ASU Scholarship Showcase

Syntrophic interactions between organohalide-respiring and fermentative microorganisms are critical for effective bioremediation of halogenated compounds. This work investigated the effect of ammonium concentration (up to 4 g liter^-1 NH4+-N) on trichloroethene-reducing Dehalococcoides mccartyi and Geobacteraceae in microbial communities fed lactate and methanol. We found that production of ethene by D. mccartyi occurred in mineral medium containing ≤2 g liter^-1 NH4+-N and in landfill leachate. For the partial reduction of trichloroethene (TCE) to cis-dichloroethene (cis-DCE) at ≥1 g liter^-1 NH4+-N, organohalide-respiring dynamics shifted from D. mccartyi and Geobacteraceae to mainly D. mccartyi. An increasing concentration of ammonium was coupled to lower metabolic rates, longer lag times, and lower gene abundances for all microbial processes studied. The methanol fermentation pathway to acetate and H₂ was conserved, regardless of the ammonium concentration provided. However, lactate fermentation shifted from propionic to acetogenic at concentrations of ≥2 g liter^-1 NH4+-N. Our study findings strongly support a tolerance of D. mccartyi to high ammonium concentrations, highlighting the feasibility of organohalide respiration in ammonium-contaminated subsurface environments.

Low fluid shear force, including that encountered in microgravity models, induces bacterial responses, but the range of bacteria capable of responding to this signal remains poorly characterized. We systematically analyzed a range of Gram negative Enterobacteriaceae for conservation of the low-shear modeled microgravity (LSMMG) response using phenotypic assays, qPCR, and targeted mutations. Our results indicate LSMMG response conservation across Enterobacteriacae with potential variance in up- or down-regulation of a given response depending on genus. Based on the data, we analyzed the role of the trp operon genes and the TrpR regulator in the LSMMG response using targeted mutations in these genes in S. Typhimurium and E. coli. We found no alteration of the LSMMG response compared to WT in these mutant strains under the conditions tested here. To our knowledge, this study is first-of-kind for Citrobacter, Enterobacter, and Serratia, presents novel data for Escherichia, and provides the first analysis of trp genes in LSMMG responses. This impacts our understanding of how LSMMG affects bacteria and our ability to modify bacteria with this condition in the future.

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection.

Butyrate is a common fatty acid produced in important fermentative systems, such as the human/animal gut and other H₂ production systems. Despite its importance, there is little information on the partnerships between butyrate producers and other bacteria. The objective of this work was to uncover butyrate-producing microbial communities and possible metabolic routes in a controlled fermentation system aimed at butyrate production. The butyrogenic reactor was operated at 37°C and pH 5.5 with a hydraulic retention time of 31 h and a low hydrogen partial pressure (PH₂). High-throughput sequencing and metagenome functional prediction from 16S rRNA data showed that butyrate production pathways and microbial communities were different during batch (closed) and continuous-mode operation. Lactobacillaceae, Lachnospiraceae, and Enterococcaceae were the most abundant phylotypes in the closed system without PH₂ control, whereas Prevotellaceae, Ruminococcaceae, and Actinomycetaceae were the most abundant phylotypes under continuous operation at low PH₂. Putative butyrate producers identified in our system were from Prevotellaceae, Clostridiaceae, Ruminococcaceae, and Lactobacillaceae. Metagenome prediction analysis suggests that nonbutyrogenic microorganisms influenced butyrate production by generating butyrate precursors such as acetate, lactate, and succinate. 16S rRNA gene analysis suggested that, in the reactor, a partnership between identified butyrogenic microorganisms and succinate (i.e., Actinomycetaceae), acetate (i.e., Ruminococcaceae and Actinomycetaceae), and lactate producers (i.e., Ruminococcaceae and Lactobacillaceae) took place under continuous-flow operation at low PH₂.

Salmonella enterica serovar Typhimurium strains belonging to sequence type ST313 are a major cause of fatal bacteremia among HIV-infected adults and children in sub-Saharan Africa. Unlike “classical” non-typhoidal Salmonella (NTS), gastroenteritis is often absent during ST313 infections and isolates are most commonly recovered from blood, rather than from stool. This is consistent with observations in animals, in which ST313 strains displayed lower levels of intestinal colonization and higher recovery from deeper tissues relative to classic NTS isolates. A better understanding of the key environmental factors regulating these systemic infections is urgently needed. Our previous studies using dynamic Rotating Wall Vessel (RWV) bioreactor technology demonstrated that physiological levels of fluid shear regulate virulence, gene expression, and stress response profiles of classic S. Typhimurium. Here we provide the first demonstration that fluid shear alters the virulence potential and pathogenesis-related stress responses of ST313 strain D23580 in a manner that differs from classic NTS.

Astronauts are exposed to a unique combination of stressors during spaceflight, which leads to alterations in their physiology and potentially increases their susceptibility to disease, including infectious diseases. To evaluate the potential impact of the spaceflight environment on the regulation of molecular pathways mediating cellular stress responses, we performed a first-of-its-kind pilot study to assess spaceflight-related gene-expression changes in the whole blood of astronauts. Using an array comprised of 234 well-characterized stress-response genes, we profiled transcriptomic changes in six astronauts (four men and two women) from blood preserved before and immediately following the spaceflight. Differentially regulated transcripts included those important for DNA repair, oxidative stress, and protein folding/degradation, including HSP90AB1, HSP27, GPX1, XRCC1, BAG-1, HHR23A, FAP48, and C-FOS. No gender-specific differences or relationship to number of missions flown was observed. This study provides a first assessment of transcriptomic changes occurring in the whole blood of astronauts in response to spaceflight.

Anode-respiring bacteria (ARB) generate electric current in microbial electrochemical cells (MXCs) by channeling electrons from the oxidation of organic substrates to an electrode. Production of high current densities by monocultures in MXCs has resulted almost exclusively from the activity of Geobacter sulfurreducens, a neutrophilic freshwater Fe(III)-reducing bacterium and the highest-current-producing member documented for the Geobacteraceae family of the Deltaproteobacteria. Here we report high current densities generated by haloalkaliphilic Geoalkalibacter spp., thus broadening the capability for high anode respiration rates by including other genera within the Geobacteraceae. In this study, acetate-fed pure cultures of two related Geoalkalibacter spp. produced current densities of 5.0 to 8.3 and 2.4 to 3.3 A m^-2 under alkaline (pH 9.3) and saline (1.7% NaCl) conditions, respectively. Chronoamperometric studies of halophilic Glk. subterraneus DSM 23483 and alkaliphilic Glk. ferrihydriticus DSM 17813 suggested that cells performed long-range electron transfer through electrode-attached biofilms and not through soluble electron shuttles. Glk. ferrihydriticus also oxidized ethanol directly to produce current, with maximum current densities of 5.7 to 7.1 A m^-2 and coulombic efficiencies of 84 to 95%. Cyclic voltammetry (CV) elicited a sigmoidal response with characteristic onset, midpoint, and saturation potentials, while CV performed in the absence of an electron donor suggested the involvement of redox molecules in the biofilm that were limited by diffusion. These results matched those previously reported for actively respiring Gb. sulfurreducens biofilms producing similar current densities (~5 to 9 A m^-2).