology impacts chronic wound pathology and healing.

DNA from uncultured organisms as a source of 2,5-diketo-D-gluconic acid reductases.

ABSTRACT Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-d-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressed in Escherichia coli. The purified gene products catalyzed the reduction of 2,5-diketo-d-gluconic acid to 2-keto-l-gulonic acid. Compared to previously described DKGRs isolated fromCorynebacterium spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higherkcat/Kmvalues than those previously determined, primarily as a result of better binding of substrate. The Kmvalues for the two new reductases were 57 and 67 μM, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.

The composition, structure and stability of a group II chaperonin are temperature regulated in a hyperthermophilic archaeon

The hyperthermoacidophilic archaeon Sulfolobus shibatae contains group II chaperonins, known as rosettasomes, which are two nine‐membered rings composed of three different 60 kDa subunits (TF55 alpha, beta and gamma). We sequenced the gene for the gamma subunit and studied the temperature‐dependent changes in alpha, beta and gamma expression, their association into rosettasomes and their phylogenetic relationships. Alpha and beta gene expression was increased by heat shock (30 min, 86°C) and decreased by cold shock (30 min, 60°C). Gamma expression was undetectable at heat shock temperatures and low at normal temperatures (75–79°C), but induced by cold shock. Polyacrylamide gel electrophoresis indicated that in vitro alpha and beta subunits form homo‐oligomeric rosettasomes, and mixtures of alpha, beta and gamma form hetero‐oligomeric rosettasomes. Transmission electron microscopy revealed that beta homo‐oligomeric rosettasomes and all hetero‐oligomeric rosettasomes associate into filaments. In vivo rosettasomes were hetero‐oligomeric with an average subunit ratio of 1α:1β:0.1γ in cultures grown at 75°C, a ratio of 1α:3β:1γ in cultures grown at 60°C and a ratio of 2α:3β:0γ after 86°C heat shock. Using differential scanning calorimetry, we determined denaturation temperatures (Tm) for alpha, beta and gamma subunits of 95.7°C, 96.7°C and 80.5°C, respectively, and observed that rosettasomes containing gamma were relatively less stable than those with alpha and/or beta only. We propose that, in vivo, the rosettasome structure is determined by the relative abundance of subunits and not by a fixed geometry. Furthermore, phylogenetic analyses indicate that archaeal chaperonin subunits underwent multiple duplication events within species (paralogy). The independent evolution of these paralogues raises the possibility that chaperonins have functionally diversified between species.

Potential impact of biofouling on the photobioreactors of the Offshore Membrane Enclosures for Growing Algae (OMEGA) system.

The influence of PBR composition [clear polyurethane (PolyU) vs. clear linear low-density polyethylene (LLDPE) (top) and black opaque high-density polyethylene (bottom)] and shape (rectangular vs. tubular) on biofouling and the influence of biofouling on algae productivity were investigated. In 9-week experiments, PBR biofouling was dominated by pennate diatoms and clear plastics developed macroalgae. LLDPE exhibited lower photosynthetic-active-radiation (PAR) light transmittance than PolyU before biofouling, but higher transmittance afterwards. Both rectangular and tubular LLDPE PBRs accumulated biofouling predominantly along their wetted edges. For a tubular LLDPE PBR after 12 weeks of biofouling, the correlation between biomass, percent surface coverage, and PAR transmittance was complex, but in general biomass inversely correlated with transmittance. Wrapping segments of this biofouled LLDPE around an algae culture reduced CO2 and NH3-N utilization, indicating that external biofouling must be controlled.

Heat shock and cold shock in Deinococcus radiodurans.

