ASU Scholarship Showcase

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.

The probiotic effects of Lactobacillus reuteri have been speculated to partly depend on its capacity to produce the antimicrobial substance reuterin during the reduction of glycerol in the gut. In this study, the potential of this process to protect human intestinal epithelial cells against infection with Salmonella enterica serovar Typhimurium was investigated. We used a three-dimensional (3-D) organotypic model of human colonic epithelium that was previously validated and applied to study interactions between S. Typhimurium and the intestinal epithelium that lead to enteric salmonellosis. Using this model system, we show that L. reuteri protects the intestinal cells against the early stages of Salmonella infection and that this effect is significantly increased when L. reuteri is stimulated to produce reuterin from glycerol. More specifically, the reuterin-containing ferment of L. reuteri caused a reduction in Salmonella adherence and invasion (1 log unit), and intracellular survival (2 log units). In contrast, the L. reuteri ferment without reuterin stimulated growth of the intracellular Salmonella population with 1 log unit. The short-term exposure to reuterin or the reuterin-containing ferment had no observed negative impact on intestinal epithelial cell health. However, long-term exposure (24 h) induced a complete loss of cell-cell contact within the epithelial aggregates and compromised cell viability. Collectively, these results shed light on a potential role for reuterin in inhibiting Salmonella-induced intestinal infections and may support the combined application of glycerol and L. reuteri. While future in vitro and in vivo studies of reuterin on intestinal health should fine-tune our understanding of the mechanistic effects, in particular in the presence of a complex gut microbiota, this the first report of a reuterin effect on the enteric infection process in any mammalian cell type.

Extra-intestinal pathogenic E. coli (ExPEC), including avian pathogenic E. coli (APEC), pose a considerable threat to both human and animal health, with illness causing substantial economic loss. APEC strain χ7122 (O78∶K80∶H9), containing three large plasmids [pChi7122-1 (IncFIB/FIIA-FIC), pChi7122-2 (IncFII), and pChi7122-3 (IncI₂)]; and a small plasmid pChi7122-4 (ColE2-like), has been used for many years as a model strain to study the molecular mechanisms of ExPEC pathogenicity and zoonotic potential. We previously sequenced and characterized the plasmid pChi7122-1 and determined its importance in systemic APEC infection; however the roles of the other pChi7122 plasmids were still ambiguous. Herein we present the sequence of the remaining pChi7122 plasmids, confirming that pChi7122-2 and pChi7122-3 encode an ABC iron transport system (eitABCD) and a putative type IV fimbriae respectively, whereas pChi7122-4 is a cryptic plasmid. New features were also identified, including a gene cluster on pChi7122-2 that is not present in other E. coli strains but is found in Salmonella serovars and is predicted to encode the sugars catabolic pathways. In vitro evaluation of the APEC χ7122 derivative strains with the three large plasmids, either individually or in combinations, provided new insights into the role of plasmids in biofilm formation, bile and acid tolerance, and the interaction of E. coli strains with 3-D cultures of intestinal epithelial cells. In this study, we show that the nature and combinations of plasmids, as well as the background of the host strains, have an effect on these phenomena. Our data reveal new insights into the role of extra-chromosomal sequences in fitness and diversity of ExPEC in their phenotypes.

Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.

We know very little about how soil-borne pollutants such as selenium (Se) can impact pollinators, even though Se has contaminated soils and plants in areas where insect pollination can be critical to the functioning of both agricultural and natural ecosystems. Se can be biotransferred throughout the food web, but few studies have examined its effects on the insects that feed on Se-accumulating plants, particularly pollinators. In laboratory bioassays, we used proboscis extension reflex (PER) and taste perception to determine if the presence of Se affected the gustatory response of honey bee (Apis mellifera L., Hymenoptera: Apidae) foragers. Antennae and proboscises were stimulated with both organic (selenomethionine) and inorganic (selenate) forms of Se that commonly occur in Se-accumulating plants. Methionine was also tested. Each compound was dissolved in 1 M sucrose at 5 concentrations, with sucrose alone as a control. Antennal stimulation with selenomethionine and methionine reduced PER at higher concentrations. Selenate did not reduce gustatory behaviors. Two hours after being fed the treatments, bees were tested for sucrose response threshold. Bees fed selenate responded less to sucrose stimulation. Mortality was higher in bees chronically dosed with selenate compared with a single dose. Selenomethionine did not increase mortality except at the highest concentration. Methionine did not significantly impact survival. Our study has shown that bees fed selenate were less responsive to sucrose, which may lead to a reduction in incoming floral resources needed to support coworkers and larvae in the field. If honey bees forage on nectar containing Se (particularly selenate), reductions in population numbers may occur due to direct toxicity. Given that honey bees are willing to consume food resources containing Se and may not avoid Se compounds in the plant tissues on which they are foraging, they may suffer similar adverse effects as seen in other insect guilds.

