ASU Scholarship Showcase

A structurally and compositionally well-defined and spectrally tunable artificial light-harvesting system has been constructed in which multiple organic dyes attached to a three-arm-DNA nanostructure serve as an antenna conjugated to a photosynthetic reaction center isolated from Rhodobacter sphaeroides 2.4.1. The light energy absorbed by the dye molecules is transferred to the reaction center, where charge separation takes place. The average number of DNA three-arm junctions per reaction center was tuned from 0.75 to 2.35. This DNA-templated multichromophore system serves as a modular light-harvesting antenna that is capable of being optimized for its spectral properties, energy transfer efficiency, and photostability, allowing one to adjust both the size and spectrum of the resulting structures. This may serve as a useful test bed for developing nanostructured photonic systems.

Time-resolved fluorescence spectroscopy was used to explore the pathway and kinetics of energy transfer in photosynthetic membrane vesicles (chromatophores) isolated from Rhodobacter (Rba.) sphaeroides cells harvested 2, 4, 6 or 24 hours after a transition from growth in high to low level illumination. As previously observed, this light intensity transition initiates the remodeling of the photosynthetic apparatus and an increase in the number of light harvesting 2 (LH2) complexes relative to light harvesting 1 (LH1) and reaction center (RC) complexes. It has generally been thought that the increase in LH2 complexes served the purpose of increasing the overall energy transmission to the RC. However, fluorescence lifetime measurements and analysis in terms of energy transfer within LH2 and between LH2 and LH1 indicate that, during the remodeling time period measured, only a portion of the additional LH2 generated are well connected to LH1 and the reaction center. The majority of the additional LH2 fluorescence decays with a lifetime comparable to that of free, unconnected LH2 complexes. The presence of large LH2-only domains has been observed by atomic force microscopy in Rba. sphaeroides chromatophores (Bahatyrova et al., Nature, 2004, 430, 1058), providing structural support for the existence of pools of partially connected LH2 complexes. These LH2-only domains represent the light-responsive antenna complement formed after a switch in growth conditions from high to low illumination, while the remaining LH2 complexes occupy membrane regions containing mixtures of LH2 and LH1–RC core complexes. The current study utilized a multi-parameter approach to explore the fluorescence spectroscopic properties related to the remodeling process, shedding light on the structure-function relationship of the photosynthetic assembles. Possible reasons for the accumulation of these largely disconnected LH2-only pools are discussed.

Biological Soil Crusts (BSCs) are organosedimentary assemblages comprised of microbes and minerals in topsoil of terrestrial environments. BSCs strongly impact soil quality in dryland ecosystems (e.g., soil structure and nutrient yields) due to pioneer species such as Microcoleus vaginatus; phototrophs that produce filaments that bind the soil together, and support an array of heterotrophic microorganisms. These microorganisms in turn contribute to soil stability and biogeochemistry of BSCs. Non-cyanobacterial populations of BSCs are less well known than cyanobacterial populations. Therefore, we attempted to isolate a broad range of numerically significant and phylogenetically representative BSC aerobic heterotrophs. Combining simple pre-treatments (hydration of BSCs under dark and light) and isolation strategies (media with varying nutrient availability and protection from oxidative stress) we recovered 402 bacterial and one fungal isolate in axenic culture, which comprised 116 phylotypes (at 97% 16S rRNA gene sequence homology), 115 bacterial and one fungal. Each medium enriched a mostly distinct subset of phylotypes, and cultivated phylotypes varied due to the BSC pre-treatment. The fraction of the total phylotype diversity isolated, weighted by relative abundance in the community, was determined by the overlap between isolate sequences and OTUs reconstructed from metagenome or metatranscriptome reads. Together, more than 8% of relative abundance of OTUs in the metagenome was represented by our isolates, a cultivation efficiency much larger than typically expected from most soils. We conclude that simple cultivation procedures combined with specific pre-treatment of samples afford a significant reduction in the culturability gap, enabling physiological and metabolic assays that rely on ecologically relevant axenic cultures.