On the basis of acquired thermotolerance and cryotolerance, the optimal heat shock and cold shock temperatures have been determined for Deinococcus radiodurans. A heat shock at 42°C maximized survival at the lethal temperature of 52°C and a cold shock at 20°C maximized survival after repeated freeze-thawing. Enhanced survival from heat shock was found to be strongly dependent on growth stage, with its greatest effect shortly after phase. Increased synthesis of a total of 67 proteins during heat shock and 42 proteins during cold shock were observed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and autoradiography. Eight of the most highly induced heat shock proteins shown by 2D PAGE were identified by MALDI-MS as Hsp20, GroEL, DnaK, SodA, Csp, Protease I and two proteins of unknown function.

Intracellular localization of a group II chaperonin indicates a membrane-related function

Chaperonins are protein complexes that are believed to function as part of a protein folding system in the cytoplasm of the cell. We observed, however, that the group II chaperonins known as rosettasomes in the hyperthermophilic archaeon Sulfolobus shibatae, are not cytoplasmic but membrane associated. This association was observed in cultures grown at 60°C and 76°C or heat-shocked at 85°C by using immunofluorescence microscopy and in thick sections of rapidly frozen cells grown at 76°C by using immunogold electron microscopy. We observed that increased abundance of rosettasomes after heat shock correlated with decreased membrane permeability at lethal temperature (92°C). This change in permeability was not seen in cells heat-shocked in the presence of the amino acid analogue azetidine 2-carboxylic acid, indicating functional protein synthesis influences permeability. Azetidine experiments also indicated that observed heat-induced changes in lipid composition in S. shibatae could not account for changes in membrane permeability. Rosettasomes purified from cultures grown at 60°C and 76°C or heat-shocked at 85°C bind to liposomes made from either the bipolar tetraether lipids of Sulfolobus or a variety of artificial lipid mixtures. The presence of rosettasomes did not significantly change the transition temperature of liposomes, as indicated by differential scanning calorimetry, or the proton permeability of liposomes, as indicated by pyranine fluorescence. We propose that these group II chaperonins function as a structural element in the natural membrane based on their intracellular location, the correlation between their functional abundance and membrane permeability, and their potential distribution on the membrane surface.

Research Spotlight: The future of biofuels: is it in the bag?

How and where it will be possible to produce biofuels at a scale that can compete with fossil fuels, without competing with agriculture for water, fertilizer and land, is a fundamental unanswered question. We propose that the answer could be offshore membrane enclosures for growing algae. Microalgae are the fastest growing biomass and best oil producers known; by cultivating microalgae offshore using wastewater as a source of water and nutrients in floating photobioreactors (PBRs), the system would not compete with agriculture. Furthermore, freshwater microalgae clean the wastewater, capture CO2 and, if they accidentally escape, they cannot become invasive species because they cannot thrive in seawater. The seawater supports the PBRs, controls temperature and can be used for forward osmosis to concentrate nutrients and facilitate harvesting. Algae products, wastewater treatment, carbon sequestration and compatible aquaculture support the economics of the system as a whole. The completion of a 2-year feasibility study on prototype PBRs, control systems, biofouling, wastewater treatment, life cycle analysis and energy return on investment sets the stage for future offshore studies.

An intrinsically fluorescent recognition ligand scaffold based on chaperonin protein and semiconductor quantum-dot conjugates.

Genetic engineering of a novel protein-nanoparticle hybrid system with great potential for biosensing applications and for patterning of various types of nanoparticles is described. The hybrid system is based on a genetically modified chaperonin protein from the hyperthermophilic archaeon Sulfolobus shibatae. This chaperonin is an 18-subunit double ring, which self-assembles in the presence of Mg ions and ATP. Described here is a mutant chaperonin (His-beta-loopless, HBLL) with increased access to the central cavity and His-tags on each subunit extending into the central cavity. This mutant binds water-soluble semiconductor quantum dots, creating a protein-encapsulated fluorescent nanoparticle. The new bioconjugate has high affinity, in the order of strong antibody-antigen interactions, a one-to-one protein-nanoparticle stoichiometry, and high stability. By adding selective binding sites to the solvent-exposed regions of the chaperonin, this protein-nanoparticle bioconjugate becomes a sensor for specific targets.