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.

Octopamine plays an important role in many behaviors in invertebrates. It acts via binding to G protein coupled receptors located on the plasma membrane of responsive cells. Several distinct subtypes of octopamine receptors have been found in invertebrates, yet little is known about the expression pattern of these different receptor subtypes and how each subtype may contribute to different behaviors. One honey bee (Apis mellifera) octopamine receptor, AmOA1, was recently cloned and characterized. Here we continue to characterize the AmOA1 receptor by investigating its distribution in the honey bee brain. We used two independent antibodies produced against two distinct peptides in the carboxyl-terminus to study the distribution of the AmOA1 receptor in the honey bee brain. We found that both anti-AmOA1 antibodies revealed labeling of cell body clusters throughout the brain and within the following brain neuropils: the antennal lobes; the calyces, pedunculus, vertical (alpha, gamma) and medial (beta) lobes of the mushroom body; the optic lobes; the subesophageal ganglion; and the central complex. Double immunofluorescence staining using anti-GABA and anti-AmOA1 receptor antibodies revealed that a population of inhibitory GABAergic local interneurons in the antennal lobes express the AmOA1 receptor in the cell bodies, axons and their endings in the glomeruli. In the mushroom bodies, AmOA1 receptors are expressed in a subpopulation of inhibitory GABAergic feedback neurons that ends in the visual (outer half of basal ring and collar regions) and olfactory (lip and inner basal ring region) calyx neuropils, as well as in the collar and lip zones of the vertical and medial lobes. The data suggest that one effect of octopamine via AmOA1 in the antennal lobe and mushroom body is to modulate inhibitory neurons.

Neural representations of odors are subject to computations that involve sequentially convergent and divergent anatomical connections across different areas of the brains in both mammals and insects. Furthermore, in both mammals and insects higher order brain areas are connected via feedback connections. In order to understand the transformations and interactions that this connectivity make possible, an ideal experiment would compare neural responses across different, sequential processing levels. Here we present results of recordings from a first order olfactory neuropile – the antennal lobe (AL) – and a higher order multimodal integration and learning center – the mushroom body (MB) – in the honey bee brain. We recorded projection neurons (PN) of the AL and extrinsic neurons (EN) of the MB, which provide the outputs from the two neuropils. Recordings at each level were made in different animals in some experiments and simultaneously in the same animal in others. We presented two odors and their mixture to compare odor response dynamics as well as classification speed and accuracy at each neural processing level. Surprisingly, the EN ensemble significantly starts separating odor stimuli rapidly and before the PN ensemble has reached significant separation. Furthermore the EN ensemble at the MB output reaches a maximum separation of odors between 84–120 ms after odor onset, which is 26 to 133 ms faster than the maximum separation at the AL output ensemble two synapses earlier in processing. It is likely that a subset of very fast PNs, which respond before the ENs, may initiate the rapid EN ensemble response. We suggest therefore that the timing of the EN ensemble activity would allow retroactive integration of its signal into the ongoing computation of the AL via centrifugal feedback.

Investigation into the causes underlying the rapid, global amphibian decline provides critical insight into the effects of changing ecosystems. Hypothesized and confirmed links between amphibian declines, disease, and environmental changes are increasingly represented in published literature. However, there are few long-term amphibian studies that include data on population size, abnormality/injury rates, disease, and habitat variables to adequately assess changes through time. We cultured and identified microorganisms isolated from abnormal/injured and repressed tissue regeneration sites of the endangered Ozark Hellbender, Cryptobranchus alleganiensis bishopi, to discover potential causative agents responsible for their significant decline in health and population. This organism and our study site were chosen because the population and habitat of C. a. bishopi have been intensively studied from 1969–2009, and the abnormality/injury rate and apparent lack of regeneration were established.

Although many bacterial and fungal isolates recovered were common environmental organisms, several opportunistic pathogens were identified in association with only the injured tissues of C.a. bishopi. Bacterial isolates included Aeromonas hydrophila, a known amphibian pathogen, Granulicetella adiacens, Gordonai terrae, Stenotrophomonas maltophilia, Aerococcus viridans, Streptococcus pneumoniae and a variety of Pseudomonads, including Pseudomonas aeruginosa, P. stutzeri, and P. alcaligenes. Fungal isolates included species in the genera Penicillium, Acremonium, Cladosporium, Curvularia, Fusarium, Streptomycetes, and the Class Hyphomycetes. Many of the opportunistic pathogens identified are known to form biofilms. Lack of isolation of the same organism from all wounds suggests that the etiological agent responsible for the damage to C. a. bishopi may not be a single organism. To our knowledge, this is the first study to profile the external microbial consortia cultured from a Cryptobranchid salamander. The incidence of abnormalities/injury and retarded regeneration in C. a. bishopi may have many contributing factors including disease and habitat degradation. Results from this study may provide insight into other amphibian population declines.