The heterocyclic indole-alkaloid scytonemin is a sunscreen found exclusively among cyanobacteria. An 18-gene cluster is responsible for scytonemin production in Nostoc punctiforme ATCC 29133. The upstream genes scyABCDEF in the cluster are proposed to be responsible for scytonemin biosynthesis from aromatic amino acid substrates. In vitro studies of ScyA, ScyB, and ScyC proved that these enzymes indeed catalyze initial pathway reactions. Here we characterize the role of ScyD, ScyE, and ScyF, which were logically predicted to be responsible for late biosynthetic steps, in the biological context of N. punctiforme. In-frame deletion mutants of each were constructed (ΔscyD, ΔscyE, and ΔscyF) and their phenotypes studied. Expectedly, ΔscyE presents a scytoneminless phenotype, but no accumulation of the predicted intermediaries. Surprisingly, ΔscyD retains scytonemin production, implying that it is not required for biosynthesis. Indeed, scyD presents an interesting evolutionary paradox: it likely originated in a duplication event from scyE, and unlike other genes in the operon, it has not been subjected to purifying selection. This would suggest that it is a pseudogene, and yet scyD is highly conserved in the scytonemin operon of cyanobacteria. ΔscyF also retains scytonemin production, albeit exhibiting a reduction of the production yield compared with the wild-type. This indicates that ScyF is not essential but may play an adjuvant role for scytonemin synthesis. Altogether, our findings suggest that these downstream genes are not responsible, as expected, for the late steps of scytonemin synthesis and we must look for those functions elsewhere. These findings are particularly important for biotechnological production of this sunscreen through heterologous expression of its genes in more tractable organisms.

There are an increasing variety of applications in which peptides are both synthesized and used attached to solid surfaces. This has created a need for high throughput sequence analysis directly on surfaces. However, common sequencing approaches that can be adapted to surface bound peptides lack the throughput often needed in library-based applications. Here we describe a simple approach for sequence analysis directly on solid surfaces that is both high speed and high throughput, utilizing equipment available in most protein analysis facilities. In this approach, surface bound peptides, selectively labeled at their N-termini with a positive charge-bearing group, are subjected to controlled degradation in ammonia gas, resulting in a set of fragments differing by a single amino acid that remain spatially confined on the surface they were bound to. These fragments can then be analyzed by MALDI mass spectrometry, and the peptide sequences read directly from the resulting spectra.

Background: Chemistry and particularly enzymology at surfaces is a topic of rapidly growing interest, both in terms of its role in biological systems and its application in biocatalysis. Existing protein immobilization approaches, including noncovalent or covalent attachments to solid supports, have difficulties in controlling protein orientation, reducing nonspecific absorption and preventing protein denaturation. New strategies for enzyme immobilization are needed that allow the precise control over orientation and position and thereby provide optimized activity.

Methodology/Principal Findings: A method is presented for utilizing peptide ligands to immobilize enzymes on surfaces with improved enzyme activity and stability. The appropriate peptide ligands have been rapidly selected from high-density arrays and when desirable, the peptide sequences were further optimized by single-point variant screening to enhance both the affinity and activity of the bound enzyme. For proof of concept, the peptides that bound to β-galactosidase and optimized its activity were covalently attached to surfaces for the purpose of capturing target enzymes. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activity and stability, as well as controlled protein orientation.

Conclusions/Significance: A simple method for immobilizing enzymes through specific interactions with peptides anchored on surfaces has been developed. This approach will be applicable to the immobilization of a wide variety of enzymes on surfaces with optimized orientation, location and performance, and provides a potential mechanism for the patterned self-assembly of multiple enzymes on surfaces.

N₂ fixation and ammonia oxidation (AO) are the two most important processes in the nitrogen (N) cycle of biological soil crusts (BSCs). We studied the short-term response of acetylene reduction assay (ARA) rates, an indicator of potential N₂ fixation, and AO rates to temperature (T, -5°C to 35°C) in BSC of different successional stages along the BSC ecological succession and geographic origin (hot Chihuahuan and cooler Great Basin deserts). ARA in all BSCs increased with T until saturation occurred between 15 and 20°C, and declined at 30–35°C. Culture studies using cyanobacteria isolated from these crusts indicated that the saturating effect was traceable to their inability to grow well diazotrophically within the high temperature range. Below saturation, temperature response was exponential, with Q₁₀ significantly different in the two areas (~ 5 for Great Basin BSCs; 2–3 for Chihuahuan BSCs), but similar between the two successional stages. However, in contrast to ARA, AO showed a steady increase to 30–35°C in Great Basin, and Chihuhuan BSCs showed no inhibition at any tested temperature. The T response of AO also differed significantly between Great Basin (Q₁₀ of 4.5–4.8) and Chihuahuan (Q₁₀ of 2.4–2.6) BSCs, but not between successional stages. Response of ARA rates to T did not differ from that of AO in either desert. Thus, while both processes scaled to T in unison until 20°C, they separated to an increasing degree at higher temperature. As future warming is likely to occur in the regions where BSCs are often the dominant living cover, this predicted decoupling is expected to result in higher proportion of nitrates in soil relative to ammonium. As nitrate is more easily lost as leachate or to be reduced to gaseous forms, this could mean a depletion of soil N over large landscapes globally.

Background: Medical and public health scientists are using evolution to devise new strategies to solve major health problems. But based on a 2003 survey, medical curricula may not adequately prepare physicians to evaluate and extend these advances. This study assessed the change in coverage of evolution in North American medical schools since 2003 and identified opportunities for enriching medical education.

Methods: In 2013, curriculum deans for all North American medical schools were invited to rate curricular coverage and perceived importance of 12 core principles, the extent of anticipated controversy from adding evolution, and the usefulness of 13 teaching resources. Differences between schools were assessed by Pearson’s chi-square test, Student’s t-test, and Spearman’s correlation. Open-ended questions sought insight into perceived barriers and benefits.

Results: Despite repeated follow-up, 60 schools (39%) responded to the survey. There was no evidence of sample bias. The three evolutionary principles rated most important were antibiotic resistance, environmental mismatch, and somatic selection in cancer. While importance and coverage of principles were correlated (r = 0.76, P < 0.01), coverage (at least moderate) lagged behind importance (at least moderate) by an average of 21% (SD = 6%). Compared to 2003, a range of evolutionary principles were covered by 4 to 74% more schools. Nearly half (48%) of responders anticipated igniting controversy at their medical school if they added evolution to their curriculum. The teaching resources ranked most useful were model test questions and answers, case studies, and model curricula for existing courses/rotations. Limited resources (faculty expertise) were cited as the major barrier to adding more evolution, but benefits included a deeper understanding and improved patient care.

Conclusion: North American medical schools have increased the evolution content in their curricula over the past decade. However, coverage is not commensurate with importance. At a few medical schools, anticipated controversy impedes teaching more evolution. Efforts to improve evolution education in medical schools should be directed toward boosting faculty expertise and crafting resources that can be easily integrated into existing curricula.

Cyanobacteria are considered good models for biohydrogen production because they are relatively simple organisms with a demonstrable ability to generate H₂ under certain physiological conditions. However, most produce only little H₂, revert readily to H₂ consumption, and suffer from hydrogenase sensitivity to O₂. Strains of the cyanobacteria Lyngbya aestuarii and Microcoleus chthonoplastes obtained from marine intertidal cyanobacterial mats were recently found to display much better H₂ production potential. Because of their ecological origin in environments that become quickly anoxic in the dark, we hypothesized that this differential ability may have evolved to serve a role in the fermentation of the photosynthate. Here we show that, when forced to ferment internal substrate, these cyanobacteria display desirable characteristics of physiological H₂ production. Among them, the strain L. aestuarii BL J had the fastest specific rates and attained the highest H₂ concentrations during fermentation of photosynthate, which proceeded via a mixed acid fermentation pathway to yield acetate, ethanol, lactate, H₂, CO₂, and pyruvate. Contrary to expectations, the H₂ yield per mole of glucose was only average compared to that of other cyanobacteria. Thermodynamic analyses point to the use of electron donors more electronegative than NAD(P)H in Lyngbya hydrogenases as the basis for its strong H₂ production ability. In any event, the high specific rates and H₂ concentrations coupled with the lack of reversibility of the enzyme, at the expense of internal, photosynthetically generated reductants, makes L. aestuarii BL J and/or its enzymes, a potentially feasible platform for large-scale H₂ production.

Soil surface temperature, an important driver of terrestrial biogeochemical processes, depends strongly on soil albedo, which can be significantly modified by factors such as plant cover. In sparsely vegetated lands, the soil surface can be colonized by photosynthetic microbes that build biocrust communities. Here we use concurrent physical, biochemical and microbiological analyses to show that mature biocrusts can increase surface soil temperature by as much as 10 °C through the accumulation of large quantities of a secondary metabolite, the microbial sunscreen scytonemin, produced by a group of late-successional cyanobacteria. Scytonemin accumulation decreases soil albedo significantly. Such localized warming has apparent and immediate consequences for the soil microbiome, inducing the replacement of thermosensitive bacterial species with more thermotolerant forms. These results reveal that not only vegetation but also microorganisms are a factor in modifying terrestrial albedo, potentially impacting biosphere feedbacks on past and future climate, and call for a direct assessment of such effects at larger scales.